Glioma Cell Lines Research Articles

IMMU-16. PUBLIC TUMOR-WIDE NEOANTIGENS DERIVED FROM CLONAL ABERRANT SPLICING EVENTS DRIVE TUMOR-SPECIFIC T-CELL RESPONSES

Abstract BACKGROUND Immunotherapy in gliomas is limited by tumor heterogeneity and low mutational burden. We developed a novel comprehensive in silico pipeline for detecting tumor-specific splicing events (neojunctions) across multiple cancer types, and in gliomas, we successfully identified tumor-wide, public, alternatively spliced neoantigens (ASNs) that elicit CD8+ T-cell-mediated immune responses. METHODS Our pipeline identified recurring public neojunctions expressed in TCGA RNA-seq (positive sample rate (PSR) > 10%) and not in GTEx normal tissue RNA-seq data (PSR < 1%) across six cancer types. To characterize intratumorally-conserved neojunctions, we utilized multi-site RNA sequencing, and with gliomas, we analyzed approximately 10 maximally-distanced intratumoral sites within 56 patients (n=535). Tumor-wide public neojunction expression was subsequently validated in RNA-sequencing and mass spectrometry (MS) data from patient-derived cell lines (n=68) and samples (n=99). Two independent algorithms then predicted peptide processing likelihood and HLA-binding affinity of ASN candidates. In vitro sensitization (IVS) of healthy donor-derived CD8+ T-cells against high-confidence ASN candidates, followed by 10x VDJ scRNA-seq, was performed to identify ASN-specific TCR sequences. TCRs were transduced into triple-reporter Jurkat76s and co-cultured with ASN and HLA-expressing COS7 cells and glioma cell lines to evaluate TCR functionality. RESULTS Our glioma-specific analysis identified 249 public neojunctions with varying intratumoral heterogeneity. 4 ASNs were concurrently identified in transcriptomic and proteomic glioma data and predicted to be presented by HLA-A*02:01 with high confidence. IVS and subsequent 10x VDJ scRNA-seq on expanded CD8+ populations identified TCR clonotypes reactive against neojunctions in RPL22 (n=7) and GNAS (n=1), the latter being highly intratumorally-conserved (detected in > 90% of spatially-mapped biopsies across 17/56 patients (26.78%)). TCR-transduced T-cells demonstrated recognition and immunogenic activation against endogenously processed and presented neoantigens in glioma and transfected COS7 cells. CONCLUSION Our unique integrative pipeline identified novel public tumor-wide splice-derived neoantigen candidates and TCRs, offering a promising off-the-shelf immunotherapy approach for gliomas.

EPCO-23. EFFECTS OF H3.3G34V MUTATION ON GENOMIC H3K36 AND H3K27 METHYLATION PATTERNS IN ISOGENIC PEDIATRIC GLIOMA CELLS

Abstract BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas (pHGGs). This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase). Consequently, reduced H3K36me3 is observed on H3.3G34V nucleosomes relative to wild-type, contributing to genomic instability and driving a distinct gene expression signature associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3.3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3.3G34V mutant protein in pediatric glioma on H3K36me3, H3K27me3 and H3.3 enrichment in vitro. METHODS Experiments were conducted using an established H3.3G34V mutant patient-derived pediatric high-grade glioma cell line (KNS42), and Histone H3.3 wild-type human astrocytes (NHAs). Lentiviral delivery of a doxycycline-inducible RNAi vector targeted against H3F3A was transduced to KNS42 cells to knock down H3.3G34V protein expression. Cell lines were then compared via ChIP-Seq, Western Blot, proliferation and viability studies. RESULTS We found that the doxycycline-inducible shRNA knockdown of mutant H3F3A encoding the H3.3G34V protein resulted in loss of H3.3G34V enrichment and increased H3K36me3 enrichment throughout the genome. After knockdown, H3.3G34V enrichment was preserved at loci observed to have the greatest H3.3G34V and H3K36me3 enrichment prior to knockdown. Induced expression of mutant H3.3G34V protein in vitro was insufficient to induce genomic H3K36me3 enrichment patterns observed in H3.3G34V mutant glioma cells. We also observed strong co-enrichment of H3.3G34V and wild-type H3.3 protein, as well as greater H3K27me3 enrichment, in cells expressing H3.3G34V. CONCLUSION Our study demonstrates the effects of H3.3G34V mutant protein on genomic H3K36me3, H3K27me3 and H3.3 enrichment patterns in isogenic cell lines.

