CT-2A Cells Research Articles

Abstract A20: A novel CXCR4 antagonist interferes with antivascular endothelial growth factor therapy-induced glioma dissemination

Abstract Background: Resistance to antiangiogenic therapy (AT) in patients with glioblastoma (GBM) treated with bevacizumab (BEV) is characterized by local recurrence and distant dissemination of gliomas associated with remodeling of tumor vessels and pronounced hypoxia known to promote glioma cell invasion. Expression of the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1α (SDF-1α) is enhanced in invading tumor cells (CXCR4) and neurons and blood vessels (SDF-1α) in GBM and associated with tumor hypoxia, proliferation, invasion and angiogenesis. Using Protein Epitope Mimetics (PEM) technology (Robinson JA et al., 2008), Polyphor Ltd. has developed selective, highly potent CXCR4 antagonists (De Marco SJ et al., 2006) (U.S. Patent no. 8,716,242), such as POL5551. To address the problem of resistance to AT, we sought to determine whether combined therapy (CTx) with POL5551 and the murine equivalent of BEV (antibody B20-4.1.1) could inhibit the invasion and associated pathologic characteristics of gliomas in vivo. Methods: Adult C57BL/6 mice implanted orthotopically with syngeneic CT-2A or GL261 glioma cells were randomized on day 14 into 4 groups: 1) control, 2) POL5551 (5 mg/kg s.c.), 3) anti-murine vascular endothelial growth factor (VEGF) monoclonal antibody B20-4.1.1 (5 mg/kg i.p.; Genentech Inc.) (Bagri A et al., 2010) and 4) combined POL5551 and B20-4.1.1 (CTx). On day 28, brain tissues were processed, sections analyzed for tumor volume and invasiveness (Sottoriva A et al., 2010) (H&E), hypoxia (Hypoxyprobe), proliferation (pHH3, phosphohistone H3), vascular density (CD105), apoptosis (cleaved caspase 3) and microglial (IBA1, ionized calcium binding adaptor molecule 1) and stem (nestin) cellular fractions and the results analyzed by analysis of variance (ANOVA). The tissue concentrations of POL5551 were measured by mass spectrometry. Results: Following B20-4.1.1, both glioma models recapitulated the increased invasive phenotype seen in human GBM associated with increased tumor hypoxia and reduced cellular proliferation and vascular density. Following POL5551 alone, tumor proliferation was slightly diminished. Nestin immunopositivity in CT-2A glioma was markedly reduced following POL5551, and to an intermediate level with CTx, consistent with a previous report (Barone A et al., 2014). In both models, CTx reduced tumor invasiveness (by up to 39.3% compared with B20-4.1.1 alone) and vascular density together with increased apoptosis. In CT-2A glioma, CTx also diminished IBA1 immunopositivity. The concentration of POL5551 was higher in GL261 glioma (45.8 nM) than in brain adjacent to tumor (23.7 nM) or normal brain (11.1 nM) and sufficient to account for its biological effects given the high binding affinity (0.5 nM) of POL5551 for CXCR4. In preliminary work, POL5551 reduced the rate of migration of GL261 glioma cells in vitro. Conclusions: Following anti-VEGF antibody treatment, both murine glioma models recapitulated the increased invasive phenotype seen in human GBM associated with a more hypoxic tumor microenvironment. In both tumor models, CTx with POL5551 and B20-4.1.1 may be beneficial in overcoming AT-induced glioma dissemination. Importantly, POL5551 crossed the blood-brain barrier and accumulated to biologically active levels in glioma tissue. Our results suggest the potential clinical utility of PEM CXCR4 antagonists as adjunct therapy for patients with GBM. Citation Format: Jean-Pierre Gagner, Yasmeen Sarfraz, Fawaz M. Alotaibi, Valerio Ortenzi, Awab T. Tayyib, Luis A. Chiriboga, Garry J. Douglas, Eric Chevalier, Barbara Romagnoli, Gerald Tuffin, Klaus Dembowsky, David Zagzag. A novel CXCR4 antagonist interferes with antivascular endothelial growth factor therapy-induced glioma dissemination. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A20.

