Patient-derived Glioblastoma Cell Lines Research Articles

Intratumoral Cell Heterogeneity in Patient-Derived Glioblastoma Cell Lines Revealed by Single-Cell RNA-Sequencing.

Glioblastoma cell lines derived from different patients are widely used in tumor biology research and drug screening. A key feature of glioblastoma is the high level of inter- and intratumor heterogeneity that accounts for treatment resistance. Our aim was to investigate whether intratumor heterogeneity is maintained in cell models. Single-cell RNA sequencing was used to investigate the cellular composition of a tumor sample and six patient-derived glioblastoma cell lines. Three cell lines preserved the mutational profile of the original tumor, whereas three others differed from their precursors. Copy-number variation analysis showed significantly rearranged genomes in all the cell lines and in the tumor sample. The tumor had the most complex cell composition, including cancer cells and microenvironmental cells. Cell lines with a conserved genome had less diverse cellularity, and during cultivation, a relative increase in the stem-cell-derived progenitors was noticed. Cell lines with genomes different from those of the primary tumors mainly contained neural progenitor cells and microenvironmental cells. The establishment of cell lines without the driver mutations that are intrinsic to the original tumors may be related to the selection of clones or cell populations during cultivation. Thus, patient-derived glioblastoma cell lines differ substantially in their cellular profile, which should be taken into account in translational studies.

Metabolic Plasticity of Glioblastoma Cells in Response to DHODH Inhibitor BAY2402234 Treatment.

Glioblastoma (IDH-wildtype) represents a formidable challenge in oncology, lacking effective chemotherapeutic or biological interventions. The metabolic reprogramming of cancer cells is a hallmark of tumor progression and drug resistance, yet the role of metabolic reprogramming in glioblastoma during drug treatment remains poorly understood. The dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 is a blood-brain barrier penetrant drug showing efficiency in in vivo models of many brain cancers. In this study, we investigated the effect of BAY2402234 in regulating the metabolic phenotype of EGFRWT and EGFRvIII patient-derived glioblastoma cell lines. Our findings reveal the selective cytotoxicity of BAY2402234 toward EGFRWT glioblastoma subtypes with minimal effect on EGFRvIII patient cells. At sublethal doses, BAY2402234 induces triglyceride synthesis at the expense of membrane lipid synthesis and fatty acid oxidation in EGFRWT glioblastoma cells, while these effects are not observed in EGFRvIII glioblastoma cells. Furthermore, BAY2402234 reduced the abundance of signaling lipid species in EGFRWT glioblastoma. This study elucidates genetic mutation-specific metabolic plasticity and efficacy in glioblastoma cells in response to drug treatment, offering insights into therapeutic avenues for precision medicine approaches.

Evaluating soluble Axl as a biomarker for glioblastoma: A pilot study.

With current imaging, discriminating tumor progression from treatment effect following immunotherapy or oncolytic virotherapy of glioblastoma (GBM) is challenging. A blood based diagnostic biomarker would therefore be helpful. Axl is a receptor tyrosine kinase that is highly expressed by many cancers including GBM. Axl expression is regulated through enzymatic cleavage of its extracellular domain. The resulting fragment can be detected in serum as soluble Axl (sAxl). sAxl levels can distinguish patients with melanoma, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma from healthy controls. This is a pilot study to determine if sAxl is a candidate biomarker for GBM. The sAxl levels in the serum of 40 healthy volunteers and 20 GBM patients were determined using an enzyme-linked immunosorbent assay (ELISA). Pre- and post- operative sAxl levels were obtained. Volumetric MRI evaluation provided GBM tumor volume metrics. There was no significant difference in the sAxl levels of the volunteers (30.16±1.88 ng/ml) and GBM patients (30.74±1.96 ng/ml) p = 0.27. The postoperative sAxl levels were significantly higher than preoperative levels (32.32±2.26 ng/ml vs 30.74±1.96 ng/ml, p = 0.03). We found no correlation between tumor volume and sAxl levels. Axl expression was low or absent in 6 of 11 (55%) patient derived GBM cell lines. Given the wide range of Axl expression by GBM tumors, sAxl may not be a reliable indicator of GBM. However, given the small sample size in this study, a larger study may be considered.

Upregulation of the Renin-Angiotensin System Is Associated with Patient Survival and the Tumour Microenvironment in Glioblastoma.

