Intracranial Glioma Model Research Articles

Abstract 4436: The down-regulation of ferritin heavy chain as an adjuvant therapy in human glioma.

Abstract Increased iron requirements and heightened iron metabolism is among the key characteristics of highly proliferative cancer cells. As the major iron storage protein, ferritin expression is elevated in cancer cells and we have previously shown that ferritin down-regulation in human glioblastoma cells increases their sensitivity to chemotherapeutic agent both in vitro and in vivo. In this study, we report that the sensitization of H-ferritin down-regulation in glioma cells against radiation and suggest the potential of H-ferritin down-regulation as an adjuvant therapy in human glioma. Down-regulation of H-ferritin was performed in human glioma cell line U251 through a nanotechnology-based transfection platform and effective knockdown efficiency was achieved. A transient decrease in transferrin receptor level was also observed, suggesting a release of intracellular iron with loss of ferritin. This iron release in turn produced intracellular oxidative stress, demonstrated by protein oxidation damage, as well as a decrease in the stability of hypoxia-inducible factors (HIFs), which is an indicator of radioresistance. In U251 cells, exposure to radiation resulted in protein oxidation which could be exacerbated by H-ferritin down-regulation. Previously we have demonstrated a DNA protection role for H-ferritin. Here we report that the down-regulation of H-ferritin is associated with increased DNA vulnerability when cells were exposed to ionizing radiation. Moreover, the activation of DNA repair mechanisms was impaired when H-ferritin was absent. The in vitro data suggest a synergistic effect of radiation and H-ferritin down-regulation. Additionally, we expand our research into CD133-positive glioma stem cells (GSCs), which are notoriously resistant to anti-cancer treatment. Down-regulation of H-ferritin in these cells inhibited cell proliferation in vitro. With an intracranial glioma model established by GSC implantation, we demonstrated that the survival was significantly prolonged by H-ferritin siRNA transfection through intravenous injection, in tumor bearing mice treated with first-line drug Temodar. This study supports the potential of H-ferritin siRNA as an adjuvant therapy in glioma treatment. Citation Format: Min Pang, A.B. Madhankumar, Becky Webb, Xiaoli Liu, James R. Connor. The down-regulation of ferritin heavy chain as an adjuvant therapy in human glioma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4436. doi:10.1158/1538-7445.AM2013-4436

Carbenoxolone Enhances TRAIL-Induced Apoptosis Through the Upregulation of Death Receptor 5 and Inhibition of Gap Junction Intercellular Communication in Human Glioma

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been used extensively in cancer therapy. However, more than half of glioblastoma multiforme are insensitive to the apoptotic effect of TRAIL. Improvement in therapeutic modalities that enhances the efficacy of TRAIL in glioma is much sought after. In this study, we combined the tumor selectivity of TRAIL and tumor-homing properties of mesenchymal stem cells (MSC) with gap junction (GJ) inhibitory effect of carbenoxolone (CBX) to target orthotopic glioma. MSC were engineered to express TRAIL (MSC-TRAIL) by incorporating the secretable trimeric form of TRAIL into a Herpes Simplex Virus (HSV) type I amplicon vector. Our results showed that combined treatment of MSC-TRAIL and CBX enhanced glioma cell death, especially in three primary human glioma isolates, of which two of those are marginally sensitive to TRAIL. CBX enhanced TRAIL-induced apoptosis through upregulation of death receptor 5, blockade of GJ intercellular communication, and downregulation of connexin 43. Dual arm therapy using TRAIL and CBX prolonged the survival of treated mice by ~27% when compared with the controls in an intracranial glioma model. The enhanced efficacy of TRAIL in combination with CBX coupled with the minimal cytotoxic nature of CBX suggested a favorable clinical usage of this treatment regimen.

