Human Glioblastoma Cell Cultures Research Articles

The combination therapy using tyrosine kinase receptors inhibitors and repurposed drugs to target patient-derived glioblastoma stem cells

The lesson from many studies investigating the efficacy of targeted therapy in glioblastoma (GBM) showed that a future perspective should be focused on combining multiple target treatments. Our research aimed to assess the efficacy of drug combinations against glioblastoma stem cells (GSCs). Patient-derived cells U3042, U3009, and U3039 were obtained from the Human Glioblastoma Cell Culture resource. Additionally, the study was conducted on a GBM commercial U251 cell line. Gene expression analysis related to receptor tyrosine kinases (RTKs), stem cell markers and genes associated with significant molecular targets was performed, and selected proteins encoded by these genes were assessed using the immunofluorescence and flow cytometry methods. The cytotoxicity studies were preceded by analyzing the expression of specific proteins that serve as targets for selected drugs. The cytotoxicity study using the MTS assay was conducted to evaluate the effects of selected drugs/candidates in monotherapy and combinations. The most cytotoxic compounds for U3042 cells were Disulfiram combined with Copper gluconate (DSF/Cu), Dacomitinib, and Foretinib with IC50 values of 52.37 nM, 4.38 µM, and 4.54 µM after 24 h incubation, respectively. Interactions were assessed using SynergyFinder Plus software. The analysis enabled the identification of the most effective drug combinations against patient-derived GSCs. Our findings indicate that the most promising drug combinations are Dacomitinib and Foretinib, Dacomitinib and DSF/Cu, and Foretinib and AZD3759. Since most tested combinations have not been previously examined against glioblastoma stem-like cells, these results can shed new light on designing the therapeutic approach to target the GSC population.

Combined ion beam irradiation platform and 3D fluorescence microscope for cellular cancer research.

To improve particle radiotherapy, we need a better understanding of the biology of radiation effects, particularly in heavy ion radiation therapy, where global responses are observed despite energy deposition in only a subset of cells. Here, we integrated a high-speed swept confocally-aligned planar excitation (SCAPE) microscope into a focused ion beam irradiation platform to allow real-time 3D structural and functional imaging of living biological samples during and after irradiation. We demonstrate dynamic imaging of the acute effects of irradiation on 3D cultures of U87 human glioblastoma cells, revealing characteristic changes in cellular movement and intracellular calcium signaling following ionizing irradiation.

Cytotoxic effect of the VVGMCSF-Lact oncolytic virus against 3D cultures of human glioblastoma cells U-87 MG

Background. One of the promising methods of treating tumors is virotherapy, which is based on direct lysis of cancer cells by a virus and a virus-mediated antitumor immune response of the body. For the recombinant vaccinia virus strain VVGMCSF-Lact, producing human GMCSF and the oncotoxic protein lactaptin, cytotoxic and antitumor effects were shown in experiments in vitro and in vivo, respectively, when using adhesive cultures of U-87 MG human glioblastoma cells. 3D cultures are a more relevant tumor model than adhesive models, as they more fully reflect the realistic scenario of cancer development, as well as the response of the tumor to anticancer therapy.The aim. To evaluate the cytotoxic effect of the oncolytic virus VV-GMCSF-Lact against 3D cultures of human glioblastoma U-87 MG.Materials and methods. The following methods were used in the work: cultivation of 3D cell cultures, cytofluorometry, microscopic analysis, virus titration, statistical analysis.Results. U-87 MG cells were transduced with a lentiviral vector carrying the GFP reporter gene. The cytotoxicity of the VV-GMCSF-Lact virus (IC50) against the studied cells was 0.024 PFU/cell. U-87 MG cells were cultured under conditions for the formation of 3D structures. Microscopic analysis showed the oncolytic effect of the virus on the cells of 3D cultures as early as 24 hours after the start of incubation. Flow cytometry showed an increase in the granularity of glioblastoma cells under the action of the virus, which indicates active replication of the virus in the cells. The virus titer was 0.44 PFU/cell.Conclusions. The recombinant VV-GMCSF-Lact virus has a cytotoxic effect on 3D human glioblastoma U-87 MG cell cultures and actively replicates in them. In the future, to test the oncolytic effect of VV-GMCSF-Lact, it is planned to use not only 3D human glioblastoma cultures, but also cerebral organelles obtained in the process of cocultivation of glioblastoma cells and induced human pluripotent cells.