Glioblastoma is a highly aggressive disease with poor survival outcomes. An emerging body of literature links the role of the renin-angiotensin system (RAS), well-known for its function in the cardiovascular system, to the progression of cancers. We studied the expression of RAS-related genes (ATP6AP2, AGTR1, AGTR2, ACE, AGT, and REN) in The Cancer Genome Atlas (TCGA) glioblastoma cohort, their relationship to patient survival, and association with tumour microenvironment pathways. The expression of RAS genes was then examined in 12 patient-derived glioblastoma cell lines treated with chemoradiation. In cases of glioblastoma within the TCGA, ATP6AP2, AGTR1, ACE, and AGT had consistent expressions across samples, while AGTR2 and REN were lowly expressed. High expression of AGTR1 was independently associated with lower progression-free survival (PFS) (p = 0.01) and had a non-significant trend for overall survival (OS) after multivariate analysis (p = 0.095). The combined expression of RAS receptors (ATP6AP2, AGTR1, and AGTR2) was positively associated with gene pathways involved in hypoxia, microvasculature, stem cell plasticity, and the molecular characterisation of glioblastoma subtypes. In patient-derived glioblastoma cell lines, ATP6AP2 and AGTR1 were upregulated after chemoradiotherapy and correlated with an increase in HIF1A expression. This data suggests the RAS is correlated with changes in the tumour microenvironment and associated with glioblastoma survival outcomes.

382 Precision and Individualized Medicine: Quantification of Brain Cancer Cell Invasion in Confined Microfluidic Spaces Predicts Prognosis

INTRODUCTION: Clinical prognosis of patients with glioblastoma (GBM) is calculated with clinical scores, extent of resection, molecular markers, and tumor subtypes. However, these are often hampered by patient comorbidities, drug toxicity, and other confounding factors. We designed a confined 3D microfluidic technology based on the brain cytoarchitecture by mimicking the diameters of the perivascular spaces and white matter tracts utilized by GBM to invade. METHODS: Patient-derived GBM cell lines (n = 13) were obtained from patients undergoing surgery. After cell culture, the GBM cells’ abilities to migrate and proliferate with MAqCI were evaluated. MAqCI score was calculated via logistic regression. Patients were categorized into low- and high-PFS. Predicted scores were correlated with the clinical status of patients. Cells were treated with gold standard and experimental drugs, and scores were calculated to predict response to treatment. RESULTS: MAqCI Predicted PFS with 83% accuracy, 84.6% sensitivity, and 75% specificity and allowed for the classification of short- and long-PFS (*p = 0.035). Survival based on MAqCI scores separated by short-term (n = 5) and long-term (n = 8) PFS, showed a significant difference between groups (**p = 0.002). After treatment with gold-standard therapy, responder patients were identified based on changes in the MAqCI score. RNAseq of highly motile cells isolated from MAqCI allowed for the identification of druggable targets. An FDA-approved antidepressant against the adrenomedullin gene was selected for MAqCI testing. Changes in MAqCI score after treatment allowed for the identification of responder patients. CONCLUSIONS: Cell invasion properties are directly correlated with patient prognosis. MAqCI could be utilized clinically as a companion assay for predicting tumor recurrence and selecting drugs for individualized GBM treatment.

390 Modelling Glioma Associated Macrophages Using Human Embryonic Stem Cell Derived Microglia

INTRODUCTION: Glioma associated macrophages/microglia (GAMs) are the most abundant immune cell type in glioblastoma and play a key role in immunotherapeutic resistance. The tumor microenvironment (TME) is thought to modulate GAM phenotype and function leading to a more pro-tumorigenic immune milieu however the exact nature of this crosstalk is unclear. Existing systems to study human GAMs are suboptimal as primary human GAMs are difficult to culture. There is therefore a need to develop human model systems of sufficient fidelity to probe GAM function. Here we report on the development of a human embryonic stem cell (hES)-based platform to derive microglia without recourse to immortalizing oncogenic transduction. This technology can assess the impact of tumor derived factors on the microglial phenotype and represents a novel, scalable model system to investigate GAM function. METHODS: hES-microglia were generated using a stepwise cytokine-based differentiation protocol. Upon exposure to conditioned media obtained from cultures of a panel of patient-derived glioblastoma cell lines (n=6 biological replicates), hES-GAMs were analyzed using flow cytometry, single cell RNA sequencing (scRNA). Functional assays included phagocytosis of pH rhodo particles, wound scratch migration assays and intracellular proteome analysis. RESULTS: Exposure to tumor media leads to transcriptional changes similar to those seen in primary GAMs with increased chemotactic genes such as CCL2(MCP1), CCL3 and CCL4, with corresponding changes in cytokine profile at the protein level. Other upregulated cytokines of note include VEGF, OSM, CCL4 and IL10RB. Functional impact of co-culture with tumor media included a dramatic increase in GAM motility (>30 fold) and a statistically significant decrease in phagocytic ability (29.1% p < 0.001). CONCLUSIONS: The hES-derived GAM model is a promising novel platform to dissect the impact of the TME on tumor-derived GAMs.