NIMG-43. A 57-YEAR-OLD MALE WITH PRIMARY GLIOBLASTOMA TREATED WITH LASER INTERSTITIAL THERMAL THERAPY: IMAGING AND AUTOPSY FINDINGS

Abstract INTRODUCTION: Glioblastoma (GBM) is an aggressive primary brain cancer with significant resistance to the current therapeutic approach of chemotherapy and radiotherapy, jointly known as chemoradiation (CRT). Magnetic hyperthermia therapy (MHT) is a promising therapy for GBM that can be used to perform multiple sessions of non-invasive, localized hyperthermia by activating locally delivered magnetic iron oxide nanoparticles (MIONPs) with an external alternating magnetic field (AMF). In this study, MHT-mediated enhancement of CRT was evaluated in murine and human glioma cell lines both in cell culture and in rodents.

EPCO-34. IMAGE-BASED PHENOTYPIC HIGH-CONTENT CRISPR-CAS9 SCREEN FOR CHARACTERIZING CANCER DRIVERS IN PEDIATRIC HIGH-GRADE GLIOMA

Abstract The genomic landscape of pediatric high-grade glioma is characterized by a high level of aneuploidy but few focal amplifications or homozygous deletions. Cancer aneuploidies are the principal source of tumor heterogeneity driving tumor growth and therapy resistance. Here, we deployed an image-based phenotypic high-content CRISPR-Cas9 screen to identify aneuploidy-associated pediatric glioma driver genes. Through integrated computational analyses combining whole-genome sequencing datasets from 106 pediatric gliomas and 81 canine gliomas, we identified a set of 460 genes present in regions of aneuploidy in both diseases. We designed a custom guideRNA library to systemically knockout the 460 gene set using an arrayed CRISPR-Cas9 knockout screen in two pediatric high-grade glioma cell lines. By deploying 72 of 384-well plates on automated confocal Opera Phenix High-Content Screening System and dyes staining cell membrane, nucleus, and apoptotic cells, we established 5 million live-cell images across three timepoints (day 7, 8, 9 post-transduction). Each gene was randomized to 32 randomized wells across four 384-well plates to gather robust morphological profiles. Through image-based nucleus and cell segmentation workflow, we characterized 50 million single cells across 27,648 wells over three time points. Using semi-supervised machine learning methods, we derived 1,050 cell morphological features to characterize knockout phenotypes, e.g., viability, growth, and morphology (size, shape, texture of nucleus and cells) across cells. The top ranked genes were then linked to oncogenes and tumor suppressors based on pathway and ontology analysis as well as functional in vitro and in silico validation using Pediatric Cancer Dependency Map. We find convergence of the most impactful molecular abnormalities - based on their knockout phenotypes - on candidate signaling pathways for the development of new drugs and repurposing of existing drugs for pediatric high-grade glioma.

TMET-19. TARGETING AMINO ACID METABOLIC VULNERABILITIES IN IDH-MUTANT AND IDH-WILDTYPE GLIOMAS

Abstract IDH-wildtype and IDH-mutant glioma have different genetical and metabolic background although their histological appearances are similar. The aim of the study is to reveal the difference in metabolites between IDH-wildtype glioma and IDH-mutant glioma, and to find the effective treatment according to the status of IDH in gliomas. Two artificial cell lines made from human astrocyte were used: NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant). Capillary electrophoresis- and ion chromatography–coupled mass spectrometry revealed that the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells. L-asparaginase, which converts asparagine into aspartate, was more effective in former cells than latter cells. L-asparaginase induced autophagy and inhibition of autophagy by 3-MA suppressed L-asparaginase-induced antitumor effect, which meant that the antitumor effect was at least partially due to the L-asparaginase-induced autophagy. Metabolic assay also showed the lower amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. Pharmacological or genetical inhibition of GLUD1 which converts glutamate to 2-oxoglutarate, suppressed proliferation of the cells by inducing ROS and apoptosis in NHAE6E7hTERTIDHmut cells. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells, revealing that cytotoxicity by GLUD1 inhibitor was at least partially due to the inhibitor-induced ROS. Mutant IDH1 overexpressed glioma cell line showed similar sensitivity to GLUD1 inhibitor to NHAE6E7hTERTIDHmut, which suggested that the difference of sensitivity to GLUD1 inhibitor was due to the status of mutant IDH. In addition, GLUD1 inhibitor suppressed the growth of xenografted glioma cells with overexpressed mutant IDH1. In conclusion, L-asparaginase and Glud1 inhibitor will be new therapeutic option for IDH-wildtype glioma and IDH-mutant glioma, respectively.