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.

The PYK2 inhibitor PF-562271 enhances the effect of temozolomide on tumor growth in a C57Bl/6-Gl261 mouse glioma model

BackgroundThe development of resistance to temozolomide (TMZ), a standard chemotherapeutic, limits the effective treatment of glioblastoma (GBM). Focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (Pyk2) regulate proliferation and invasion of GBM cells. We found that TMZ activates FAK and Pyk2 signaling in GBM. We hypothesized that pharmacological inhibitors of Pyk2/FAK together with TMZ can enhance the inhibitory effect of TMZ on tumor growth and dispersal and improve the treatment outcome.MethodsPrimary human GBM cell cultures and a C57Bl/6-GL261 mouse glioma implantation model were used. Pyk2 (Tyr579/580) and FAK (Tyr925) phosphorylation was analyzed by western blotting. Viability, cell cycle, migration, invasion and invadopodia formation were investigated in vitro. Animal survival, tumor size and invasion, TUNEL apoptotic cell death and the Ki67 proliferation index were evaluated in vivo upon treatment with TMZ (50 mg/kg, once/day, orally) and the Pyk2/FAK inhibitor PF-562271 (once/daily, 50 mg/kg, orally) vs. TMZ monotherapy.ResultsIn vitro studies revealed significantly reduced viability, cell cycle progression, invasion and invadopodia with TMZ (100 µM) + PF-562271 (16 nM) compared with TMZ alone. In vivo studies demonstrated that combinatorial treatment led to prominent reductions in tumor size and invasive margins, extensive signs of apoptosis and a reduced proliferation index, together with a 15% increase in the survival rate in animals, compared with TMZ monotherapy.ConclusionTMZ + PF-562271 eliminates TMZ-related Pyk2/FAK activation in GBM and improves the treatment efficacy.

Systemic Delivery of an Adjuvant CXCR4-CXCL12 Signaling Inhibitor Encapsulated in Synthetic Protein Nanoparticles for Glioma Immunotherapy.

Glioblastoma (GBM) is an aggressive primary brain cancer, with a 5 year survival of ∼5%. Challenges that hamper GBM therapeutic efficacy include (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME), and (iv) the blood-brain barrier (BBB). The C-X-C motif chemokine ligand-12/C-X-C motif chemokine receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it has poor pharmacokinetic properties, and unfavorable bioavailability has hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4+ monocytic myeloid-derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long-term survival, with ∼60% of GBM tumor-bearing mice remaining tumor free after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.

IGlioSub: an integrative transcriptomic and epigenomic classifier for glioblastoma molecular subtypes

BackgroundGlioblastoma (GBM) is the most aggressive and prevalent primary brain tumor, with a median survival of 15 months. Advancements in multi-omics profiling combined with computational algorithms have unraveled the existence of three GBM molecular subtypes (Classical, Mesenchymal, and Proneural) with clinical relevance. However, due to the costs of high-throughput profiling techniques, GBM molecular subtyping is not currently employed in clinical settings.MethodsUsing Random Forest and Nearest Shrunken Centroid algorithms, we constructed transcriptomic, epigenomic, and integrative GBM subtype-specific classifiers. We included gene expression and DNA methylation (DNAm) profiles from 304 GBM patients profiled in the Cancer Genome Atlas (TCGA), the Human Glioblastoma Cell Culture resource (HGCC), and other publicly available databases.ResultsThe integrative Glioblastoma Subtype (iGlioSub) classifier shows better performance (mean AUC = 95.9%) stratifying patients than gene expression (mean AUC = 91.9%) and DNAm-based classifiers (AUC = 93.6%). Also, to expand the understanding of the molecular differences between the GBM subtypes, this study shows that each subtype presents unique DNAm patterns and gene pathway activation.ConclusionsThe iGlioSub classifier provides the basis to design cost-effective strategies to stratify GBM patients in routine pathology laboratories for clinical trials, which will significantly accelerate the discovery of more efficient GBM subtype-specific treatment approaches.