Abstract 5846: Inhibiting elements of the proteasome recovery pathway sensitizes glioblastoma to proteasome inhibitors

Abstract Bacground: Glioblastoma (GBM) is the most common primary brain tumor, accounting for 15% of all central nervous system related tumors. Despite the aggressive standard of care treatment including chemotherapy, radiotherapy, and maximally safe surgical resection, patient outcomes are abysmal: with 95% of patients relapsing and a median overall survival of 15 months. Thus, this necessitates the rapid query for personalized therapeutics and agents that can potentiate the clinical effects of currently approved treatments. One such pathway that demonstrates aberrant functioning in cancer and is a targetable mechanism is the ubiquitin-proteosome pathway (UPP). Studies have shown that UPP related proteolysis remains constitutively active in cancer cells leading to the rapid degradation of proteins that regulate tumor suppressor genes and oncogenes. Despite robust preclinical evaluations for the usage of proteosome inhibitors against GBM, most tested proteosome inhibitors failed in phase II and III trials, indicating resistance. Methods: We conducted an unbiased genome wide CRISPR-Cas9 screen in human HAP1 cells treated with the proteosome inhibitor, Bortezomib (BTZ), to identify genes that may lead to a BTZ-resistant phenotype. We identified several genes that when perturbed, sensitized cells to BTZ including N-glycanase-1 (NGLY-1), Nuclear factor Erythroid 2-Like-1 (NFE2L1), and DNA damage inducible 1 homolog 2 (DDI2). Herein we sought to evaluate the effects of perturbing components of the proteosome rescue pathway in our Singh lab patient derived GBM cell lines by utilizing CRISPR/Cas9 knockout technology in vitro and in vivo. Results: The generation of NGLY-1, DDI2, and NFE2L1 KO cell lines demonstrated functional sensitivity to the proteosome inhibitor Marizomib (MZB) as observed through a significant reduction of the IC50 in the KO cell lines compared to a control. Functional evaluation also revealed a reduction in proliferation capacity as well as sphere formation in these genetically modified GBM lines. Ongoing in vivo work will aim to evaluate the mitigation of this resistant pathway in our NSG mouse models orthotopically transplanted with these patient derived KO cell lines. In an ongoing collaborative effort, we are working to identify and functionally assess a novel small molecule NGLY-1 inhibitor to evaluate preclinical sensitivity to MZB in our in vitro and in vivo models. Citation Format: Alisha Anand, Muhammad Vaseem Shaikh, Chirayu Chokshi, Benjamin Brakel, William Maich, Shan Grewal, Chitra Venugopal, Sheila Singh. Inhibiting elements of the proteasome recovery pathway sensitizes glioblastoma to proteasome inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5846.