PATH-21. GPX4 IS CRUCIALLY IMPLICATED IN THE PATHOBIOLOGY OF GLIOBLASTOMA AND A PUTATIVE ACTIONABLE TARGET

Abstract BACKGROUND Glioblastoma(GBM) is the most common malignant primary brain tumor with a dismal prognosis. Glutathione-mediated inhibition of the ferroptotic form of cell death is crucially implicated in various malignancies, which is principally regulated by glutathione peroxidase 4 (GPX4). However, there is limited data on GBM. The present study evaluated the expression of GPX4 in GBM and its implications in tumor biology, clinical outcome, and therapeutic potentials. METHODS The mRNA and protein expression was assessed by qRT-PCR and immunohistochemistry, respectively, in 75 cases of adult hemispheric GBMs (IDH wild type). For comparison, 21 lower-grade astrocytomas (IDH-mutant, grade-2/3) [LGA] and 20 cases were also evaluated. In-vitro analysis was performed on U87MG and LN229 glioma cell line. RESULTS The mRNA expression of GPX-4 was significantly higher in GBM than LGA, and normal brain (NB) [p-values 0.028, and 0.001, respectively]. Normal brain cases were negative for GPX4. Almost half of the GBMs (49.3%) showed strong immunoreactivity, which was higher than LGAs (23.8%) [p-value -0.04]. Inhibition of GPX-4 in-vitro by 1S-RSL3 & FIN56 was associated with decreased cell survival, proliferation, migration, invasion, and increased cell death. GPX-4 expression was not significantly associated with overall survival in GBM. CONCLUSION GPX-4 is crucial for the tumor cells' survival and aggressive biological behavior. Considering its immune-positivity only in the neoplastic cell and strong positivity in GBM it may be a useful adjunct to the diagnosis. The present study emphasized its role as a potential therapeutic target in the form of 1S-RSL3 & FIN56.

DDDR-24. SINGLE-CELL FUNCTIONAL TISSUE PROFILING IDENTIFIES PROMISING CANDIDATES FOR DRUG REPURPOSING AGAINST GLIOBLASTOMA

Abstract There remains an unmet need for novel treatment strategies against glioblastoma that are more effective than the current standard of care. However, the complex tumor microenvironment and genetic heterogeneity of these tumors require novel technologies enabling high-throughput assessment of cellular responses to treatments on a functional level in complex tumor samples with an intact cellular microenvironment. Here, we used a novel single-cell functional tissue profiling platform based on high—throughput microscopy followed by computer vision and machine learning algorithms to screen 70 clinically-approved drugs with good blood-brain-barrier penetration in 35 glioblastoma patient samples that were freshly obtained from surgery. This revealed prognostically relevant subtypes of glioma cell morphologies and promising candidates for drug repurposing. Among the drugs with potent anti-glioma activity, we identified the antidepressant Vortioxetine, that had strong anti-glioma activity across patients independent of the promoter methylation status of O6-methylguanine-DNA methyltransferase (MGMT). Complementrary in vitro studies using patient-derived glioma cell lines and orthotopic glioma xenograft mouse models confirmed the strong anti-glioma activity of Vortioxetine, leading to a notable fraction of long-term surviving mice. Mechanistically, multiplexed transcriptomics and proteomics unveiled that Vortioxetine induced an upregulation of the Activator protein 1 (AP-1) transcription factor family, along with the B-cell translocation gene (BTG) tumor suppressor genes. These findings serve as the cornerstone for future clinical applications of the single-cell functional tissue profiling platform and the potential clinical translation of Vortioxetine as a promising drug-repurposing candidate for glioblastoma patients.