Norepinephrine inhibits migration and invasion of human glioblastoma cell cultures possibly via MMP-11 inhibition

PurposeGrowing evidence has shown that the stress hormones affect tumor progression. Patients with surgery to remove tumor often have increased norepinephrine during the perioperative period. However, the effect of norepinephrine on the progression of glioblastoma has not yet studied. Therefore, the present study aimed at investigating the effects of norepinephrine on the migration and invasion of the human glioblastoma U87 and U251 cell lines and the mechanism for the effects. MethodsThe U87 and U251 cells were treated with 0, 0.1, 1, 5, 10 or 50 μM norepinephrine. A scratch wound healing assay and a transwell invasion assay were used to investigate cell migration and invasion, respectively. The Human Tumor Metastasis RT2 Profiler PCR Array was used to detect the expression of 84 genes known to be involved in metastasis. ResultsFollowing norepinephrine treatment, the ability of the U87 and U251 cells to migrate and invade was significantly decreased. Human Tumor Metastasis RT2 Profiler PCR Array assay showed that matrix metallopeptidase-11 (MMP-11) was decreased following norepinephrine treatment. The β-adrenergic receptor blocker (AR) propranolol blunted the suppressive effect of norepinephrine on the migration and invasion of U251 cells but did not have such an effect on the invasion of U87 cells. MMP-11 silencing inhibited the migration and invasion of U87 and U251 cells. The Cancer Genome Atlas data showed that patients with higher expression of MMP-11 in the glioblastoma tissues had poorer prognosis. ConclusionOur results indicate that norepinephrine inhibits the migration and invasion of human glioblastoma cells. This effect may be mediated by the decrease of MMP-11. β-AR may be a regulatory factor for this effect in U251 cells.

Preliminary assessment of low-intensity transcranial magnetic stimulation (TMS) during the treatment for brain glioblastomas.

2545 Background: The standard treatment of malignant brain gliomas, including surgical and radiation therapies, does not provide recovery and a long-time favorable prognosis. The development of technologies and international guidelines on the introduction of electric (TTF) and electromagnetic (TMS) fields in combination treatment for glioblastomas aims to improve immediate results, as shown in experiments on human glioblastoma cell culture. The TMS protocol requires further refinement in parameters of frequency, intensity, and exposure with an assessment of the immediate results of combined treatment. Methods: The study included 60 patients diagnosed with MBG receiving osteoplastic craniotomy with radical (within visible unchanged tissues) tumor removal. Starting from the second day after the surgery, patients of group 1 (n = 30) received 10 sessions of magnetotherapy in the double exposure mode. For the first morning exposure, we used an ultra-low-frequency magnetic field (ULFMF) (0.03 to 9.0 Hz) on the hypothalamus projection area to induce a general antistress reaction. After 2.5-3 hours, local (on the surgical site) TMS exposure with the Neuro-MSD system (Russia) was applied in the pulse algorithm, up to 1 GHz and 5 Hz, 15 mT, 3 min. The induction was reduced exponentially (C = 0.8). The control group 2 (n = 30) did not receive ULFMF or TMS. Magnetic resonance imaging (MRI) was used to determine the volume of tumors (Vt, cm3) and perifocal edema (Ve, cm3) calculated according to the Shrek’s formula for an ellipsoid (V = a×b×c×π/6). Results: Before surgery, Vt = 54.7±5.7cm3 in group 1, in group 2 - Vt = 60.9±8.5cm3 (no statistical differences). After surgery and the subsequent course of ULFMF and TMS, residual tumor volumes in group 1 were 2.5 times lower than in controls (p < 0.05). The difference between Ve values before and after treatment was on average 80.7 cm3 in group 1 and 41.8 cm3 in group 2 (p < 0.05). Conclusions: The inclusion of sequential ULFMF and TMS exposures into postoperative therapy for gliomas, taking into account various vectors of the influence on the projection of centers of homeostasis regulation and the surgical field, as well as the development of programmed modes of biotropic exposure parameters, improves antitumor and anti-edematous effects.

