Human Glioblastoma Cells Research Articles

Exosome-transmitted ANGPTL1 suppresses angiogenesis in glioblastoma by inhibiting the VEGFA/VEGFR2/Akt/eNOS pathway

ObjectiveGlioblastoma (GBM) is a highly vascularized malignancy that relies on new vessel generation, and thus targeting angiogenesis has been a promising anti-GBM approach. ANGPTL1 is well-known for its anti-angiogenic property; nevertheless, its role in GBM is yet to be explored. Recently, the crucial role of exosomes (Exos) as intercellular communication mediators has gained prominence in GBM therapy. This work aimed to explore the role of exosomal ANGPTL1 in GBM angiogenesis and its mechanisms. MethodsBioinformatic analysis was performed to evaluate ANGPTL expression in GBM. Human GBM cell lines (U87 and U251) and a xenograft mouse model were employed. Exos were isolated from oe-NC- and oe-ANGPTL-transfected bone mesenchymal stem cells and identified. Cell proliferation, migration, and apoptosis were detected. Immunofluorescence, qRT-PCR, western blotting, co-immunoprecipitation, and immunohistochemistry were used to determine the molecular mechanisms underlying exosomal ANGPTL1 against GBM angiogenesis. Besides, tube generation and transmission electron microscope assays were conducted to assess GBM angiogenesis. ResultsLow ANGPTL1 expression was observed in GBM tumor tissues and cells. Functionally, e-ANGPTL-Exos inhibited GBM malignant progression and angiogenesis in vitro and in vivo. Mechanically, e-ANGPTL-Exos reduced VEGFA expression and blocked the VEGFR2/Akt/eNOS pathway in GBM cells and tumor tissues. Co-immunoprecipitation revealed a link between ANGPTL1 and VEGFA in GBM cells. Notably, oe-VEGFA abolished the suppressive functions of e-ANGPTL-Exos in GBM progression and angiogenesis and the VEGFR2/Akt/eNOS axis. The VEGFR2 inhibitor, vandetanib, eliminated the promotive effects of oe-VEGFA on GBM angiogenesis with suppressed VEGFR2/Akt/eNOS pathway. ConclusionsExosomal ANGPTL1 suppressed GBM angiogenesis by inhibiting the VEGFA/VEGFR2/Akt/eNOS axis.

10168-BOT-2 ANTITUMOR AROMA THERAPY WITH LEMONGRASS ESSENTIAL OIL FOR GLIOBLASTOMA

Abstract Aromatic essential oils have been used in cancer treatment to enhance immunity, induce sedation, and reduce the side effects of chemotherapy. Recently, essential oils have been reported to have anti-tumor effects. We have been investigating anti-tumor aroma of essential oils as a novel treatment for glioblastoma and have found that hiba essential oil has a proliferation inhibitory effect. In this study, we investigated the antitumor effect of lemongrass essential oil for glioblastoma and its antitumor aromatherapy. Human glioblastoma cells (U87, T98G) and mouse brain tumor cells (RSV-M) were used. Lemongrass essential oil was added to each tumor cell line, and growth inhibition was observed in all cell lines. In addition, lemon grass essential oil showed growth inhibition effect when the tumor cells were separated from the oil, as was the case with direct administration, suggesting that the lemongrass essential oil components that evaporated acted on tumor cells in the surrounding area. The antitumor component of lemongrass essential oil was isolated and identified as citral, an antitumor factor, by gas chromatography. Analysis of the effects of citral showed that it strongly inhibited MARK4 activity and growth in three cell lines. The mechanism revealed that citral decreased the phosphorylation of Tau protein. MARK4 is highly expressed in glioblastoma and has been reported to be involved in cell proliferation. This study suggests a potential mechanism for citral to inhibit tumor cell proliferation via MARK4 in malignant gliomas. Citral is a volatile monoterpene and may exert its antitumor effect by evaporation diffusion. Lemongrass essential oil contains citral, which shows antitumor effects, and is expected to be applied to a new glioblastoma therapy using "aroma".