Abstract 690: Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma

Abstract Glioblastoma (GBM) is a highly aggressive primary brain tumor. Treatment consists of maximal safe tumor resection, chemotherapy (temozolomide) and radiotherapy; however, this aggressive treatment offers only a modest improvement in outcomes, with average life expectancy of approximately 12 months. Consequently, novel, effective and easily translatable therapies are urgently required. While radiotherapy is an effective treatment for many solid tumors, GBM displays an innate radio-resistance that contributes to treatment failure. Therefore, there is a need to identify chemotherapeutics that can be used as radiosensitizers to increase efficacy. Notably, a key pathway involved in therapeutic resistance is apoptotic evasion, which is driven by upregulation of pro-survival BCL-2 proteins that inhibit the pro-apoptotic proteins necessary for the apoptotic response. Recent advances in drug development have produced a range of BH3 mimetics - novel chemotherapeutics with potential to reinstate apoptosis by targeting specific pro-survival molecules. We hypothesized that the radiation sensitivity of GBM cells would be enhanced by using BH3 mimetics to inhibit pro-survival molecules and release the apoptotic block. A panel of BH3 mimetics was tested in combination with radiotherapy across three patient-derived GBM cell lines (G7, E2 and R15), using a 3D clonogenic culture system that recapitulates in vivo growth patterns and responses. We found that the BCL-XL inhibitor, A1331852, significantly radiosensitized all 3 GBM lines. In addition, Western blot analysis revealed A1331852 to induce PARP1 cleavage in G7 cells when used as a single agent. Subsequent in vivo studies were conducted in our well characterized orthotopic G7 xenograft murine model of GBM. Immunohistochemical studies showed that the pro-survival proteins MCL-1 and BCL-XL were upregulated in irradiated tumors. Furthermore, a pharmacokinetic study in the same orthotopic murine model, allowing for drug delivery evaluation, and optimized treatment scheduling. Importantly, A1331852 was found to be blood brain barrier penetrant at 25 mg/kg, with increased drug detected in tumor vs normal brain tissue. From these data, a survival study investigating A1331852 as a radiosensitizing agent was carried out in the same G7 GBM orthotopic xenograft model. Kaplan-Meier analysis showed an increase in survival of mice treated with A133852 plus radiotherapy compared with the radiotherapy only group (p = 0.0309). Overall, we have demonstrated that dependence on BCL-2 family proteins is a vulnerability that can be targeted to improve radiotherapy response in GBM. Furthermore, we have identified a promising BH3 mimetic, A1331852, to use in combination with RT and have completed a proof-of-concept in vivo study that confirms the potential of BH3 mimetics to improve outcomes for patients with GBM. Citation Format: Louise R. Dutton, Clara Mullen, Anna L. Koessinger, Mark Jackson, Katrina Stevenson, Anthony J. Chalmers, Stephen Tait, Kirsteen J. Campbell, Joanna L. Birch. Investigation of BH-3 mimetics as radiosensitizing agents in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 690.

Abstract 5238: uPAR as a novel immunotherapeutic target in recurrent glioblastoma

Abstract Glioblastoma (GBM) is the common malignant brain tumor in adults, accounting for approximately 15% of all CNS tumors, and 48.6% of malignant brain tumors, with a median survival of approximately 15 months, and minimal clinical progress having been made in the past two decades. GBM is characterized by extensive inter- and intra-tumoral heterogeneity as well as an extremely immunosuppressive tumor microenvironment. Following Standard-of-Care (SoC) surgical resection and chemoradiotherapy at primary diagnosis, few therapeutic avenues exist at recurrence, owing in part due to a lack of clinically relevant targets. Data from our multi-institutional target pipeline shows that the extracellular urokinase plasminogen activator receptor (uPAR) is significantly upregulated at recurrence on putative GBM brain tumor initiating cells (BTICs), which are believed to drive de novo tumor formation, tumor recurrence, and therapeutic resistance. uPAR plays an important role in the plasminogen activation system, and in the context of cancer, has been implicated in numerous pro-tumorigenic processes such as invasion, proliferation, epithelial-to-mesenchymal transition, and therapy resistance. We used CRISPR-Cas9 to genetically delete uPAR from our in-house patient-derived GBM cell lines, and found that in vitro, knockout of uPAR expression in recurrent GBM cells significantly reduces proliferation and sphere formation capacity, two stem-like traits associated with BTICs. Additionally, we found that uPAR knockout significantly reduced the frequency of BTICs in a given population. Additionally, uPAR knockout cells display increased sensitivity to standard-of-care chemoradiotherapy, implicating uPAR expression in therapy resistance, as seen in recurrent disease. We orthotopically injected immunocompromised mice to generate patient-derived xenograft models of GBM, and found that knockout of uPAR significantly increases survival, and decreases tumor burden. Further, we generated novel anti-uPAR single domain antibodies (sdAbs) and found that they specifically bind uPAR at low nanomolar concentrations in vitro. Using these sdAbs, we created an anti-uPAR CAR T which showed potent cytotoxicity in vitro, and drastically reduced tumor burden and extended survival in vivo. From this work we believe uPAR to be a clinically relevant target in recurrent GBM, and further investigation into therapeutic strategies to target uPAR-positive GBMs should be investigated further. Citation Format: William T. Maich, Muhammad Vaseem Shaikh, Anish Puri, Alisha Anand, Chirayu Chokshi, Sabra Salim, Neil Savage, Chitra Venugopal, Martin Rossotti, Thomas Kislinger, Kevin Henry, Sheila Singh. uPAR as a novel immunotherapeutic target in recurrent glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5238.