EPCO-49. MYC AMPLIFICATION AT DIAGNOSIS REGULATES THERAPY-INDUCED HYPERMUTATION AT RECURRENT GLIOMA

Abstract Cancer progression and resistance to therapy are driven by clonal evolution. Recent studies have shown that adult diffuse gliomas have divergent clonal dynamics. However, the genomic and epigenomic factors that influence molecular changes after treatment are not well understood. To address this, we analyzed sequencing and clinical data from 544 adult diffuse glioma patients to identify predictors of cancer evolution. Using machine learning methods CELLO2, we predict grade progression and treatment-induced hypermutation based on tumor characteristics and clinical features collected at diagnosis. We found that MYC copy number gain is more prevalent as a founding alteration in the East Asian gliomas and it is the most significant predictor of hypermutation under temozolomide treatment. Further experiments using patient-derived gliomaspheres and established glioma cell lines confirmed the critical role of MYC amplification and pathway activation in hypermutagenesis. We also showed that c-Myc binding to open chromatin and transcriptionally active regions increases the vulnerability of genomic regions to TMZ-induced mutagenesis. This study highlights MYC as an early predictor of cancer evolution and provides a machine learning platform to improve patient management by predicting cancer dynamics.

DDDR-09. FUNCTIONAL GENOMICS AND CHEMOTHERAPY RESISTANCE SCREENING IDENTIFIES BIOMARKERS AND TARGETABLE MECHANISMS OF CCNU RESISTANCE IN GLIOMAS

Abstract The bifunctional alkylator lomustine (CCNU) remains widely but heterogeneously used as one of the few effective treatments in glioblastoma (GBM) and IDH mutant gliomas. Despite its widespread use, mechanisms of response and resistance to this agent are controversial and patients who benefit are poorly understood. To address this with next-generation patient models and functional genomics approaches, we generated and characterized CCNU response in a large cohort of 71 patient-derived glioma cell lines (PDCLs, spheroids/organoids). Models had fully defined clinical and molecular characteristics (WES, RNAseq, and patient and model MGMT). Patient models, including those derived from patients who acquired resistance to CCNU, exhibited a surprisingly wide range of differential response to CCNU (median IC50: 21.66uM; range: 1.52uM-159uM) using metabolic (CTG) as well as cell death readouts, with no lines showing complete resistance (EC100 < 291uM). MGMT promoter methylation status but not mismatch repair (MMR) trended, but did not reach, statistical significance as a predictor of CCNU sensitivity (p=0.053), in striking contrast to TMZ (p=0.01). Chronic CCNU exposure of two PDCLs led to acquired resistance to CCNU (IC50 increase from 5uM to 50uM on average). RNA sequencing showed 194 significantly differentially expressed genes while no mutations acquired in resistant models were identified in DNA sequencing. PRISM pooled screening of over 900 barcoded cancer cell lines and correlation with DepMap whole genome CRISPR-knockout data showed unique patterns of CCNU response and strong upregulation of DNA repair targets in the resistant state, which differed from TMZ. Shared targets were also identified in a CRISPR-activator screen performed on a CCNU sensitive cell line in the presence and absence of CCNU and converged on multiple mTOR pathway genes as top enriched targets and biomarkers of resistance in CCNU-resistant models. These druggable targets are currently being investigated for combination therapies with CCNU and potential to overcome resistance.

Retracted] Effect of CCL2 siRNA on proliferation and apoptosis in the U251 human glioma cell line.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the VEGF western blotting data shown in Fig.4A on p.3392 were strikingly similar to data appearing in different form in other articles written by different authors at different research institutes. Owing to the fact that the contentious data in the above article had already been published elsewhere prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office never received a reply.The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 16: 3387‑3394, 2017; DOI: 10.3892/mmr.2017.6995].

Molecular Mechanism of Inhibition of Glioma by Targeting Autophagy Microtubule-Associated Protein 1A/1B-Light Chain 3 Family via miR-16 with Novel Liposome Nanoparticles

MiR-16 and other several known oncogenes co-exist in various solid tumors and play carcinogenic roles in many tumors. This study explores whether miR-16 regulates autophagy expression and analyzes the role of targeted nanoparticle intervention in glioma. miR-16 and LC3 expressions were examined by reverse transcription-polymerase chain reaction (RT-PCR). They were assessed in normal lymphocytes, low-metastatic glioma, and high-metastatic glioma cell lines as well. The glioma cell line U251 was used to detect and compare the expression of LC3. Flow cytometry detected cell proliferation and the number of cell invasion and metastasis was detected by Transwell. LC3 mRNA in glioma tissues was evidently increased. The later the Tumor Node Metastasis (TNM) stage, the lower expression of miR-16 and the higher expression of LC3, which is related to TNM stage. LC3 mRNA in glioma cells was obviously higher than normal cells while miR-16 was lower than the latter. The expression of LC3 in glioma cell line U251 was higher, while miR-16 was lower. Transfection of siRNA-LC3 and targeted nanoparticles could effectively down-regulate the level of LC3 in the glioma cell line U251. In conclusion, miR-16 is related to the increased expression of LC3 and the enhanced ability of glioma cells to invade and metastasize.