10073-CBMS-6 CURCUMIN ANALOG B EXHIBIT ANTI-TUMOR ACTIVITY AGAINST GLIOBLASTOMAAT LOWER CONCENTRATIONS THAN CURCUMIN

Abstract PURPOSE Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and resistance to the standard treatment, temozolomide. Therefore, the development of noble therapeutic agents against GBM is required. This study investigated the anti-tumor activity of the curcumin (Cur) analog Compound B (ComB) against GBM. METHODS AND RESULTS To evaluate anti-tumor activity against GBM, we performed an MTT assay. The human glioblastoma cell lines U87-MG and U251 were pre-treated with Cur or ComB, and then cell viability was examined using a cell counting kit, and IC50 values were calculated. The IC50 value for U87-MG of Cur was 9.78 μM, and that of ComB was1.28 μM. The IC50 value for U251 of Cur was 9.50 μM, and that of ComB was 0.64 μM. To examine the effects of ComB on normal cells, the same MTT assay was performed on primary cultured astrocytes prepared from neonatal rats. ComB did not reduce cell viability in astrocytes at concentrations that had an anti-tumor effect on GBM cells. Next, a cell cycle analysis was performed with Propidium Iodide (PI) staining and an apoptosis assay with Annexin V/PI staining using flow cytometry. ComB induced cell cycle G2/M arrest and apoptosis at lower concentrations than Cur. qPCR showed that mRNA expression levels of CDK1 and CyclinB1, which play an essential role in the progression from the G2 phase to the M phase, decreased by ComB at lower concentrations than Cur. DISCUSSION These results suggest that ComB, at lower concentrations than Cur, has an anti-tumor effect without affecting normal cells by inducing cell cycle arrest and apoptosis against GBM. Further detailed analysis and in vivo studies are expected to lead to the development of novel therapeutic agents for GBM.

10051-CBMS-4 CURCUMIN ANALOG COMPOUND A INHIBITS THE GROWTH OF GLIOBLASTOMA BY INDUCING G2/M PHASE ARREST AND APOPTOSIS AT A LOWER CONCENTRATION THAN CURCUMIN

Abstract PURPOSE Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Over 15,000 patients are diagnosed with GBM yearly, with a median survival expectancy of<15 months and<10% survival rate beyond five years, despite applying conventional multimodal treatment containing surgical resection, chemotherapy, and radiation therapy. The efficiency of conventional therapies is limited due to the lack of specificity and the accuracy of eradicating aggressive diffusing GBM cells. Developing new therapeutic agents against GBM is strongly recommended. The purpose of this study was to investigate whether the curcumin (Cur) analogs Compound A (ComA) possess anti-tumor activity against GBM. METHODS AND RESULTS To evaluate the anti-tumor activity of ComA against GBM, we performed an MTT assay. The human GBM cell lines U87-MG and U251 were pre-treated with Cur or ComA, cell viability was examined using a cell counting kit, and then IC50 values were evaluated. The IC50 values for U87-MG were: Cur, 9.78 μM; ComA, 2.42 μM. Those for U251 were: Cur, 9.50 μM; ComA, 2.27 μM. To examine the effects of ComA on normal cells, the same MTT assay was performed on primary cultured astrocytes from neonatal rats. ComA did not significantly reduce the cell viability in astrocytes at concentrations that had an anti-tumor effect on GBM cells (ComA, 3 μM). Next, a cell cycle analysis was performed with PI staining and an apoptosis assay with Annexin V/PI staining using flowcytometry. ComA, at lower concentrations than Cur, induced G2/M phase arrest and apoptosis. qPCR showed that ComA decreases CDK1 and CyclinB1 mRNA levels, the cell cycle-related proteins, at a lower concentration than Cur. DISCUSSION These results indicate that ComA has an anti-tumor effect by inducing cell cycle arrest and apoptosis against GBM at lower concentrations than Cur.