Fabrication of 3D calcium-alginate scaffolds for human glioblastoma modeling and anticancer drug response evaluation.

The three-dimensional (3D) cell culture model has been increasingly used to study cancer biology and screen for anticancer agents due to its close mimicry to in vivo tumor biopsies. In this study, 3D calcium(Ca)-alginate scaffolds were developed for human glioblastoma cell culture and an investigation of the responses to two anticancer agents, doxorubicin and cordycepin. Compared to the 2D monolayer culture, glioblastoma cells cultured on these 3D Ca-alginate scaffolds showed reduced cell proliferation, increased tumor spheroid formation, enhanced expression of cancer stem cell genes (CD133, SOX2, Nestin, and Musashi-1), and improved expression of differentiation potential-associated genes (GFAP and β-tubulin III). Additionally, the vascularization potential of the 3D glioblastoma cells was increased, as indicated by a higher expression of tumor angiogenesis biomarker (VEGF) than in the cells in 2D culture. To highlight the application of Ca-alginate scaffolds, the 3D glioblastomas were treated with anticancer agents, including doxorubicin and cordycepin. The results demonstrated that the 3D glioblastomas presented a greater resistance to the tested anticancer agents than that of the cells in 2D culture. In summary, the 3D Ca-alginate scaffolds for glioblastoma cells that were developed in this study offer a promising platform for anticancer agent screening and the discovery of drug-resistant mechanisms of cancer.

Achievable Central Nervous System Concentrations of the Green Tea Catechin EGCG Induce Stress in Glioblastoma Cells in Vitro

The polyphenolic compounds present in green tea are preventative against cancer in several animal tumor models. However, direct cytotoxic effects on cancer cells have also been reported. In order to determine whether drinking of green tea has chemopreventive or cytotoxic effects on brain cancer cells, we investigated the effect of the major green tea polyphenol EGCG as a pure substance and as tea extract dietary supplement on primary human glioblastoma cell cultures at the CNS-achievable concentration of 100 nM reported in the literature. We compared this with the effect of the cytotoxic concentration of 500 μM determined to be specific for the investigated primary glioblastoma cultures. After treatment with 500 µM EGCG, strong induction of autophagy and apoptosis was observed. Under treatment with 100 nM EGCG, glioblastoma cells proliferated over the entire observation period of 6 days without any detectable signs of cell death. Only within the first 12 h of treatment was increased accumulation of autophagic vacuoles and increased reactive oxygen species production as a stress response demonstrated. Mild forms of stress, such as treatment with 100 nM EGCG, activate different endogenous repair mechanisms to protect cells. Our data imply that drinking of green tea may have chemopreventive effects, but no direct cytotoxic properties.

A comparative study of the replicative properties of antitumor recombinant vaccinia viruses in cultivated U87 human glioblastoma cells and simian CV-1 kidney cells

At present, virotherapy is one of the rapidly developing areas in the treatment of cancer, and its advantage is the selective destruction of cancer cells with the minimal destructive effect on the healthy cells of the body. Orthopoxviruses provide a promising basis for the design of oncolytic drugs. They have a number of advantages over other viral vectors, and one of these advantages is the large capacity of their genomes. It allows them to be furnished with genes encoding proteins with antitumor properties. In this study, we compare the replicative properties of ten variants of the vaccinia virus (LIVP strain of VACV) tested on cultures of human glioblastoma cells and simian CV-1 kidney cells. Some of these viruses have additional genes, such as those encoding the granulocyte-macrophage colony-stimulating factor, apoptosis-inducing protein TRAIL, and green fluorescent protein. We have also studied VACV with five virulence genes deleted, namely, the genes encoding hemagglutinin, the γ-interferon-binding protein, thymidine kinase, the complement-binding protein, and the Bcl2-like inhibitor of apoptosis. The reactogenicity and neurovirulence of this disarmed derivative are much less than in the original LIVP strain. It has been found that derivatives of the vaccinia virus with the deleted thymidine kinase gene most intensely replicate in the glioblastoma cell culture.

A molecular cascade modulates MAP1B and confers resistance to mTOR inhibition in human glioblastoma.