ET-51 * COMBINED THERAPY WITH A NOVEL CXCR4 ANTAGONIST AND AN ANTI-VEGF ANTIBODY IMPAIRS THE GROWTH OF GLIOMAS

BACKGROUND: Expression of chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1α (SDF-1α) is enhanced in glioblastoma, including at invasive edges, and associated with proliferation, invasion and angiogenesis (Zagzag, 2008; Ehtesham, 2013; Gagliardi, 2014). Using Protein Epitope Mimetics technology (Robinson, 2008), Polyphor Ltd. has developed selective, highly potent CXCR4 antagonists (De Marco, 2006), e.g., POL5551 (Ki: 0.5 nM). To address the problem of resistance to antiangiogenic therapy, we sought to determine whether POL5551 could inhibit the growth and/or invasion of gliomas in vivo. METHODS: Female adult C57BL/6 mice were implanted orthotopically with 1 x 105 CT-2A or GL261 glioma cells. On day 14, mice were randomized into 4 groups: 1) control, 2) POL5551 (5 mg/kg s.c.) twice daily, 3) anti-murine VEGF monoclonal antibody B20-4.1.1 (Genentech Inc.) (5 mg/kg i.p.) twice weekly (Bagri, 2010) and 4) combined POL5551 and B20-4.1.1. On day 28, brain tissues were processed, sections analyzed for tumor volume and invasion (HE 45.8 nM) than in brain adjacent to tumor (45 ± 4.8; 23.7) or normal brain (21 ± 1.5; 11.1) and sufficient to account for its biological effects. CONCLUSIONS: Although combined therapy did not reduce significantly glioma invasion, it did impair the growth of CT-2A and GL261 gliomas by 69.8% and 59.9%, respectively. Our results suggest the potential utility of POL5551 as adjunct therapy for glioblastoma.

Production of Bioactive Soluble Interleukin-15 in Complex with Interleukin-15 Receptor Alpha from a Conditionally-Replicating Oncolytic HSV-1

Oncolytic type-1 herpes simplex viruses (oHSVs) lacking the γ134.5 neurovirulence gene are being evaluated for treatment of a variety of malignancies. oHSVs replicate within and directly kill permissive cancer cells. To augment their anti-tumor activity, oHSVs have been engineered to express immunostimulatory molecules, including cytokines, to elicit tumor-specific immune responses. Interleukin-15 (IL-15) holds potential as an immunotherapeutic cytokine because it has been demonstrated to promote both natural killer (NK) cell-mediated and CD8+ T cell-mediated cytotoxicity against cancer cells. The purpose of these studies was to engineer an oHSV producing bioactive IL-15. Two oHSVs were constructed encoding murine (m)IL-15 alone (J100) or with the mIL-15 receptor α (mIL-15Rα, J100D) to determine whether co-expression of these proteins is required for production of bioactive mIL-15 from oHSV. The following were demonstrated: i) both oHSVs retain replication competence and cytotoxicity in permissive tumor cell lines. ii) Enhanced production of mIL-15 was detected in cell lysates of neuro-2a cells following J100D infection as compared to J100 infection, suggesting that mIL-15Rα improved mIL-15 production. iii) Soluble mIL-15 in complex with mIL-15Rα was detected in supernates from J100D-infected, but not J100-infected, neuro-2a, GL261, and CT-2A cells. These cell lines vary in permissiveness to oHSV replication and cytotoxicity, demonstrating soluble mIL-15/IL-15Rα complex production from J100D was independent of direct oHSV effects. iv) The soluble mIL-15/IL-15Rα complex produced by J100D was bioactive, stimulating NK cells to proliferate and reduce the viability of syngeneic GL261 and CT-2A cells. v) J100 and J100D were aneurovirulent inasmuch as no neuropathologic effects were documented following direct inoculation into brains of CBA/J mice at up to 1x107 plaque forming units. The production of mIL-15/mIL-15Rα from multiple tumor lines, as well as the lack of neurovirulence, renders J100D suitable for investigating the combined effects of oHSV and mIL-15/IL-15Rα in various cancer models.