Synergistic effect of cryptotanshinone and temozolomide treatment against human glioblastoma cells

Glioblastoma multiforme (GBM) is a complex disease to treat owing to its profound chemoresistance. Therefore, we evaluated the combined effect and therapeutic efficacy of temozolomide (TMZ), a potent alkylating agent and the current gold standard therapy for GBM, and cryptotanshinone (CTS), which inhibits glioma cell proliferation in GBM cells. Using LN229 and U87-MG human GBM cells in a short-term stimulation in vitro model, the cytotoxic and anti-proliferative effects of single and combined treatment with 4 μM CTS and 200 μM TMZ were investigated. Furthermore, cell viability, DNA damage, apoptosis rate, and signal transducer and activator of transcription 3 (STAT3) protein were measured using cytotoxic assay, comet assay, flow cytometry, and western blotting analysis, respectively. The two drugs’ synergistic interaction was validated using the synergy score. We found that the anti-proliferative effects of combination therapy using the two drugs were greater than that of each agent used alone (CTS or TMZ). Western blot analysis indicated that treatment of GBM cells with CTS combined with TMZ more significantly decreased the expression of MGMT and STAT3, than that with TMZ alone. Combined treatment with CTS and TMZ might be an effective option to overcome the chemoresistance of GBM cells in a long-term treatment strategy.

Radio-chemotherapy of glioblastoma cells promotes phagocytosis by macrophages in vitro

Background and PurposeImmunotherapy is actively explored in glioblastoma (GBM) to improve patient prognosis. Tumor-associated macrophages (TAMs) are abundant in GBM and harnessing their function for anti-tumor immunity is of interest. They are plastic cells that are influenced by the tumor microenvironment, by radio-chemotherapy and by their own phagocytic activity. Indeed, the engulfment of necrotic cells promotes pro-inflammatory (and anti-tumoral) functions while the engulfment of apoptotic cells promotes anti-inflammatory (and pro-tumoral) functions through efferocytosis. Materials and MethodsTo model the effect of radio-chemotherapy on the GBM microenvironment, we exposed human macrophages to supernatant of treated GBM cells in vitro. Macrophages were derived from human monocytes and GBM cells from patient-resected tumors. GBM cells were exposed to therapeutically relevant doses of irradiation and chemotherapy. Apoptosis and phagocytic activity were assessed by flow cytometry. ResultsThe phagocytic activity of macrophages was increased, and it was correlated with the proportion of apoptotic GBM cells producing the supernatant. Whether uptake of apoptotic tumor cells could occur would depend upon the expression of efferocytosis-associated receptors. Indeed, we showed that efferocytosis-associated receptors, such as AXL, were upregulated. Conclusions and PerspectivesWe showed that macrophage phagocytic activity increased when exposed to supernatant from GBM cells treated by radio-chemotherapy. However, as efferocytosis-associated receptors were up-regulated, this effect could be deleterious for the anti-GBM immune response. We speculate that by inducing GBM cell apoptosis in parallel to an increase in efferocytosis receptor expression, the impact of radio-chemotherapy on phagocytic activity could promote anti-inflammatory and pro-tumoral TAM functions.

Retracted] Role of the AKT pathway in microRNA expression of human U251 glioblastoma cells.

Following the publication of the above article, a concerned reader drew to the Editor's attention that, regarding the western blots featured in Fig.3B on p.670, the bands featured in the U251 and U251‑MC lanes for the miR‑21 and U6 experiments appeared to be duplicates of each other. Moreover, certain of these data were strikingly similar to data that appeared in another article published at around the same time featuring some of the same authors (again, with apparent duplications of bands within the same gel slices, as they were presented). After having conducted an internal investigation of this matter, the Editor of International Journal of Oncology has judged that the apparently anomalous grouping of the data could not have been attributed to pure coincidence. Therefore, the Editor has decided that this article should be retracted from the publication on the grounds of an overall lack of confidence in the data. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor sincerely apologizes to the readership for any incovenience caused, and we thank the reader for bringing this matter to our attention. [International Journal of Oncology 36: 665‑672, 2010; DOI: 10.3892/ijo_00000542].

A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway.