Abstract 6867: TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis

Abstract Glioblastoma remains difficult to treat with immunotherapy due to the immunosuppressive tumor microenvironment and the ability of glioma cells to invade diffusely into normal brain parenchyma. While it is known that glioblastoma-associated myeloid cells (GAMs) comprise a large proportion of the tumor microenvironment and can suppress adaptive and innate immune responses through various mechanisms, detailed mapping of myeloid lineages in glioblastoma is still missing. Further characterization of pro-tumoral myeloid-lineages in glioblastoma is important for identifying myeloid-based drug targets that may synergize with T cell-based therapies and produce a more meaningful anti-tumor immune response. Towards this end, we examined numerous scRNAseq datasets derived from glioblastoma patients and identified expression of TNFRSF12A, the gene encoding the TNF- family receptor, Fn14, in a subset of GAMs enriched for expression of cytokines such as CCL3, CCL5, IL-6, and IL-8. This result is intriguing since TWEAK (TNFSF12), the ligand for Fn14, is abundantly expressed within numerous patient-derived glioblastoma cell lines, namely GBM6, GBM8, GBM22, and GBM39. To validate Fn14 protein expression in GAMs, we isolated murine bone-marrow derived macrophages and treated them with glioma-conditioned media, which induced Fn14 gene expression. We then conducted in vitro experiments utilizing the human microglia line HMC3, the murine microglia line SIM-A9, and primary murine bone-marrow-derived macrophages to assess the effects of Fn14 activation in myeloid cells. We found that treatment of Fn14+ myeloid cells with TWEAK ligand upregulates CCL3, CCL5, IL-6, and IL-8 expression. We tested the conditioned media generated from TWEAK-treated Fn14+ myeloid cell lines and found that it promotes glioblastoma invasion in a CCL5-dependent manner. This aligns with previous reports demonstrating the CCL5-CCR5 signaling in glioma cells promotes invasion through calcium-dependent matrix metalloproteinase 2. To assess the spatial distribution of this myeloid subpopulation, we performed RNAscope hybridization against AIF1, TMEM119, CCR5, CCL5, and Fn14 transcripts on sections of murine gliomas generated using the RCAS/tv-a model of human glioblastoma. We found an abundance of Fn14+ plus AIF1+ as well as Fn14+plus TMEM119+ myeloid cells throughout the tumor core and rim of murine tumors and identified CCL5 expression in pseudo-palisades bordering necrotic regions. Our results to date suggest that Fn14 expression in GAMs can promote the invasion of tumor cells into normal brain parenchyma by release of CCL5. Currently, we are utilizing orthotopic patient-derived xenograft models co-implanted with Fn14+/- myeloid cells to test the hypothesis that TWEAK-Fn14 signaling in GAMs provides strong pro-tumoral signals in the glioblastoma microenvironment. Citation Format: Angad Beniwal, Matthew Dufault, Pranjali Kanvinde, David Nascari, Ryan Eghlimi, Adarsha Malla, Dolores Hambardzumyan, Henrique Borges da Silva, Graeme Woodworth, Jeffrey Winkles, Nhan Tran. TWEAK-Fn14 signaling in glioblastoma-associated myeloid cells promotes tumor invasion via CCL5-CCR5 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6867.

Abstract A009: Plinabulin, a novel tubulin targeting agent, collapses the tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival

Abstract A009: Plinabulin, a novel tubulin targeting agent, collapses the tumor microtube network in primary and recurrent patient derived glioblastoma cell lines to inhibit neurosphere invasion and survival

Kinome-Wide Synthetic Lethal Screen Identifies PANK4 as a Modulator of Temozolomide Resistance in Glioblastoma.

Temozolomide (TMZ) represents the cornerstone of therapy for glioblastoma (GBM). However, acquisition of resistance limits its therapeutic potential. The human kinome is an undisputable source of druggable targets, still, current knowledge remains confined to a limited fraction of it, with a multitude of under-investigated proteins yet to be characterized. Here, following a kinome-wide RNAi screen, pantothenate kinase 4 (PANK4) isuncovered as a modulator of TMZ resistance in GBM. Validation of PANK4 across variousTMZ-resistant GBM cell models, patient-derivedGBM cell lines, tissue samples, as well as in vivo studies, corroborates the potential translational significance of these findings. Moreover, PANK4 expression is induced during TMZ treatment, and its expression is associated with a worse clinical outcome. Furthermore, a Tandem Mass Tag (TMT)-based quantitative proteomic approach, reveals that PANK4 abrogation leads to a significant downregulation of a host of proteins with central roles in cellular detoxification and cellular response to oxidative stress. More specifically, as cells undergo genotoxic stress during TMZ exposure, PANK4 depletion represents a crucial event that can lead to accumulation of intracellular reactive oxygen species (ROS) and subsequent cell death. Collectively, a previously unreported role for PANK4 in mediating therapeutic resistance to TMZ in GBM is unveiled.