Stemness of the CT-2A Immunocompetent Mouse Brain Tumor Model: Characterization In Vitro

Evidence has pointed to brain tumor stem cells (BTSC) as culprits behind human high-grade glioma (hHGG) resistance to standard therapy. Pre-clinical rodent models are the mainstay for testing of new therapeutic strategies. The typical model involves the intracranial injection of human glioma cells into immunocompromised hosts, hindering the evaluation of tumor-host responses and resulting in non-infiltrative tumors. The CT-2A model is an immunocompetent mouse model with potential to overcome these disadvantages. In this study, we confirmed the highly infiltrative nature of intracranial CT-2A tumors and optimized reproducible injection parameters. We then generated neurospheres and established, for the first time, the stemness of this model. CT-2A expression of the BTSC marker, CD133, increased from 2% in monolayer cells to 31% in fully-formed neurospheres. Investigation of three stem cell markers (Oct4, Nanog and Nestin) revealed a distinct stemness signature with monolayer cells expressing Oct4 and Nestin (no Nanog), and neurospheres expressing all three. Additionally, CT-2A cells were more proliferative and invasive than U87 cells, while CT-2A neurospheres were significantly more proliferative and invasive than either monolayer cells in vitro. Taken together, our results show that this model is a valuable tool for pre-clinical testing of novel therapeutics against hHGG and also affords the opportunity for investigation of BTSC in an immunocompetent setting.

In Vitro Growth Environment Produces Lipidomic and Electron Transport Chain Abnormalities in Mitochondria from Non-Tumorigenic Astrocytes and Brain Tumours

The mitochondrial lipidome influences ETC (electron transport chain) and cellular bioenergetic efficiency. Brain tumours are largely dependent on glycolysis for energy due to defects in mitochondria and oxidative phosphorylation. In the present study, we used shotgun lipidomics to compare the lipidome in highly purified mitochondria isolated from normal brain, from brain tumour tissue, from cultured tumour cells and from non-tumorigenic astrocytes. The tumours included the CT-2A astrocytoma and an EPEN (ependymoblastoma), both syngeneic with the C57BL/6J (B6) mouse strain. The mitochondrial lipidome in cultured CT-2A and EPEN tumour cells were compared with those in cultured astrocytes and in solid tumours grown in vivo. Major differences were found between normal tissue and tumour tissue and between in vivo and in vitro growth environments for the content or composition of ethanolamine glycerophospholipids, phosphatidylglycerol and cardiolipin. The mitochondrial lipid abnormalities in solid tumours and in cultured cells were associated with reductions in multiple ETC activities, especially Complex I. The in vitro growth environment produced lipid and ETC abnormalities in cultured non-tumorigenic astrocytes that were similar to those associated with tumorigenicity. It appears that the culture environment obscures the boundaries of the Crabtree and the Warburg effects. These results indicate that in vitro growth environments can produce abnormalities in mitochondrial lipids and ETC activities, thus contributing to a dependency on glycolysis for ATP production.

Up-regulation of NG2 proteoglycan and interferon-induced transmembrane proteins 1 and 3 in mouse astrocytoma: A membrane proteomics approach

Although brain tumors are classified as if their lineage were well understood, the relationship between the molecular events that specify neural cell lineage and brain tumors remains enigmatic. Traditionally, cell surface membrane antigens have served as biomarkers that distinguish brain tumor origin and malignancy. In this study, membrane proteins were identified from a terminally differentiated mouse astrocyte (AC) and CT-2A astrocytoma (CT-2A) cell line using liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS). A total of 321 and 297 protein groups with at least one unique peptide were identified in the AC and CT-2A cells. Using a label-free quantitative MS approach, 25 plasma membrane proteins in CT-2A were found significantly up- or down-regulated compared with those in AC. Three of the up-regulated proteins, chondroitin sulfate proteoglycan-4 (Cspg4), interferon-induced transmembrane protein-2 (IFITM2) and -3 (IFITM3) were further validated by semi-quantitative RT-PCR analysis. In addition, a third member of the IFITM family, interferon-induced transmembrane protein-1 (IFITM1) was also analyzed. Expression of Cspg4, IFITM1 and IFITM3 was significantly greater in the CT-2A cells than that in the AC cells. Interestingly, Cspg4, also known as neuronal/glial 2 (NG2) proteoglycan in human, is an oligodendrocyte progenitor marker. Therefore, our data suggest that the CT-2A tumor may be derived from NG2 glia rather than from fully differentiated astrocytes. Moreover, the CT-2A cells also express a series of interferon-induced signature proteins that may be specific to this tumor. These data highlight the utility of LC-MS/MS for the identification of brain tumor membrane biomarkers.