PTP1B plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify molecular targets of PTP1B that mediate its role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify Cdk3 as a novel PTP1B substrate. Substrate trapping experiments and docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at tyrosine residue 15 invitro and interacts with it in human glioblastoma cells. Next, we found that pharmacological inhibition of PTP1B or its depletion with siRNA leads to cell cycle arrest with diminished activity of Cdk3, hypophosphorylation of Rb, and the downregulation of E2F target genes Cdk1, Cyclin A, and Cyclin E1. Finally, we observed that the expression of a constitutively active Cdk3 mutant bypasses the requirement of PTP1B for cell cycle progression and expression of E2F target genes. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells.

Micro-Vessels-Like 3D Scaffolds for Studying the Proton Radiobiology of Glioblastoma-Endothelial Cells Co-Culture Models.

Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. While X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can damage cancer cells while sparing the surrounding healthy tissue. However, the effects of protons on in vitro GBM models at the cellular level, especially when co-cultured with endothelial cells, the building blocks of brain micro-vessels, are still unexplored. In this work, novel 3D-engineered scaffolds inspired by the geometry of brain microvasculature are designed, where GBM cells cluster and proliferate. The architectures are fabricated by two-photon polymerization (2PP), pre-cultured with endothelial cells (HUVECs), and then cultured with a human GBM cell line (U251). The micro-vessel structures enable GBM in vivo-like morphologies, and the results show a higher DNA double-strand breakage in GBM monoculture samples when compared to the U251/HUVECs co-culture, with cells in 2D featuring a larger number of DNA damage foci when compared to cells in 3D. The discrepancy in terms of proton radiation response indicates a difference in the radioresistance of the GBM cells mediated by the presence of HUVECs and the possible induction of stemness features that contribute to radioresistance and improved DNA repair.

Therapeutic Potential of Resveratrol for Glioma: A Systematic Review and Meta-Analysis of Animal Model Studies

Gliomas are aggressive malignant brain tumors, with poor prognosis despite available therapies, raising the necessity for finding new compounds with therapeutic action. Numerous preclinical investigations evaluating resveratrol’s anti-tumor impact in animal models of glioma have been reported; however, the variety of experimental circumstances and results have prevented conclusive findings about resveratrol’s effectiveness. Several databases were searched during May 2023, ten publications were identified, satisfying the inclusion criteria, that assess the effects of resveratrol in murine glioma-bearing xenografts. To determine the efficacy of resveratrol, tumor volume and animal counts were retrieved, and the data were then subjected to a random effects meta-analysis. The influence of different experimental conditions and publication bias on resveratrol efficacy were evaluated. Comparing treated to untreated groups, resveratrol administration decreased the tumor volume. Overall, the effect’s weighted standardized difference in means was −2.046 (95%CI: −3.156 to −0.936; p-value < 0.001). The efficacy of the treatment was observed for animals inoculated with both human glioblastoma or rat glioma cells and for different modes of resveratrol administration. The combined administration of resveratrol and temozolomide was more effective than temozolomide alone. Reducing publication bias did not change the effectiveness of resveratrol treatment. The findings suggest that resveratrol slows the development of tumors in animal glioma models.

Inhibitory Effects of Urolithins, Bioactive Gut Metabolites from Natural Polyphenols, against Glioblastoma Progression

We previously reported that proinflammatory cytokines, particularly tumor necrosis factor (TNF)-α, promoted tumor migration, invasion, and proliferation, thus worsening the prognosis of glioblastoma (GBM). Urolithins, the potent metabolites produced by the gut from pomegranate polyphenols, have anticancer properties. To develop an effective therapy for GBM, this study aimed to study the effects of urolithins against GBM. Urolithin A and B significantly reduced GBM migration, reduced epithelial–mesenchymal transition, and inhibited tumor growth. Moreover, urolithin A and B inhibited TNF-α-induced vascular cell adhesion molecule (VCAM)-1 and programmed death ligand 1 (PD-L1) expression, thereby reducing human monocyte (HM) binding to GBM cells. Aryl hydrocarbon receptor (AhR) level had higher expression in patients with glioma than in healthy individuals. Urolithins are considered pharmacological antagonists of AhR. We demonstrated that the inhibition of AhR reduced TNF-α-stimulated VCAM-1 and PD-L1 expression. Furthermore, human macrophage condition medium enhanced expression of PD-L1 in human GBM cells. Administration of the AhR antagonist attenuated the enhancement of PD-L1, indicating the AhR modulation in GBM progression. The modulatory effects of urolithins in GBM involve inhibiting the Akt and epidermal growth factor receptor pathways. The present study suggests that urolithins can inhibit GBM progression and provide valuable information for anti-GBM strategy.