Toxicity of Carboplatin-Niosomal Nanoparticles in a Brain Cancer Cell Line.

Cancer poses a significant challenge in modern medicine, standing as the primary cause of death in many countries, second only to cardiovascular diseases. Among the various treatments available, carboplatin, a chemotherapy drug, is employed for specific cancer types, including brain carcinoma. The main objective of this investigation is to enhance the therapeutic efficacy of carboplatin by utilizing niosomal nanocarriers. We synthesized nanoniosomal carboplatin using the reverse-phase evaporation technique and conducted an assessment of its particle size, zeta potential, and drug-release properties. Subsequently, we evaluated the cytotoxicity of nanoniosomal carboplatin using the C6 rat glioma cell line. Our research revealed that these niosomal nanoparticles possessed a particle size of 290.5±5.5 nm and a zeta potential of -21.7±7.4 mV. The amount of encapsulated drug and drug loading level were found to be 60.2±2.3% and 2.5±1.1%, respectively. Importantly, the cytotoxic impact of these nanoniosomes on the C6 rat glioma cell line exhibited a significant increase compared to the free drug (P<0.05). Based on our discoveries, it is evident that carboplatin niosomal nanocarriers hold potential as an innovative approach to chemotherapy for brain cancer therapy.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

SUMOylation of RALY promotes vasculogenic mimicry in glioma cells via the FOXD1/DKK1 pathway

Human malignant gliomas are the most common and aggressive primary malignant tumors of the human central nervous system. Vasculogenic mimicry (VM), which refers to the formation of a tumor blood supply system independently of endothelial cells, contributes to the malignant progression of glioma. Therefore, VM is considered a potential target for glioma therapy. Accumulated evidence indicates that alterations in SUMOylation, a reversible post-translational modification, are involved in tumorigenesis and progression. In the present study, we found that UBA2 and RALY were upregulated in glioma tissues and cell lines. Downregulation of UBA2 and RALY inhibited the migration, invasion, and VM of glioma cells. RALY can be SUMOylated by conjugation with SUMO1, which is facilitated by the overexpression of UBA2. The SUMOylation of RALY increases its stability, which in turn increases its expression as well as its promoting effect on FOXD1 mRNA. The overexpression of FOXD1 promotes DKK1 transcription by activating its promoter, thereby promoting glioma cell migration, invasion, and VM. Remarkably, the combined knockdown of UBA2, RALY, and FOXD1 resulted in the smallest tumor volumes and the longest survivals of nude mice in vivo. UBA2/RALY/FOXD1/DKK1 axis may play crucial roles in regulating VM in glioma, which may contribute to the development of potential strategies for the treatment of gliomas.

CircIQGAP1 regulates RCAN1 and RCAN2 through the mechanism of ceRNA and promotes the growth of malignant glioma

Circular RNA (circRNA) is one of non-coding RNA with specific circular structure, which has been found to be involved in regulating a series of malignant biological behaviors in many malignant tumors. In this study, based on the IDH1 molecular typing of gliomas, we identified a significant downregulation of circRNA (circIQGAP1) expression in IDH1 mutant gliomas by high-throughput sequencing. In 79 tissue samples, we confirmed that circIQGAP1 expression was significantly downregulated in IDH1 mutant gliomas, and that low circIQGAP1 expression was positively associated with better prognosis. Knockdown of circIQGAP1 in glioma cell lines inhibited glioma cell malignancy and conversely overexpression of circIQGAP1 promoted glioma malignancy. circIQGAP1 regulated glioma cell migration, proliferation, invasion and apoptosis through miR-1256/RCAN1/Bax/Bcl-2/Caspase3 and miR-622/RCAN2/Bax/Bcl-2/Caspase3 axes. These results suggest that circIQGAP1 plays an important role in glioma development, promotes tumor growth, and is a potential therapeutic target for glioma.