The prospective application of a hypoxic radiosensitizer, doranidazole to rat intracranial glioblastoma with blood brain barrier disruption

BackgroundGlioblastoma is one of the intractable cancers and is highly resistant to ionizing radiation. This radioresistance is partly due to the presence of a hypoxic region which is widely found in advanced malignant gliomas. In the present study, we evaluated the effectiveness of the hypoxic cell sensitizer doranidazole (PR-350) using the C6 rat glioblastoma model, focusing on the status of blood brain barrier (BBB).MethodsReproductive cell death in the rat C6 glioma cell line was determined by means of clonogenic assay. An intracranial C6 glioma model was established for the in vivo experiments. To investigate the status of the BBB in C6 glioma bearing brain, we performed the Evans blue extravasation test. Autoradiography with [14C]-doranidazole was performed to examine the distribution of doranidazole in the glioma tumor. T2-weighted MRI was employed to examine the effects of X-irradiation and/or doranidazole on tumor growth.ResultsDoranidazole significantly enhanced radiation-induced reproductive cell death in vitro under hypoxia, but not under normoxia. The BBB in C6-bearing brain was completely disrupted and [14C]-doranidazole specifically penetrated the tumor regions. Combined treatment with X-irradiation and doranidazole significantly inhibited the growth of C6 gliomas.ConclusionsOur results revealed that BBB disruption in glioma enables BBB-impermeable radiosensitizers to penetrate and distribute in the target region. This study is the first to propose that in malignant glioma the administration of hydrophilic hypoxic radiosensitizers could be a potent strategy for improving the clinical outcome of radiotherapy without side effects.

Inhibition of glioblastoma and enhancement of survival via the use of mibefradil in conjunction with radiosurgery

The survival of patients with high-grade gliomas remains unfavorable. Mibefradil, a T-type calcium channel inhibitor capable of synchronizing dividing cells at the G1 phase, has demonstrated potential benefit in conjunction with chemotherapeutic agents for gliomas in in vitro studies. In vivo study of mibefradil and radiosurgery is lacking. The authors used an intracranial C6 glioma model in rats to study tumor response to mibefradil and radiosurgery. Two weeks after implantation of C6 cells into the animals, each rat underwent MRI every 2 weeks thereafter for 8 weeks. After tumor was confirmed on MRI, the rats were randomly assigned to one of the experimental groups. Tumor volumes were measured on MR images. Experimental Group 1 received 30 mg/kg of mibefradil intraperitoneally 3 times a day for 1 week starting on postoperative day (POD) 15; Group 2 received 8 Gy of cranial radiation via radiosurgery delivered on POD 15; Group 3 underwent radiosurgery on POD 15, followed by 1 week of mibefradil; and Group 4 received mibefradil on POD 15 for 1 week, followed by radiosurgery sometime from POD 15 to POD 22. Twenty-seven glioma-bearing rats were analyzed. Survival was compared between groups using Kaplan-Meier methodology. Median survival in Groups 1, 2, 3, and 4 was 35, 31, 43, and 52 days, respectively (p = 0.036, log-rank test). Two animals in Group 4 survived to POD 60, which is twice the expected survival of untreated animals in this model. Analysis of variance and a post hoc test indicated no tumor volume differences on PODs 15 and 29. However, significant volume differences were found on POD 43; mean tumor volumes for Groups 1, 2, 3, and 4 were 250, 266, 167, and 34 mm(3), respectively (p = 0.046, ANOVA). A Cox proportional hazards regression test showed survival was associated with tumor volume on POD 29 (p = 0.001) rather than on POD 15 (p = 0.162). In vitro assays demonstrated an appreciable and dose-dependent increase in apoptosis between 2- and 7-μM concentrations of mibefradil. Mibefradil response is schedule dependent and enhances survival and reduces glioblastoma when combined with ionizing radiation.

Fasting Enhances the Response of Glioma to Chemo- and Radiotherapy

BackgroundGlioma, including anaplastic astrocytoma and glioblastoma multiforme (GBM) are among the most commonly diagnosed malignant adult brain tumors. GBM is a highly invasive and angiogenic tumor, resulting in a 12 to 15 months median survival. The treatment of GBM is multimodal and includes surgical resection, followed by adjuvant radio-and chemotherapy. We have previously reported that short-term starvation (STS) enhances the therapeutic index of chemo-treatments by differentially protecting normal cells against and/or sensitizing tumor cells to chemotoxicity.Methodology and Principal FindingsTo test the effect of starvation on glioma cells in vitro, we treated primary mouse glia, murine GL26, rat C6 and human U251, LN229 and A172 glioma cells with Temozolomide in ad lib and STS mimicking conditions. In vivo, mice with subcutaneous or intracranial models of GL26 glioma were starved for 48 hours prior to radio- or chemotherapy and the effects on tumor progression and survival were measured. Starvation-mimicking conditions sensitized murine, rat and human glioma cells, but not primary mixed glia, to chemotherapy. In vivo, starvation for 48 hours, which causes a significant reduction in blood glucose and circulating insulin-like growth factor 1 (IGF-1) levels, sensitized both subcutaneous and intracranial glioma models to radio-and chemotherapy.ConclusionStarvation-induced cancer sensitization to radio- or chemotherapy leads to extended survival in the in vivo glioma models tested. These results indicate that fasting and fasting-mimicking interventions could enhance the efficacy of existing cancer treatments against aggressive glioma in patients.