Iron inhibits glioblastoma cell migration and polarization.

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate-a key question, especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

Elucidation of GPR55-Associated Signaling behind THC and LPI Reducing Effects on Ki67-Immunoreactive Nuclei in Patient-Derived Glioblastoma Cells.

GPR55 is involved in many physiological and pathological processes. In cancer, GPR55 has been described to show accelerating and decelerating effects in tumor progression resulting from distinct intracellular signaling pathways. GPR55 becomes activated by LPI and various plant-derived, endogenous, and synthetic cannabinoids. Cannabinoids such as THC exerted antitumor effects by inhibiting tumor cell proliferation or inducing apoptosis. Besides its effects through CB1 and CB2 receptors, THC modulates cellular responses among others via GPR55. Previously, we reported a reduction in Ki67-immunoreactive nuclei of human glioblastoma cells after GPR55 activation in general by THC and in particular by LPI. In the present study, we investigated intracellular mechanisms leading to an altered number of Ki67+ nuclei after stimulation of GPR55 by LPI and THC. Pharmacological analyses revealed a strongly involved PLC-IP3 signaling and cell-type-specific differences in Gα-, Gβγ-, RhoA-ROCK, and calcineurin signaling. Furthermore, immunochemical visualization of the calcineurin-dependent transcription factor NFAT revealed an unchanged subcellular localization after THC or LPI treatment. The data underline the cell-type-specific diversity of GPR55-associated signaling pathways in coupling to intracellular G proteins. Furthermore, this diversity might determine the outcome and the individual responsiveness of tumor cells to GPR55 stimulation by cannabin oids.

Molecular Targeted Therapies in Glioblastoma Multiforme: A Systematic Overview of Global Trends and Findings.

This systematic review assesses current molecular targeted therapies for glioblastoma multiforme (GBM), a challenging condition with limited treatment options. Using PRISMA methodology, 166 eligible studies, involving 2526 patients (61.49% male, 38.51% female, with a male-to-female ratio of 1.59/1), were analyzed. In laboratory studies, 52.52% primarily used human glioblastoma cell cultures (HCC), and 43.17% employed animal samples (mainly mice). Clinical participants ranged from 18 to 100 years, with 60.2% using combined therapies and 39.8% monotherapies. Mechanistic categories included Protein Kinase Phosphorylation (41.6%), Cell Cycle-Related Mechanisms (18.1%), Microenvironmental Targets (19.9%), Immunological Targets (4.2%), and Other Mechanisms (16.3%). Key molecular targets included Epidermal Growth Factor Receptor (EGFR) (10.8%), Mammalian Target of Rapamycin (mTOR) (7.2%), Vascular Endothelial Growth Factor (VEGF) (6.6%), and Mitogen-Activated Protein Kinase (MEK) (5.4%). This review provides a comprehensive assessment of molecular therapies for GBM, highlighting their varied efficacy in clinical and laboratory settings, ultimately impacting overall and progression-free survival in GBM management.