Glioma-derived S100A9 polarizes M2 microglia to inhibit CD8+T lymphocytes for immunosuppression via αvβ3 integrin/AKT1/TGFβ1

Our previous studies showed that S100A9 was overexpressed in glioma and promoted tumor growth. However, S100A9 can also be secreted by tumor cells to regulate the tumor microenvironment (TME). In this study, we aimed to explore the functions of glioma derived-S100A9 in microglial M2 polarization, resulting in inhibition of CD8+ T lymphocytes and promotion of immunosuppression. We first showed that glioma exhibited higher expression and secretion of S100A9 than astrocytes. After knocking down S100A9 in two glioma cell lines, the secretion of S100A9 was repressed. Then, the medium was collected and considered as conditioned medium (CM), which was incubated with microglia. We found that glioma-derived S100A9 drove microglial M2 polarization and increased TGFβ1 secretion. These molecular mechanisms were related to the interaction of S100A9 with αvβ3 integrin and the subsequent activation of AKT1 in microglia. Furthermore, we demonstrated that S100A9-induced M2 microglia negatively affected cell viability, IL-2 and IFN-γ secretion, together with increased early apoptosis in CD8+T lymphocytes via TGFβ1. Additionally, glioma cells were implanted into mouse brains, and we confirmed that S100A9 stimulated microglial M2 polarization, enhanced TGFβ1 levels and repressed CD8+ T lymphocytes in orthotopically transplanted tumors. In human glioma samples, S100A9 expression was positively associated with CD206 expression, but negatively correlated with CD8+T lymphocyte accumulation in the TME. Our data indicated that glioma-derived S100A9 has a promising ability to manipulate non-malignant cells and promote immune evasion in the TME, providing valuable insight into the mechanism by which S100A9 participates in the progression of glioma.

Synthesis and Bioevaluation of 3-(Arylmethylene)indole Derivatives: Discovery of a Novel ALK Modulator with Antiglioblastoma Activities.

Glioblastoma is the most common brain tumor, with high recurrence and low survival rates. An integrative bioinformatics analysis demonstrated that anaplastic lymphoma kinase (ALK) is a promising therapeutic target for glioblastoma. We designed and synthesized a series of 3-(arylmethylene)indole derivatives, which were further evaluated for antiproliferative activity using glioma cell lines. Among them, compound 4a significantly inhibited the viability of glioblastoma cells. With favorable pharmacokinetic characteristics and blood-brain barrier permeability, 4a improved the survival rate and inhibited the growth of orthotopic glioblastoma. The Phospho-Totum system revealed that ALK was a potential target for the antiglioblastoma activity of 4a. Further experiments indicated that 4a might be a novel ALK modulator, which interacted with the extracellular ligand-binding domain of ALK, thus selectively induced ERK-mediated autophagy and apoptosis. Our findings provide an alternative ALK-based targeting strategy and a new drug candidate for glioblastoma therapy.

Ceramide Is Involved in Temozolomide Resistance in Human Glioblastoma U87MG Overexpressing EGFR.

Glioblastoma multiforme (GBM) is the most frequent and deadly brain tumor. Many sphingolipids are crucial players in the regulation of glioma cell growth as well as in the response to different chemotherapeutic drugs. In particular, ceramide (Cer) is a tumor suppressor lipid, able to induce antiproliferative and apoptotic responses in different types of tumors including GBM, most of which overexpress the epidermal growth factor receptor variant III (EGFRvIII). In this paper, we investigated whether Cer metabolism is altered in the U87MG human glioma cell line overexpressing EGFRvIII (EGFR+ cells) to elucidate their possible interplay in the mechanisms regulating GBM survival properties and the response to the alkylating agent temozolomide (TMZ). Notably, we demonstrated that a low dose of TMZ significantly increases Cer levels in U87MG cells but slightly in EGFR+ cells (sensitive and resistant to TMZ, respectively). Moreover, the inhibition of the synthesis of complex sphingolipids made EGFR+ cells sensitive to TMZ, thus involving Cer accumulation/removal in TMZ resistance of GBM cells. This suggests that the enhanced resistance of EGFR+ cells to TMZ is dependent on Cer metabolism. Altogether, our results indicate that EGFRvIII expression confers a TMZ-resistance phenotype to U87MG glioma cells by counteracting Cer increase.