Imaging Biomarker Dynamics in an Intracranial Murine Glioma Study of Radiation and Antiangiogenic Therapy

There is a growing need for noninvasive biomarkers to guide individualized spatiotemporal delivery of radiation therapy (RT) and antiangiogenic (AA) therapy for brain tumors. This study explored early biomarkers of response to RT and the AA agent sunitinib (SU), in a murine intracranial glioma model, using serial magnetic resonance imaging (MRI). Mice with MRI-visible tumors were stratified by tumor size into 4 therapy arms: control, RT, SU, and SU plus RT (SURT). Single-fraction conformal RT was delivered using MRI and on-line cone beam computed tomography (CT) guidance. Serial MR images (T2-weighted, diffusion, dynamic contrast-enhanced and gadolinium-enhanced T1-weighted scans) were acquired biweekly to evaluate tumor volume, apparent diffusion coefficient (ADC), and tumor perfusion and permeability responses (K(trans), K(ep)). Mice in all treatment arms survived longer than those in control, with a median survival of 35 days for SURT (P<.0001) and 30 days for RT (P=.009) and SU (P=.01) mice vs 26 days for control mice. At Day 3, ADC rise was greater with RT than without (P=.002). Sunitinib treatment reduced tumor perfusion/permeability values with mean K(trans) reduction of 27.6% for SU (P=.04) and 26.3% for SURT (P=.04) mice and mean K(ep) reduction of 38.1% for SU (P=.01) and 27.3% for SURT (P=.02) mice. The magnitude of individual mouse ADC responses at Days 3 and 7 correlated with subsequent tumor growth rate R values of -0.878 (P=.002) and -0.80 (P=.01), respectively. Early quantitative changes in diffusion and perfusion MRI measures reflect treatment responses soon after starting therapy and thereby raise the potential for these imaging biomarkers to guide adaptive and potentially individualized therapy approaches in the future.

Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood-brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin-Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.

The impact of bevacizumab on temozolomide concentrations in intracranial U87 gliomas

An important question in the sequencing of anti-cancer therapies in patients with glioblastoma (GBM) is whether concurrent anti-angiogenesis therapies improve or impair brain concentrations of concomitantly administered cytotoxic therapies. The purpose of this study is to assess the intratumoral disposition of temozolomide (TMZ) via microdialysis before and after bevacizumab in an intracranial GBM xenograft model. Microdialysis probes were placed within tumor and contralateral brain in athymic rats bearing U87 intracerebral gliomas. TMZ (50 mg/kg oral) was administered 10 days thereafter. Extracellular fluid (ECF) was collected for 6 h. BEV was administered (10 mg/kg IV), and TMZ was re-dosed (50 mg/kg oral) 36 h thereafter with additional ECF collection. All ECF samples were assessed for TMZ concentration with liquid chromatography-tandem mass spectrometry. Tumor TMZ mean area under the concentration-time curve (AUC(0-∞)) was 3.35 μg h/mL pre-BEV. Post-BEV, tumor mean TMZ AUC(0-∞) was 3.98 μg h/mL. In non-tumor brain, mean TMZ AUC(0-∞) pre-BEV was 3.22 μg h/mL and post-BEV was 3.34 μg h/mL. There were no statistically significant changes in TMZ pharmacokinetics before or after BEV in the athymic rat U87 intracranial glioma model. BEV and TMZ are being investigated as a combination therapy in several ongoing studies for patients with glioma. These data reassuringly suggest that BEV does not significantly change the ECF tumor concentrations of TMZ in either tumor-bearing or normal brain when dosed 36 h prior to TMZ.