Novel Brain-Penetrant, Small-Molecule Tubulin Destabilizers for the Treatment of Glioblastoma.

Glioblastoma (GB) is the most lethal brain cancer in adults, with a 5-year survival rate of 5%. The standard of care for GB includes maximally safe surgical resection, radiation, and temozolomide (TMZ) therapy, but tumor recurrence is inevitable in most GB patients. Here, we describe the development of a blood-brain barrier (BBB)-penetrant tubulin destabilizer, RGN3067, for the treatment of GB. RGN3067 shows good oral bioavailability and achieves high concentrations in rodent brains after oral dosing (Cmax of 7807 ng/mL (20 μM), Tmax at 2 h). RGN3067 binds the colchicine binding site of tubulin and inhibits tubulin polymerization. The compound also suppresses the proliferation of the GB cell lines U87 and LN-18, with IC50s of 117 and 560 nM, respectively. In four patient-derived GB cell lines, the IC50 values for RGN3067 range from 148 to 616 nM. Finally, in a patient-derived xenograft (PDX) mouse model, RGN3067 reduces the rate of tumor growth compared to the control. Collectively, we show that RGN3067 is a BBB-penetrant small molecule that shows in vitro and in vivo efficacy and that its design addresses many of the physicochemical properties that prevent the use of microtubule destabilizers as treatments for GB and other brain cancers.

ATR inhibition using gartisertib enhances cell death and synergises with temozolomide and radiation in patient-derived glioblastoma cell lines.

Glioblastoma cells can restrict the DNA-damaging effects of temozolomide (TMZ) and radiation therapy (RT) using the DNA damage response (DDR) mechanism which activates cell cycle arrest and DNA repair pathways. Ataxia-telangiectasia and Rad3-Related protein (ATR) plays a pivotal role in the recognition of DNA damage induced by chemotherapy and radiation causing downstream DDR activation. Here, we investigated the activity of gartisertib, a potent ATR inhibitor, alone and in combination with TMZ and/or RT in 12 patient-derived glioblastoma cell lines. We showed that gartisertib alone potently reduced the cell viability of glioblastoma cell lines, where sensitivity was associated with the frequency of DDR mutations and higher expression of the G2 cell cycle pathway. ATR inhibition significantly enhanced cell death in combination with TMZ and RT and was shown to have higher synergy than TMZ+RT treatment. MGMT promoter unmethylated and TMZ+RT resistant glioblastoma cells were also more sensitive to gartisertib. Analysis of gene expression from gartisertib treated glioblastoma cells identified the upregulation of innate immune-related pathways. Overall, this study identifies ATR inhibition as a strategy to enhance the DNA-damaging ability of glioblastoma standard treatment, while providing preliminary evidence that ATR inhibition induces an innate immune gene signature that warrants further investigation.

Involvement of the tumour necrosis factor receptor system in glioblastoma cell death induced by palbociclib-heptamethine cyanine dye conjugate

Glioblastoma is the most common and aggressive primary brain tumour in adults. The development of anti-brain cancer agents are challenged by the blood-brain barrier and the resistance conferred by the local tumour microenvironment. Heptamethine cyanine dyes (HMCDs) are a class of near-infrared fluorescence compounds that have recently emerged as promising agents for drug delivery. We conjugated palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, to an HMCD, MHI-148, and conducted drug activity analysis on primary patient-derived glioblastoma cell lines. In addition to the expected cytostatic activity, our in vitro studies revealed that palbociclib-MHI-148 conjugate resulted in an almost 100-fold increase in cytotoxicity compared to palbociclib alone. This shift of palbociclib from cytostatic to cytotoxic when conjugated to MHI-148 was due to increased DNA damage, as indicated by an increase in γH2AX foci, followed by an increased expression of key extrinsic apoptosis genes, including TP53, TNFR1, TRAIL, FADD and caspase 8. In addition, we observed a time-dependent increase in the cell surface expression of TNFR1, consistent with an observed increase in the secretion TNFα, followed by TNFR1 endocytosis at 48 h. The treatment of patient GBM cells with the palbociclib-MHI-148 conjugate prevented TNFα-induced NFκB translocation, suggesting conjugate-induced TNFR1 signalling favoured the TNFR1-mediated apoptotic response rather than the pro-inflammatory response pathway. Notably, pharmacological inhibition of endocytosis of TNFR1, and siRNA-knockdown of TNFR1 reversed the palbociclib-MHI-148-induced cell death. These results show a novel susceptibility of glioblastoma cells to TNFR1-dependent apoptosis, dependent on inhibition of canonical NFκB signalling using our previously reported palbociclib-HMCD conjugate.9u5DmcuTyYJ7m7Hnyrq7sRVideo

One-pot synthesis of tetrahydropyrimidinecarboxamides enabling in vitro anticancer activity: a combinative study with clinically relevant brain-penetrant drugs.