Differential effects of energy stress on AMPK phosphorylation and apoptosis in experimental brain tumor and normal brain

BackgroundAMP-activated protein kinase (AMPK) is a known physiological cellular energy sensor and becomes phosphorylated at Thr-172 in response to changes in cellular ATP levels. Activated AMPK acts as either an inducer or suppressor of apoptosis depending on the severity of energy stress and the presence or absence of certain functional tumor suppressor genes.ResultsHere we show that energy stress differentially affects AMPK phosphorylation and cell-death in brain tumor tissue and in tissue from contra-lateral normal brain. We compared TSC2 deficient CT-2A mouse astrocytoma cells with syngeneic normal astrocytes that were grown under identical condition in vitro. Energy stress induced by glucose withdrawal or addition of 2-deoxyglucose caused more ATP depletion, AMPK phosphorylation and apoptosis in CT-2A cells than in the normal astrocytes. Under normal energy conditions pharmacological stimulation of AMPK caused apoptosis in CT-2A cells but not in astrocytes. TSC2 siRNA treated astrocytes are hypersensitive to apoptosis induced by energy stress compared to control cells. AMPK phosphorylation and apoptosis were also greater in the CT-2A tumor tissue than in the normal brain tissue following implementation of dietary energy restriction. Inefficient mTOR and TSC2 signaling, downstream of AMPK, is responsible for CT-2A cell-death, while functional LKB1 may protect normal brain cells under energy stress.ConclusionTogether these data demonstrates that AMPK phosphorylation induces apoptosis in mouse astrocytoma but may protect normal brain cells from apoptosis under similar energy stress condition. Therefore, using activator of AMPK along with glycolysis inhibitor could be a potential therapeutic approach for TSC2 deficient human malignant astrocytoma.

Standardization of an orthotopic mouse brain tumor model following transplantation of CT-2A astrocytoma cells.

Animal models of glial-derived neoplasms are needed to study the biological mechanisms of glioma tumorigenesis and those that sustain the disease state. With the aim to develop and characterize a suitable in vivo experimental mouse model for infiltrating astrocytoma, with predictable and reproducible growth patterns that recapitulate human astrocytoma, this study was undertaken to analyze the long-term course of a syngeneic orthotopically implanted CT-2A mouse astrocytoma in C57BL/6J mice. Intracranial injection of CT-2A cells into caudate-putamen resulted in development of an aggressive tumor showing typical features of human glioblastoma multiforme, sharing close histological, immunohistochemical, proliferative, and metabolic profiles. To simulate metastatic disease to the brain, CT-2A cells were injected through the internal carotid artery. Tumors identical to those obtained by intracranial injection were obtained. Finally, CT-2A cells were re-isolated from experimental brain tumors and transcranially re-injected into the caudate-putamen of healthy mice. These cells generated new tumors that were indistinguishable from the initial ones, suggesting in vivo self-renewal of tumor cells. Small-animal models are essential for testing novel biological therapies directed against relevant molecular targets. In a preliminary study, experimental CT-2A tumors were chronically treated with the small molecule 77427, a gastrin-releasing peptide (GRP) blocker compound that inhibits angiogenesis. Treated animals developed significantly smaller tumors than controls, suggesting an antitumor action for 77427 in glioblastomas. We conclude that the orthotopic CT-2A tumor model, as described herein, is appropriate to explore the mechanisms of glioma development and for preclinical trials of promising drugs.