Abstract 1905: Autophagy inhibitor quinacrine synergistically enhances anti-angiogenic efficacy of Cediranib in intracranial mouse glioma

Abstract Despite robust vascularity of malignant gliomas, anti-angiogenic therapy (AAT), largely fails to induce durable responses. We hypothesized that AAT efficacy in treating glioma could be increased by disrupting adaptive mechanisms that enable tumors to survive AAT alone. Hypoxic/nutrient stress conditions, such as those induced by AAT, can activate autophagy in tumor cells, a degradative catabolic pathway that promotes cellular survival during metabolic stress. The anti-malarial agent quinacrine induces late-stage autophagic inhibition, which can induce cell death. We determined whether the angiogenic/anti-tumor efficacy of Cediranib, a VEGF/PDGF receptor tyrosine kinase inhibitor, could be synergistically enhanced through combined administration of quinacrine. A noninvasive, dual bolus perfusion MRI approach was used to assess tumor growth and vascular parameters in the syngeneic 4C8 mouse model of intracranial glioma. Dynamic contrast enhanced (DCE) MRI, provided high resolution maps of Ktrans, an index of vascular permeability. At the same time, dynamic susceptibility contrast (DSC) MRI determined cerebral blood flow (CBF). Once tumor growth was documented by MRI, mice were randomized to untreated (U), Cediranib (C, 6 mg/kg daily), quinacrine (Q, 50mg/kg daily), or Cediranib plus quinacrine (C+Q) groups. Tumor growth rate (days−1, mean±SE) was moderately decreased for C (0.17±0.01) in comparison to U (0.22±0.01) and Q (0.21±0.01) (p&amp;lt;0.05). Tumor growth rate was reduced substantially, with C+Q (0.11±0.004) (p&amp;lt;0.01 versus other groups). Consistent with this, survival (days from tumor growth initiation) was greatly increased for C+Q, 25.3±1.8, versus other groups (p&amp;lt;0.01): 11.0 ± 1.6 (U), 11.5 ± 1.2 (Q), and, 14.3 ± 0.7 (C). Perfusion MRI indicated that mean tumor Ktrans for C (0.13±0.01 min−1) was moderately reduced in comparison to U (0.21±0.04) and Q (0.20±0.2), during the 2nd treatment week (P&amp;lt;0.05). Dramatically reduced Ktrans was observed with C+Q (0.07±0.01; p&amp;lt;0.05 vs U, C, and Q). Consistent with this, mean tumor CBF relative to contralateral brain CBF during the 2nd treatment week, was dramatically lower for C+Q, 1.61±0.06 (p&amp;lt;0.05) compared to other groups: 3.06 ± 0.15 (U), 2.55±0.24 (Q), 3.07±0.47 (C). In vitro MTS cell viability assays of 4C8 glioma cells indicated markedly increased efficacy for combined C+Q under hypoxic conditions: 1μM C/2.5μM Q decreased cell viability by 76±6% and 33±1% with 0.5 and 21% O2, respectively. Combination indices (CI) indicated less than additive effects for C and Q with normal O2, while synergism (CI&amp;lt;1) was observed under hypoxic conditions (CI=0.58, 0.75μM C/1μM Q). In conclusion, the autophagy inhibitor quinacrine synergistically increases the anti-angiogenic/anti-tumor effect of Cediranib in 4C8 mouse glioma. Tumor microenvironment conditions such as hypoxia may play a role in the synergistic interaction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1905. doi:1538-7445.AM2012-1905

Antitumor Activity of NVP-BKM120—A Selective Pan Class I PI3 Kinase Inhibitor Showed Differential Forms of Cell Death Based on p53 Status of Glioma Cells

The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo. The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model. We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell line's sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts). Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment.

Specific Inhibition of SRC Kinase Impairs Malignant Glioma Growth In Vitro and In Vivo

Malignant glioma is a severe cancer with a poor prognosis. Local occurrence and rare metastases of malignant glioma make it a suitable target for gene therapy. Several studies have demonstrated the importance of Src kinase in different cancers. However, these studies have focused mainly on Src-deficient mice or pharmacological inhibitors of Src. In this study we have used Src small hairpin RNAs (shRNAs) in a lentiviral backbone to mimic a long-term stable treatment and determined the role of Src in tumor tissues. Efficacy of Src shRNAs was confirmed in vitro demonstrating up to 90% target gene inhibition. In a mouse malignant glioma model, Src shRNA tumors were almost 50-fold smaller in comparison to control tumors and had significantly reduced vascularity. In a syngenic rat intracranial glioma model, Src shRNA-transduced tumors were smaller and these rats had a survival benefit over the control rats. In vivo treatment was enhanced by chemotherapy and histone deacetylase inhibition. Our results emphasise the importance of Src in tumorigenesis and demonstrate that it can be efficiently inhibited in vitro and in vivo in two independent malignant glioma models. In conclusion, Src is a potential target for RNA interference-mediated treatment of malignant glioma.