CSIG-33. A 2ND GENERATION IDO1 PROTEIN DEGRADER TO OVERCOME NON-ENZYME-MEDIATED IDO1-DEPENDENT IMMUNE SUPPRESSION FOR GLIOBLASTOMA

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive rate-limiting enzyme that metabolizes the essential amino acid, tryptophan (Trp), into the downstream catabolite, kynurenine. IDO1 is expressed in >90% of patient-resected glioblastoma (GBM). We recently validated that IDO1 can suppress the anti-brain tumor immune response and ablate immunotherapeutic treatment efficacy through non-metabolic IDO1 activity. To-date, most IDO1-targeted therapy has focused on inhibiting tryptophan metabolism. This class of drugs has failed to improve the overall survival of patients with cancer. To overcome this failure, we developed a therapeutic agent that inhibits both IDO1 enzyme- and non-enzyme-activities in the form of an IDO1-Proteolysis Targeting Chimera (PROTAC). We previously published the characterization of a 1st generation lead compound (Bollu et al., 2022; J. Med. Chem) with a DC50 for IDO1 of 0.329 µM in human GBM cells. In a more recent investigation, we discovered a 2nd generation IDO1-PROTAC, NU227326, with 30× more potency and a DC50 of 10.4 nM in human GBM cells. IDO1-expressing U87, U138, and patient derived xenograft (PDX) GBM43 were treated with either IDO1-PROTAC, IDO1-mutant-PROTAC, IDO1 enzyme inhibitor, or IDO1 siRNA. Transcriptomic analysis revealed differentially expressed genes that were commonly regulated after treatment with the IDO1-PROTAC as compared to treatment with the IDO1-mutant-PROTAC or IDO1 enzyme inhibitor groups in U87, U138, and GBM43 cells. Mass spectrometry discovered 34 unique proteins that were differentially expressed inside of human GBM cells and an additional 20 unique proteins that were identified in the supernatant of cultured human GBM cells after IDO1-PROTAC treatment. Meta-analysis of the transcriptomic and proteomic analyses identified a novel factor that was unique to IDO1-PROTAC treatment. IDO1-PROTAC treatment-enriched GO terms were associated with nucleoside kinase and metallocarboxypeptidase activities. This study discovered multiple new pathways that immunosuppressive IDO1 non-metabolically regulates in human glioblastoma cells.

CSIG-23. PRIMARY CILIA DISRUPTION ALTERS THE TRANSCRITOME OF HUMAN GLIOBLASTOMA CELLS AND SUGGESTS NOVEL THERAPEUTIC STRATEGIES

Abstract BACKGROUND Glioblastoma is the most common malignant primary brain tumor. Despite standard-of-care therapy with maximal safe resection, concomitant chemoradiation followed by adjuvant chemotherapy, median survival is 18 months, and 5-year survival is 6.8%. The genetic complexity, ability to suppress host immune surveillance, and the blood-brain-barrier all contribute to the uniform fatality of the disease. Primary cilia are microtubule-based organelles that protrudes from the apical surface of all mammalian cells. They regulate several canonical signal transduction cascades important for development and pathogenesis. Glioblastoma cells are ciliated although the tumor downregulate genes associated with cilia formation and function. Contemporary studies suggest that the regulation of ciliogenesis is an important prognostic consideration. In this study, human glioblastoma cells were modified through stable lentiviral transduction to downregulate KIF3A (a gene required for ciliogenesis), or AURKA (a component of the ciliary disassembly complex). We then performed deep transcriptomic sequencing to determine how the overall transcriptome of human glioblastoma cells change in response to cilia-targeted genetic manipulation. RESULTS High-resolution confocal microscopy demonstrated that cilia were absent in KIF3A knockdown cells but abundant following loss of AURKA. Functional loss of primary cilia was confirmed with Gli2A RT-PCR following treatment with SAG (SHH pathway activator). There appeared to be rearrangement of the transcriptome with ciliary loss. Ingenuity Pathway Analysis demonstrated that ciliary loss resulted in the activation of pathways of glioblastoma and other cancer signaling pathways, tumor immune microenvironment pathways, cytokine signaling, and actin cytoskeletal rearrangement among several others. CONCLUSION Loss of primary cilia in glioblastomas activate several pro-cancer and immunosuppressive pathways. This suggests that targeting of primary cilia may lead to simultaneous loss of multiple glioblastoma phenotypes that contribute to the tumor’s evolutionary advantage. This may result in a novel paradigm of ‘ciliotherapeutics’ for glioblastomas and other cancers.