Clinical trials of therapies directed against nodes of the signaling axis of phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin (mTOR) in glioblastoma (GBM) have had disappointing results. Resistance to mTOR inhibitors limits their efficacy. To determine mechanisms of resistance to chronic mTOR inhibition, we performed tandem screens on patient-derived GBM cultures. An unbiased phosphoproteomic screen quantified phosphorylation changes associated with chronic exposure to the mTOR inhibitor rapamycin, and our analysis implicated a role for glycogen synthase kinase (GSK)3B attenuation in mediating resistance that was confirmed by functional studies. A targeted short hairpin RNA screen and further functional studies both in vitro and in vivo demonstrated that microtubule-associated protein (MAP)1B, previously associated predominantly with neurons, is a downstream effector of GSK3B-mediated resistance. Furthermore, we provide evidence that chronic rapamycin induces microtubule stability in a MAP1B-dependent manner in GBM cells. Additional experiments explicate a signaling pathway wherein combinatorial extracellular signal-regulated kinase (ERK)/mTOR targeting abrogates inhibitory phosphorylation of GSK3B, leads to phosphorylation of MAP1B, and confers sensitization. These data portray a compensatory molecular signaling network that imparts resistance to chronic mTOR inhibition in primary, human GBM cell cultures and points toward new therapeutic strategies.

PAMAM Dendrimers Attached to Collagen via a Malondialdehyde Linker

AbstractFunctionalization of polyamidoamine dendrimers (PAMAM) for use as macromolecular drug carriers with aldehydes was performed using aminoacetaldehyde dimethylacetal (AADA) and malondialdehyde (MDA). AADA was used to obtain low molecular weight product upon reaction with dimethyl succinate and PAMAM G‐0.5. The structure of succinate bis‐(dimethylacetal)acetamide was elucidated by X‐ray crystallography. This product, as well as the product of condensation of PAMAM G‐0.5 with AADA, were resistant to hydrolysis. MDA reacted with iso‐butylamine and polyamidoamine PAMAM G0 dendrimers to give 3‐[N‐(2‐methylpropyl)amino]prop‐2‐enal and PAMAM G0 with four arms ending with prop‐2‐enal groups. The compounds were characterized by NMR, MS and/or elemental analysis. PAMAM G3 was also converted to aldehyde‐terminated dendrimer with excess MDA, however, the product was water insoluble. The low molecular weight water soluble analogue prop‐2‐enal terminated PAMAM G0 dendrimer was tested for toxicity on normal human fibroblasts and glioblastoma cell cultures. The toxicity of prop‐2‐enal terminated PAMAM G0 was comparable with that of amine ending PAMAM G0. Finally, fluorescein‐labeled PAMAM G3 was covalently attached to a collagen scaffold via a MDA linker as demonstrated by confocal microcopy. This rapid method of covalent attachment of PAMAM‐drug conjugates to MDA‐activated collagen opens new pathways for delivery of drugs into tissue.