Bevacizumab With Angiostatin-armed oHSV Increases Antiangiogenesis and Decreases Bevacizumab-induced Invasion in U87 Glioma

Bevacizumab (BEV) is an antiangiogenic drug approved for glioblastoma (GBM) treatment. However, it does not increase survival and is associated with glioma invasion. Angiostatin is an antiangiogenic polypeptide that also inhibits migration of cancer cells, but is difficult to deliver. Oncolytic viruses (OV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them and deliver anticancer agents to uninfected cells. We have tested a combination treatment of BEV plus an OV expressing angiostatin (G47Δ-mAngio) in mice-bearing human GBM. Using a vascular intracranial human glioma model (U87) in athymic mice, we performed histopathological analysis of tumors treated with G47Δ-mAngio or BEV alone or in combination, followed tumor response by magnetic resonance imaging (MRI), and assessed animal survival. Our results indicate that injection of G47Δ-mAngio during BEV treatment allows increased virus spread, tumor lysis, and angiostatin-mediated inhibition of vascular endothelial growth factor (VEGF) expression and of BEV-induced invasion markers (matrix metalloproteinases-2 (MMP2), MMP9, and collagen). This leads to increased survival and antiangiogenesis and decreased invasive phenotypes. We show for the first time the possibility of improving the antiangiogenic effect of BEV while decreasing the tumor invasive-like phenotype induced by this drug, and demonstrate the therapeutic advantage of combining systemic and local antiangiogenic treatments with viral oncolytic therapy.

Abstract 2568: Combination treatment of bevacizumab and oncolytic HSV armed with angiostatin show enhanced antitumoral and antiangiogenic effects

Abstract Background: Bevacizumab (Avastin), a monoclonal antibody that inactivates VEGF, was approved by US Food and Drug Administration as a single agent for treatment of recurrent glioblastomas (GBM). It prolongs progression-free survival and controls peritumoral edema. However, its effect on prognosis and overall survival remains to be determined. Angiostatin is a naturally occurring inhibitor of angiogenesis that was shown to inhibit growth of primary and metastatic tumors, but can not be delivered efficiently to GBM. Oncolytic viruses (OV) are promising agents for the treatment of GBM, whose therapeutic potential is not fully exploited due to infiltration of anti-viral macrophages. Treatment with anti-angiogenic factors was shown to increase replication and therapeutic capacity of OV. We hypothesized that combination of Avastin with an HSV OV (G47Δ) coding for angiostatin (G47Δ-angio) would enhance GBM treatment due to increased viral spread and delivery of angiostatin. Materials &amp; Methods: G47Δ-angio was constructed using the “flip-flop” HSV-BAC system. Expression of angiostatin was confirmed by western blot; in vitro matrigel endothelial tube formation assay was performed to test its anti-angiogenic potential. We confirmed the capacity of this virus to infect cancer cells and inhibit tumor angiogenesis in vivo, and determined therapeutic efficacy using both a subcutaneous and an intracerebral glioma model in mice. A control virus not carrying the angiostatin gene (G47Δ-empty) served as the negative control for all experiments. Avastin (5mg/kg) was delivered by i.v injection in intracerebral glioma model, started at Day 5 and followed by twice a week for consecutive 4 weeks. Tumor angiogenesis was determined by CD31 and VEGF staining, macrophages infiltration was detected by F4/80 staining. The overall survival of intracerebral glioma mice was followed up in G47Δ-angio, Avastin and combination (G47Δ-angio + Avastin) treatments. Results: G47Δ-angio decreased endothelial tube formation in vitro and demonstrated potent antiangiogenic effects in vivo. In our subcutaneous tumor model, G47Δ inhibited tumor growth (n=7/group, P&amp;lt;0.05) and in the intracranial glioma model, G47Δ extended survival (n=10/group, P&amp;lt;0.05). Furthermore, the combination treatment (G47Δ-angio + Avastin) had the most significant antitumor and antiangiogenesis efficacy compared with either treatment alone (n=10/group, P&amp;lt;0.05). Conclusion: Our results demonstrate that G47Δ-angio could significantly enhance antitumor and antiangiogenic efficacy. Moreover, the combination treatment could further improve the antitumor and antiangiogenic efficacy in intracerebral glioma model in vivo. Hence, “Arming” HSV with an antiangiogenic gene that targets both the tumor cells and tumor vasculature is a promising therapy for use with Avastin treatment in clinical translation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2568. doi:10.1158/1538-7445.AM2011-2568