BIOM-11. THE ROLE OF INSULIN-LIKE GROWTH FACTOR 2 BINDING PROTEIN 3 (IGF2BP3) AS A DRIVER OF TUMORIGENESIS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with a poor median survival of fifteen months after diagnosis. GBM accounts for half of malignant and fifteen percent of all primary brain tumors diagnosed in adults. To promote targeted therapy, there are ongoing efforts to understand GBM at a molecular level. This involves characterization of these tumors via their transcriptional and mutational profile. Recent studies indicate aberrations in receptor tyrosine kinase (RTKs), including EGFR and PDGFRA, and the Pi-3 kinase (PI3K) signaling pathways are major drivers of tumorigenesis in GBM. Using a proteomics approach to locate downstream targets of these pathways, we identified IGF2 Binding Protein 3 (IGF2BP3), which is an RNA binding protein, as a downstream effector of aberrant RTK-PI3K signaling in gliomagenesis. Our subsequent follow-up studies indicate that IGF2BP3, which is overexpressed in RTK mutant human GBMs, drives survival and proliferation of RTK-PI3K-dependent human GBM cells. To understand how IGF2BP3 functions to promote RTK-PI3K driven tumorigenesis, we used cultured human GBM cells and in GBM xenograft models to test the effects of loss of IGF2BP3 function in tumorous glia as well as normal glia. Our results indicate that IGF2BP3 activity is essential for self-renewal and maintenance of neural stem-cell like features of GBM tumor cells, but dispensable for the growth of normal glia. Currently, we are using genetic approaches to identify and validate RNA targets of IGF2BP3 function in tumor cells and to understand how these targets are specifically regulated by RTK-PI3K-dependent signaling in glioma. Overall, our data suggests that IGF2BP3 may play a role in promoting GBM tumorigenesis and our future directions aim to reveal an important tumor-specific RNA target signaling pathway for consideration in potential therapeutics.

TMIC-53. TRANSGLUTAMINASE 2 EXPRESSION IN GLIOBLASTOMA: EVIDENCE FOR A ROLE IN EFFEROCYTOSIS

Abstract Glioblastoma is the most common type of brain cancer in adults. It is currently incurable and there is a clear need for more effective treatments. The heterogeneity of this cancer and its ability to promote both local and systemic immunosuppression are major obstacles to the development of effective therapies. Previous studies have explored the use of transglutaminase 2 (TGM2) inhibitors for glioblastoma therapy. These studies focussed on TGM2 that is expressed in a subset of glioblastoma cells. We show here that glioblastoma-associated macrophages are a major source of TGM2 in glioblastoma tumours. Analysis of bulk and single cell RNAseq data showed that TGM2 mRNA was expressed at high levels in glioblastoma-associated macrophages. Immunohistochemical and immunofluorescence analysis of TGM2 expression was performed in a mouse xenograft model in which human mesenchymal subtype glioblastoma cells were grown intracerebrally in nude mice. In these xenograft tumours, glioblastoma cells were negative for TGM2 expression, while infiltrating macrophages were strongly positive. The most intense staining for TGM2 was observed in macrophages in the vicinity of necrotic regions. Apoptotic cells were frequently present within the necrotic regions and highly TGM2-positive macrophages were observed to be phagocytosing these. The same patterns of TGM2 expression were also observed in samples from glioblastoma patients. Consistent with a role in phagocytosis, TGM2 co-localized with the endosomal protein CD68 in glioblastoma-associated macrophages. The phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is tolerogenic, both because it clears potentially antigenic material and because it induces an immunosuppressive phenotype in macrophages. As TGM2 has a previously established role in efferocytosis and autoimmunity suppression, our findings suggest that TGM2 has a role in maintaining the glioblastoma immunosuppressive environment and that its inhibition might enhance immunotherapy for this cancer.