In this study, we describe a one-pot three-component synthesis of bioactive tetrahydopyrimidinecarboxamide derivatives employing lanthanum triflate as a catalyst. Out of the synthesized compounds, 4f had the most potent anti-cancer activity and impeded cell cycle progression effectively. Anti-cancer bioactivity was observed in 4f against liver, breast, and lung cancers as well as primary patient-derived glioblastoma cell lines. Compound 4f effectively inhibited the 3D neurosphere formation in primary patient-derived glioma stem cells. Specifically, 4f exhibited synergistic cytotoxicity with the EGFR inhibitor that is the clinical epidermal growth factor receptor inhibitor osimertinib. 4f does not exhibit anti-kinase activity and is cytostatic in nature, and further work is needed to understand the true molecular target of 4f and its derivatives. Through our current work, we establish a promising tetrahydopyrimidinecarboxamide-based lead compound with anti-cancer activity, which may exhibit potent anti-cancer activity in combination with specific clinically relevant small molecule kinase inhibitors.

Eliciting calcium transients with UV nanosecond laser stimulation in adult patient-derived glioblastoma brain cancer cells in vitro

Objective. Glioblastoma (GBM) is the most common and lethal type of high-grade adult brain cancer. The World Health Organization have classed GBM as an incurable disease because standard treatments have yielded little improvement with life-expectancy being 6–15 months after diagnosis. Different approaches are now crucial to discover new knowledge about GBM communication/function in order to establish alternative therapies for such an aggressive adult brain cancer. Calcium (Ca2+) is a fundamental cell molecular messenger employed in GBM being involved in a wide dynamic range of cellular processes. Understanding how the movement of Ca2+ behaves and modulates activity in GBM at the single-cell level is relatively unexplored but holds the potential to yield opportunities for new therapeutic strategies and approaches for cancer treatment. Approach. In this article we establish a spatially and temporally precise method for stimulating Ca2+ transients in three patient-derived GBM cell-lines (FPW1, RN1, and RKI1) such that Ca2+ communication can be studied from single-cell to larger network scales. We demonstrate that this is possible by administering a single optimized ultra-violet (UV) nanosecond laser pulse to trigger GBM Ca2+ transients. Main results. We determine that 1.58 µJ µm−2 is the optimal UV nanosecond laser pulse energy density necessary to elicit a single Ca2+ transient in the GBM cell-lines whilst maintaining viability, functionality, the ability to be stimulated many times in an experiment, and to trigger further Ca2+ communication in a larger network of GBM cells. Significance. Using adult patient-derived mesenchymal GBM brain cancer cell-lines, the most aggressive form of GBM cancer, this work is the first of its kind as it provides a new effective modality of which to stimulate GBM cells at the single-cell level in an accurate, repeatable, and reliable manner; and is a first step toward Ca2+ communication in GBM brain cancer cells and their networks being more effectively studied.

EPCO-41. MICRORNA-MEDIATED DIRECT NEURONAL REPROGRAMMING OF GLIOBLASTOMAS

Abstract Neuron-specific microRNAs miR-9/9* and miR-124 (miR-9/9*-124) are potent neurogenic molecules whose ectopic expression in non-neuronal somatic cells effectively induces direct cell fate conversion into a neuronal fate. Our recent work indicates that the fate transition occurs in distinct steps where miR-9/9*-124 first represses core gene regulatory networks to erase the original non-neuronal cell identity and subsequently activates the neuronal program to confer neuronal fate. Extending these findings, we reason that repression of similar gene regulatory networks by miR-9/9*-124 can also induce fate erasure in cancerous cells using glioblastoma as a paradigm. Glioblastoma multiforme (GBM) is a type of aggressive brain tumor with poor prognosis. Instead of having specific mutation profiles, GBMs present a global genetic network dysregulation which gives rise to a neural stem cell or progenitor cell-like state resembling embryonic neural development. Whereas miR-9/9*-124 plays a key role in inducing mitotic exit and orchestrating neurogenesis during development and neuronal reprogramming, we hypothesize that miR-9/9*-124 will instruct neuronal reprogramming of GBMs. Our results so far demonstrate that miR-9/9*-124 indeed exerts reprogramming activities in patient-derived GBM cell lines. Ectopic expression of miR-9/9*-124 in vitro readily induces GBM cells to exit cell cycle and promotes neuronal identity over time. In GBM intracerebral xenograft mouse model, mice receiving GBM cells overexpressing miR-9/9*-124 were able to survive longer than those with GBMs overexpressing non-specific microRNAs. We are currently carrying out transcriptomic and chromatin accessibility analyses to infer the epigenetic switch as GBM cells are reprogrammed to neurons. Based on these studies, we will further examine whether the GBM cells undergo irreversible fate erasure and are reprogrammed into a stable post-mitotic neuronal state. Our studies will provide insights into the plasticity of GBM fate that can be controlled by microRNAs and the key downstream regulatory nodes underlying fate transition of GBM to permanent post-mitotic state.