Curcumol reverses temozolomide resistance in glioma cells by regulating the UTX/MGMT axis

To explore the mechanism through which curcumol reverses primary drug resistance in glioma cells. The inhibitory effect of 10, 20, and 40 μg/mL curcumol were observed in human glioma cell lines A172 and U251. UTX-overexpressing glioma cells constructed by lentiviral transfection were treated with curcumol (40 μg/mL), temozolomide (TMZ; 10 μg/mL), or both, and the changes in cell viability, clone formation capacity and apoptosis were assessed using MTT assay, cell clone formation experiment, and flow cytometry; UTX activity in the cells was determined using a UTX detection kit, and the enrichment of UTX and H3K27me3 in the MGMT promoter region was detected with ChiP-qPCR. The protein expressions in glioma cells were detected using Western blotting and immunohistochemistry. In a nude mouse model bearing glioma xenografts, the effects of curcumol (20 mg/kg), TMZ (20 mg/kg) and their combination on tumor growth and expressions of UTX, H3K27me3 and MGMT were evaluated. Curcumol significantly inhibited the proliferation (P<0.05) and promoted apoptosis of cultured glioma cells (P<0.01). Curcumol, but not TMZ, produced significant inhibitory effect on tumor growth in the tumor-bearing mice (P<0.01). Curcumol significantly inhibited UTX activity and increased the expression level of H3K27me3 protein in the glioma cells. UTX overexpression obviously decreased H3K27me3 protein expression and reversed the effects of curcumol on glioma cell proliferation and apoptosis (P<0.01). Curcumol reduced the enrichment of UTX and H3K27me3 in the MGMT promoter region (P<0.05) and decreased MGMT protein expression, which was reversed by UTX overexpression. In both the in vivo and in vitro experiments, curcumol combined with TMZ significantly increased H3K27me3 protein expression in the glioma cells, reduced the expression of its downstream target gene MGMT, and enhanced TMZ sensitivity of the glioma cells. Curcumol can enhance glioma cell sensitivity to TMZ by regulating the UTX/MGMT axis.

Expression profiles and function prediction of tRNA-derived fragments in glioma

BackgroundGlioblastoma (GBM) is the most aggressive malignant primary brain tumor. The transfer RNA-derived fragments (tRFs) are a new group of small noncoding RNAs, which are dysregulated in many cancers. Until now, the expression and function of tRFs in glioma remain unknown.MethodsThe expression profiles of tRF subtypes were analyzed using the Cancer Genome Atlas (TCGA)-low-grade gliomas (LGG)/GBM dataset. The target genes of tRFs were subjected to Gene Ontology, Kyoto Encyclopedia and Gene set enrichment analysis of Genes and Genomes pathway enrichment analysis. The protein-protein interaction enrichment analysis was performed by STRING. QRT-PCR was performed to detect the expressions of tRFs in human glioma cell lines U87, U373, U251, and human astrocyte cell line SVG p12. Western blot assay was used to detect to the expression of S100A11. The interaction between tRF-19-R118LOJX and S100A11 mRNA 3’UTR was detected by dual-luciferase reporter assay. The effects of tRF-19-R118LOJX, tRF-19-6SM83OJX and S100A11 on the glioma cell proliferation, migration and in vitro vasculogenic mimicry formation ability were examined by CCK-8 proliferation assay, EdU assay, HoloMonitor cell migration assay and tube formation assay, respectively.ResultstRF-19-R118LOJX and tRF-19-6SM83OJX are the most differentially expressed tRFs between LGG and GBM groups. The functional enrichment analysis showed that the target genes of tRF-19-R118LOJX and tRF-19-6SM83OJX are enriched in regulating blood vessel development. The upregulated target genes are linked to adverse survival outcomes in glioma patients. tRF-19-R118LOJX and tRF-19-6SM83OJX were identified to suppress glioma cell proliferation, migration, and in vitro vasculogenic mimicry formation. The mechanism of tRF-19-R118LOJX might be related to its function as an RNA silencer by targeting the S100A11 mRNA 3’UTR.ConclusiontRFs would become novel diagnostic biomarkers and therapeutic targets of glioma, and the mechanism might be related to its post-transcriptionally regulation of gene expression by targeting mRNA 3’UTR.