Efficacy of adenovirally expressed soluble TRAIL in human glioma organotypic slice culture and glioma xenografts

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in malignant cells, including gliomas, and is currently in anticancer clinical trials. However, the full-length and tagged forms of TRAIL, unlike the untagged ligand (soluble TRAIL (sTRAIL)), exhibits toxicity against normal cells. Here, we report the generation and testing of an adenovirus (AdsTRAIL) that expresses untagged sTRAIL in an intracranial xenograft model and a human glioma organotypic slice culture model. AdsTRAIL efficiently induced apoptosis in glioma cell lines, including those resistant to sTRAIL, but not in normal human astrocytes (NHAs). It inhibited anchorage-independent glioma growth and exerted a bystander effect in transwell assays. Intratumoral injections of AdsTRAIL in a rodent intracranial glioma model resulted in reduced tumor growth and improved survival compared with Ad-enhanced green fluorescent protein (EGFP)- or vehicle-treated controls without toxicity. Human glioma organotypic slices treated with AdsTRAIL demonstrated apoptosis induction and caspase activation.

An Extensive Invasive Intracranial Human Glioblastoma Xenograft Model: Role of High Level Matrix Metalloproteinase 9

The lack of an intracranial human glioma model that recapitulates the extensive invasive and hypervascular features of glioblastoma (GBM) is a major hurdle for testing novel therapeutic approaches against GBM and studying the mechanism of GBM invasive growth. We characterized a high matrix metalloproteinase-9 (MMP-9) expressing U1242 MG intracranial xenograft mouse model that exhibited extensive individual cells and cell clusters in a perivascular and subpial cellular infiltrative pattern, geographic necrosis and infiltrating tumor-induced vascular proliferation closely resembling the human GBM phenotype. MMP-9 silencing cells with short hairpin RNA dramatically blocked the cellular infiltrative pattern, hypervascularity, and cell proliferation in vivo, and decreased cell invasion, colony formation, and cell motility in vitro, indicating that a high level of MMP-9 plays an essential role in extensive infiltration and hypervascularity in the xenograft model. Moreover, epidermal growth factor (EGF) failed to stimulate MMP-9 expression, cell invasion, and colony formation in MMP-9-silenced clones. An EGF receptor (EGFR) kinase inhibitor, a RasN17 dominant-negative construct, MEK and PI3K inhibitors significantly blocked EGF/EGFR-stimulated MMP-9, cell invasion, and colony formation in U1242 MG cells, suggesting that MMP-9 is involved in EGFR/Ras/MEK and PI3K/AKT signaling pathway-mediated cell invasion and anchorage-independent growth in U1242 MG cells. Our data indicate that the U1242 MG xenograft model is valuable for studying GBM extensive invasion and angiogenesis as well as testing anti-invasive and anti-angiogenic therapeutic approaches.

Effective conversion of irinotecan to SN-38 after intratumoral drug delivery to an intracranial murine glioma model in vivo

Irinotecan (CPT-11), a topoisomerase I inhibitor, is a cytotoxic agent with activity against malignant gliomas and other tumors. After systemic delivery, CPT-11 is converted to its active metabolite, SN-38, which displays significantly higher cytotoxic potency. However, the achievement of therapeutically effective plasma levels of CPT-11 and SN-38 is seriously complicated by variables that affect drug metabolism in the liver. Thus the capacity of CPT-11 to be converted to the active SN38 intratumorally in gliomas was addressed. For in vitro studies, 2 glioma cell lines, U87 and U251, were tested to determine the cytotoxic effects of CPT-11 and SN-38 in a dose-dependent manner. In vivo studies were performed by implanting U87 intracranially into athymic/nude mice. For a period of 2 weeks, SN-38, CPT-11, or vehicle was administered intratumorally by means of an osmotic minipump. One series of experiments measured the presence of SN-38 or CPT-11 in the tumor and surrounding brain tissues after 2 weeks' exposure to the drug. In a second series of experiments, after 2 weeks' exposure to the drug, the animals were maintained, in the absence of drug, until death. The survival curves were then calculated. The results show that the animals that had CPT-11 delivered intratumorally by the minipump expressed SN-38 in vivo. Furthermore, both CPT-11 and SN-38 accumulated at higher levels in tumor tissues compared with uninvolved brain. Intratumoral delivery of CPT-11 or SN-38 extended the average survival time of tumor-bearing animals from 22 days to 46 and 65 days, respectively. These results demonstrate that intratumorally administered CPT-11 can be effectively converted to SN-38 and this method of drug delivery is effective in extending the survival time of animals bearing malignant gliomas.