CNSC-18. DIFFERENTIAL EFFECTS OF NEUROTRANSMITTERS ON THE GROWTH OF GLIOBLASTOMA SUBTYPES

Abstract Glioblastomas are aggressive brain tumors associated with the worst patient prognosis. Neurons play critical roles in modulating the growth of glioblastoma. It has been demonstrated that glutamatergic neurons form bona fide synapses with glioblastoma tumor cells that are critical for tumor cell proliferation and migration. It is less clear whether the activity of other neuronal types influences glioblastoma pathophysiology, especially among the different glioblastoma molecular subtypes. Here, we examined the effects of neurotransmitters on the growth of glioblastoma cells characterized as the classical, proneural and mesenchymal subtypes. Patient-derived glioblastoma cell lines were exposed to varying concentrations of different neurotransmitters for 72 hours and assessed for cell viability using the CCK-8 assay. RESULTS: from the CCK8 assay indicate that acetylcholine and dopamine increase glioblastoma cell growth. Consistent with previous findings, we observed an increase in cell viability in all cell lines after being incubated with L-glutamate. The classical subtype has a stronger growth response to L-glutamate compared to the other two subtypes. Transcriptomic data from TCGA showed that the expression of GRIK4 (glutamate ionotropic receptor kainate type subunit 4) is the highest in the classical subtype. By comparing GRIK4 expression with patient survival in TCGA glioblastoma cases stratified by molecular subtypes, we found that high GRIK4 expression correlates with worse prognosis in the classical subtype, but not the proneural or mesenchymal patient groups. Our findings suggest that each glioblastoma subtype may differentially leverage neuromodulatory mechanisms to support their own growth. Ongoing studies are investigating the differential neuronal responses in glioblastoma subtypes using both pharmacological and genetic approaches.

STEM-16. PROMOTING THE CANCER STEM CELL SUBPOPULATION AND PHENOTYPE IN GLIOBLASTOMA VIA EXTRACELLULAR VESICLE CROSS-TALK

Abstract INTRODUCTION Extracellular vesicles (EVs) released from cancer cells contain bioactive molecules that have been long implicated in tumorigenesis. In glioblastoma (GBM), there is mounting evidence that EVs play a role in critical processes including angiogenesis, immune suppression, and invasion. GBM secreted EVs have also been demonstrated to promote a cancer stem cell (CSC) phenotype by transporting stemness markers and proteins (I.e. Notch-1) involved in stem-cell activating pathways. Understanding the maintenance of CSCs, which contributes to tumor proliferation, treatment resistance, and re-expansion of the tumor bulk, can offer insight into new therapeutic drug targets that can disrupt this subpopulation. METHODS EVs were isolated from patient-derived primary GBM cell lines maintained as adherent cultures and free floating spheres (n = 3) using a staged ultracentrifugation technique. Normal astrocytes were used as the control group (n = 1). Protein expression profiles were then generated via high-throughput mass spectrometry for both EVs and whole cell lysate samples from the same cell line and compared against each other. RESULTS EGF-containing fibulin extracellular matrix protein 1 (EFEMP1) was shown to be upregulated in GBM derived EVs compared to control. Further analysis shows that EFEMP1 is enhanced in adherent cultures compared to free floating sphere cultures. CONCLUSION EFEMP1 is an extracellular matrix protein that has been identified as an oncogenic protein in GBM by activating Notch-1, Akt, and NF-Kb signaling pathways resulting in increased Temozolomide resistance, cellular proliferation, and invasion, all of which are hallmark characteristics of CSCs. EFEMP1 is also a downstream effector in the HIF-1 pathway, which has been strongly correlated in the maintenance of CSCs. Our results therefore demonstrate a possible EV mediated mechanism for the maintenance of the CSC population and phenotype in GBM.