Abstract 5430: GAPDH loss in a tumorigenic human glioblastoma cell line

Abstract Background: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous, multifunctional 37 kDa enzyme best known for catalyzing the sixth step in glycolysis. GAPDH expression is so consistent that it has been granted “housekeeping” status, and is used as a standard (along with β-actin) for normalizing Western blots. While increased GAPDH expression has been described in hypoxia, diabetes, and some cancers, decreased expression is rare. Here we describe a new human glioblastoma cell line that exhibits GAPDH loss, while maintaining a high degree of malignancy. Methods: Primary cultures of human glioblastoma cells were initiated and maintained in DMEM with 10% FBS. This particular cell line (designated E297) grew readily, and was passaged more than 40 times. E297 cells were confirmed to be negative for HIV, HTLV, hepatitis B and C, CMV, and mycoplasma contamination. Cells in exponential growth were stained for routine markers, and studied by DNA flow cytometry. GAPDH expression was studied by Western blot analysis using polyclonal rabbit anti-human GAPDH IgG (Abcam, Rosemont, IL), with HeLa, U138, U87 and U251 cells (as well as β-actin staining) as positive controls. For intracranial implantation into Wistar-Furth rats (male, 10-11 weeks), cells were suspended at 20 x 106 cells/ml. Using aseptic technique, rats were anesthetized, and placed into a stereotactic frame. A burr hole was drilled 3 mm right of the bregma and 25 µl of suspension injected (5 mm depth) over 10 min. by Hamilton syringe. Rats were studied by MRI and euthanized when symptomatic. Results: E297 cells have glial/epithelioid morphology, with a doubling time of 24 + 2 hours in logarithmic growth phase, with a single DNA subpopulation with 28% S phase. They stain positively for GFAP, vimentin, bFGF, c-myc and p53, but fail to express GAPDH. 10/10 animals implanted intracerebrally developed tumors, becoming symptomatic and requiring sacrifice at 25 days (mean). Loss of GAPDH expression was confirmed by Western blot analysis. Conclusions: The E297 human glioblastoma cell line is highly aggressive and proliferates rapidly. It is tumorigenic in nonimmunosuppressed rats but lacks GAPDH protein. We conclude that GAPDH expression is not essential for glioblastoma cell proliferation under routine culture conditions, and care needs to be taken when using GAPDH as a normalization standard for Western blots of cancer cells. Citation Format: Zachary Gaertner, Yi Lu, Jinsuh Kim, Gayatry Mohapatra, Ankit Mehta, Sanjani Lakka, Herbert Engelhard. GAPDH loss in a tumorigenic human glioblastoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5430. doi:10.1158/1538-7445.AM2017-5430

Safe and Effective Treatment of Experimental Neuroblastoma and Glioblastoma Using Systemically Delivered Triple MicroRNA-Detargeted Oncolytic Semliki Forest Virus.

Background: Glioblastoma multiforme and high-risk neuroblastoma are cancers with poor outcome. Immunotherapy in the form of neurotropic oncolytic viruses is a promising therapeutic approach for these malignancies. Here we evaluate the oncolytic capacity of the neurovirulent and partly IFNβ-resistant Semliki Forest virus (SFV)-4 in glioblastoma multiformes and neuroblastomas. To reduce neurovirulence we constructed SFV4miRT, which is attenuated in normal central nervous system (CNS) cells through insertion of microRNA target sequences for miR124, miR125, miR134.Methods: Oncolytic activity of SFV4miRT was examined in mouse neuroblastoma and glioblastoma multiforme cell lines and in patient-derived human glioblastoma cell cultures (HGCC). In vivo neurovirulence and therapeutic efficacy was evaluated in two syngeneic orthotopic glioma models (CT-2A, GL261) and a syngeneic subcutaneous neuroblastoma model (NXS2). The role of IFNβ in inhibiting therapeutic efficacy was investigated.Results: The introduction of miRNA target sequences reduced neurovirulence of SFV4 in terms of attenuated replication in mouse CNS cells and ability to cause encephalitis when administered intravenously. A single intravenous injection of SFV4miRT prolonged survival and cured four of eight mice (50%) with NXS2 and three of 11 mice (27%) with CT-2A, but not for GL261 tumor-bearing mice. In vivo therapeutic efficacy in different tumor models inversely correlated to secretion of IFNβ by respective cells upon SFV4 infection in vitro Similarly, killing efficacy of HGCC lines inversely correlated to IFNβ response and interferon-α/β receptor-1 expression.Conclusions: SFV4miRT has reduced neurovirulence, while retaining its oncolytic capacity. SFV4miRT is an excellent candidate for treatment of glioblastoma multiforme and neuroblastoma with low IFN-β secretion. Clin Cancer Res; 23(6); 1519-30. ©2016 AACR.

A novel 3D human glioblastoma cell culture system for modeling drug and radiation responses.