Gene‐linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma

Brain tumor growth and progression is dependent upon vascularity, and is associated with altered ganglioside composition and distribution. In this study, we examined the influence of gangliosides on growth and vascularity in a malignant mouse astrocytoma, CT-2A. Ganglioside distribution was altered in CT-2A tumor cells using an antisense construct to beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T), a key enzyme that uses the simple ganglioside GM3 as a substrate for the synthesis of the more complex gangliosides, GM2, GM1 and GD1a. GalNAc-T gene expression was significantly lower in CT-2A cells stably transfected with the antisense GalNAc-T plasmid, pcDNA3.1/TNG (CT-2A/TNG) than in either non-transfected CT-2A or mock-transfected (CT-2A/V) control tumor cells. GM3 was elevated from 16% to 58% of the total ganglioside distribution, whereas GM1 and GD1a were reduced from 17% and 49% to 10% and 17%, respectively, in CT-2A/TNG tumor cells. Growth, vascularity (blood vessel density and Matrigel assay) and vascular endothelial growth factor (VEGF) expression was significantly less in CT-2A/TNG tumors than in control CT-2A brain tumors. In addition, the expression of VEGF, hypoxia-inducible factor 1alpha (HIF-1alpha) and neuropilin-1 (NP-1) was significantly lower in CT-2A/TNG tumor cells than in control CT-2A tumor cells. These data suggest that gene-linked changes in ganglioside composition influence the growth and angiogenic properties of the CT-2A astrocytoma.

N -butyldeoxynojirimycin reduces growth and ganglioside content of experimental mouse brain tumours.

Abnormalities in glycosphingolipid (GSL) biosynthesis have been implicated in the oncogenesis and malignancy of brain tumours. GSLs comprise the gangliosides and the neutral GSLs and are major components of the cell surface glycocalyx. N -butyldeoxynojirimycin (N B-DNJ) is an imino sugar that inhibits the glucosyltransferase catalysing the first step in GSL biosynthesis. The influence of N B-DNJ was studied on the growth and ganglioside composition of two 20-methylcholanthrene-induced experimental mouse brain tumours, EPEN and CT-2A, which were grown in vitro and in vivo. N B-DNJ (200 μM) inhibited the proliferation of the EPEN and CT-2A cells by 50%, but did not reduce cell viability. The drug, administered in the diet (2400 mg kg−1) to adult syngeneic C57BL/6 mice, reduced the growth and ganglioside content of subcutaneous and intracerebral EPEN and CT-2A tumours by at least 50% compared to the untreated controls. N B-DNJ treatment also shifted the relative distribution of tumour gangliosides in accordance with the depletion of metabolic substrates. Side effects of N B-DNJ treatment were generally mild and included reductions in body and spleen weights and intestinal distension. We conclude that N B-DNJ may inhibit tumour growth through an effect on ganglioside biosynthesis and may be useful as a new chemotherapy for brain tumours. © 2001 Cancer Research Campaign http://www.bjcancer.com

Influence of ganglioside GM3 and high density lipoprotein on the cohesion of mouse brain tumor cells.

Previous findings with various murine tumor cell lines suggest an association between ganglioside GM3 and cell cohesive properties. The influence of GM3 on cohesion was studied in two mouse brain tumor cell lines: ependymoblastoma (EPEN) and CT-2A. In culture, the EPEN cells grow as islands and contain GM3 as the only ganglioside, whereas the CT-2A cells grow as a fusiform cell monolayer and contain GM2, GM1, and GD1a as major gangliosides and low amounts of GM3. To examine the role of GM3 in cohesion, both cell lines were treated with 1) C. perfringens neuraminidase, 2) anti-GM3 monoclonal antibody (mAb DH2), or 3) were grown in serum-free medium. All three treatments caused a significant increase in the number of non-cohesive and protoplasmic process-bearing cells for the EPEN, but had no effect on the morphology of the CT-2A cells. The neuraminidase treatment removed GM3 from both cell lines and caused a significant accumulation of GM1 in the CT-2A cells. EPEN cell cohesion and GM3 content returned to control levels after removal of neuraminidase. EPEN cell cohesion was restored in serum-free medium with added high density lipoprotein (HDL). The HDL effect on the EPEN cell cohesion was dose-dependent and was not seen with other lipoproteins. We suggest that EPEN cell cohesion could involve an interaction between extracellular HDL, acting as a bridge, and GM3 molecules on opposing cell surfaces.