DDDR-16. CURCUMIN ANALOGS EXHIBIT ANTI-TUMOR ACTIVITY AGAINST GLIOBLASTOMA AT LOWER CONCENTRATIONS THAN CURCUMIN

Abstract PURPOSE Glioblastoma (GBM) has a high risk of recurrence and a poor prognosis due to the difficulty of surgical resection and resistance to the standard treatment, temozolomide. Therefore, the development of new therapeutic agents against GBM is required. This study investigated the anti-tumor activity of the curcumin (Cur) analogs Compound A (ComA) and B (ComB) against GBM. METHODS AND RESULTS To evaluate anti-tumor activity against GBM, we performed a MTT assay. The human glioblastoma cell lines U87-MG and U251 were pre-treated with Cur, ComA, or ComB, then cell viability was examined using a cell counting kit, and IC50 values were calculated. The IC50 values for U87-MG were: Cur, 9.78 µM; ComA, 2.42 µM; and ComB, 1.28 µM. Those for U251 were: Cur, 9.50 µM; ComA, 2.27 µM; ComB, 0.64 µM. To examine the effects of ComA and ComB on normal cells, the same MTT assay was performed on primary cultured astrocytes from neonatal rats. Neither ComA nor ComB resulted in reduced cell viability in astrocytes at concentrations that had an anti-tumor effect on GBM cells (ComA, 3 µM; ComB, 1.5 µM). Next, a cell cycle analysis was performed with PI staining and an apoptosis assay with Annexin V/PI staining using flow cytometry. qPCR was performed to examine the mRNA expression levels of the cell cycle-related proteins CDK1 and CyclinB1. Both ComA and ComB, at lower concentrations than Cur, induced 1) G2/M phase arrest due to a decrease in the mRNA expression levels of CDK1 and CyclinB1 and 2) apoptosis. DISCUSSION These results suggest that both ComA and ComB, at lower concentrations than Cur, have an anti-tumor effect without affecting normal cells by inducing cell cycle arrest and apoptosis against GBM. Further detailed analysis and in vivo studies are expected to lead to the development of novel therapeutic agents for GBM.

MODL-02. DEVELOPMENT OF RODENT GLIOBLASTOMA MODELS OF MGMT-MEDIATED TEMOZOLOMIDE RESISTANCE

Abstract O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs O6-methylguanine DNA damage lesions generated by alkylators such as temozolomide (TMZ), the standard chemotherapeutic for glioblastoma (GBM) treatment. High MGMT expression leads to TMZ resistance and is correlated with lower survival, making it an important prognostic biomarker and potential therapeutic target. Xenograft models with human GBM cell lines require implantation into immunodeficient animals, precluding the study of MGMT-dependent alkylator sensitivity in the setting of an intact tumor immune microenvironment. In this study, we developed rat and mouse glioma models of MGMT overexpression and characterized response to alkylator therapy. RG2 rat glioma and SB28 mouse glioma cells, which have low endogenous MGMT levels, were transduced with cDNA expressing FLAG-tagged MGMT to generate an isogenic pair. RG2 MGMT+ and SB28 MGMT+ cells showed robust expression of MGMT, which was effectively abrogated by MGMT inhibitors. In vitro cell viability assays showed that MGMT overexpression resulted in increased resistance to TMZ compared to parental cells, and this resistance was reversed when cells were concurrently treated with the MGMT inhibitor lomeguatrib. To model MGMT overexpression in vivo, luciferase-expressing RG2 MGMT+ cells were implanted into the brains of syngeneic Fischer 344 rats. Rats were treated with TMZ at 50mg/kg daily x 5 days via intraperitoneal injection. Tumor growth was measured by bioluminescence imaging and rats were monitored for survival and toxicity. RG2 MGMT+ tumors were highly resistant to TMZ, as treatment did not improve survival or delay tumor growth. Overall, our newly developed isogenic rat and mouse glioma models recapitulate MGMT-dependent TMZ sensitivity and provide useful preclinical tools for studying MGMT as a biomarker and potential therapeutic target in GBM. In addition, these syngeneic models facilitate studies on the immune modulation of chemotherapeutic agents in the context of MGMT expression and allows for testing of chemoimmunotherapy combinations.