Background.Glioblastoma (GBM) is the most common primary brain tumor, with dismal prognosis. The failure of drug–radiation combinations with promising preclinical data to translate into effective clinical treatments may relate to the use of simplified 2-dimensional in vitro GBM cultures.Methods.We developed a customized 3D GBM culture system based on a polystyrene scaffold (Alvetex) that recapitulates key histological features of GBM and compared it with conventional 2D cultures with respect to their response to radiation and to molecular targeted agents for which clinical data are available.Results.In 3 patient-derived GBM lines, no difference in radiation sensitivity was observed between 2D and 3D cultures, as measured by clonogenic survival. Three different molecular targeted agents, for which robust clinical data are available were evaluated in 2D and 3D conditions: (i) temozolomide, which improves overall survival and is standard of care for GBM, exhibited statistically significant effects on clonogenic survival in both patient-derived cell lines when evaluated in the 3D model compared with only one cell line in 2D cells; (ii) bevacizumab, which has been shown to increase progression-free survival when added to standard chemoradiation in phase III clinical trials, exhibited marked radiosensitizing activity in our 3D model but had no effect on 2D cells; and (iii) erlotinib, which had no efficacy in clinical trials, displayed no activity in our 3D GBM model, but radiosensitized 2D cells.Conclusions.Our 3D model reliably predicted clinical efficacy, strongly supporting its clinical relevance and potential value in preclinical evaluation of drug–radiation combinations for GBM.

A comparative study of replicative properties of antitumor recombinant vaccinia viruses on human glioblastoma cell culture U87 and monkey kidney cell culture CV-1

In the world of today, virotherapy is one of the rapidly developing areas in the treatment of cancer, and its advantage is selective destruction of cancer cells with minimizing the destructive effect on normal cells of the body. A promising basis for the creation of oncolytic drugs is orthopoxviruses, which have a number of advantages over other viral vectors, and one of these advantages is a large capacity of the genome, which allows genes encoding proteins with antitumor properties to be cloned into their genome. In this study, we compared the replicative properties of ten variants of vaccinia virus (the strain LIVP of VACV) using human glioblastoma cell culture; some of these viruses have additional genes, such as the gene encoding granulocyte-macrophage colony stimulating factor, gene encoding apoptosis-inducing protein TRAIL and gene encoding green fluorescent protein. Furthermore, the virus with five virulence genes deleted (genes encoding hemagglutinin, γ-interferonbinding protein, thymidine kinase, complementbinding protein and Bcl2-like inhibitor of apoptosis), which has significantly lower reactogenicity and neurovirulence compared to the original strain LIVP of VACV, was studied. These data suggest that variants of vaccinia virus with a defective gene encoding thymidine kinase most actively replicate in glioblastoma cell culture.

Efficacy of systemic adoptive transfer immunotherapy targeting NY-ESO-1 for glioblastoma.

Immunotherapy is an ideal treatment modality to specifically target the diffusely infiltrative tumor cells of malignant gliomas while sparing the normal brain parenchyma. However, progress in the development of these therapies for glioblastoma has been slow due to the lack of immunogenic antigen targets that are expressed uniformly and selectively by gliomas. We utilized human glioblastoma cell cultures to induce expression of New York-esophageal squamous cell carcinoma (NY-ESO-1) following in vitro treatment with the demethylating agent decitabine. We then investigated the phenotype of lymphocytes specific for NY-ESO-1 using flow cytometry analysis and cytotoxicity against cells treated with decitabine using the xCelligence real-time cytotoxicity assay. Finally, we examined the in vivo application of this immune therapy using an intracranially implanted xenograft model for in situ T cell trafficking, survival, and tissue studies. Our studies showed that treatment of intracranial glioma-bearing mice with decitabine reliably and consistently induced the expression of an immunogenic tumor-rejection antigen, NY-ESO-1, specifically in glioma cells and not in normal brain tissue. The upregulation of NY-ESO-1 by intracranial gliomas was associated with the migration of adoptively transferred NY-ESO-1-specific lymphocytes along white matter tracts to these tumors in the brain. Similarly, NY-ESO-1-specific adoptive T cell therapy demonstrated antitumor activity after decitabine treatment and conferred a highly significant survival benefit to mice bearing established intracranial human glioma xenografts. Transfer of NY-ESO-1-specific T cells systemically was superior to intracranial administration and resulted in significantly extended and long-term survival of animals. These results reveal an innovative, clinically feasible strategy for the treatment of glioblastoma.

Intracranial Glioblastomas: New Hope for an Effective Treatment

Intracranial Glioblastomas: New Hope for an Effective Treatment