Abstract 5243: Value of 1H magnetic resonance spectroscopy compared with anatomical imaging in predicting response to standard chemotherapy and radiation in U87-MG-luc intracranial glioma model

Abstract Introduction: Luciferase enabled tumor cell lines are widely used for tracking orthotopic tumor burden in small animal models using bioluminescence imaging (BLI). Tumor burden estimated by MRI, while clinically relevant, may not correlate with patient survival. Tumor total (or phospho) choline has been shown to be an early indicator of tumor response to therapy measured using 1H (or 31P) magnetic resonance spectroscopy (MRS). In this study, correlation of response to therapy as measured by total choline (using 1H MRS) was compared with luciferase and MRI assessments of tumor burden, and lifespan. Materials and methods: U87-MG-luc human glioma cells (106 cells in 10 μl) were implanted using a stereotax frame intracranially (2mm right lateral and 1mm anterior to the coronal suture at a depth of 2mm) in female CD1 nu/nu mice. On Day 27 post implant, intracranial tumor burden was characterized using BLI and animals were randomized into 3 groups-Group 1(n=9): vehicle controls saline, QDx5, 0.2ml/20g; Group 2 (n=9): BCNU QDx5, IV, 15mg/kg; Group 3 (n=6): 15 Gy radiation; (QDx5;2off)x2wks). Pre-treatment and 1 week post treatment scans were acquired using BLI (tumor burden), T2 weighted MRI (tumor volume) and water suppressed proton MR spectroscopy (spectra fitted to obtain total choline/NAA ratio). Additional BLI scans were performed weekly and animal lifespan was also recorded. For each parameter, mean and median were analyzed. Additionally, to assess the predictive power of each parameter, the animals on study were distributed into responder or non-responder categories using median lifespan in the control group as the threshold for response determination. Mean and medians for each parameter were then calculated separately for responders and non-responders. Discussion: A Mann-Whitney rank sum test showed that the difference in median survival in both the treated groups was significantly different from controls, which had a median lifespan of 37.5 days. BCNU treatment (Group 2, p=0.021) was moderately active, resulting in a 5.5 day improvement in median lifespan. Radiation treatment (Group 3, p=0.003) was highly active, resulting in an 18.5 day improvement in median lifespan. Anatomical MRI determined tumor volumes and tumor luciferase signals at 1 week post treatment start were predictive of group median lifespan in Groups 2 and 3. Cho/NAA ratio at 1 week post-treatment start also correlated with response for Groups 2 and 3, but was less sensitive than the anatomically derived parameters. The secondary analysis based on response category revealed results consistent with the group mean and median analyses for all 3 parameters. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5243.

Applying stereotactic technique to establish C6 brain glioma models for laser interstitial thermotherapy research

Objective C6 brain glioma models were established with stereotactic technique to study laser interstitial thermotherapy (LITT) in SD rats C6 intracranial glioma models. Methods The C6 cells cultured in vitro were stereotaxically implanted into the right caudate nucleus of SD rat brain (20 μl free serum DMEM for one rat which concentration was 1×10~(11)/L). The following step was to judge MRI scan. Tumor was confirmed with staining of ⅧR, GFAP and S - 100 immunohistochemistry. After MRI scanning and correction of tumor location, the models were divided into groups according treating time and laser power from 2 to 10 W. Semiconductor laser optical fibers were inserted in tumors for LITT, simultaneously cortex's temperature conducted from center target was measured by ThermaCAM S65 type infrared thermograph, and (or) deep tissue's temperature around target was measured by thermocouple. Results Inoculated with optimized stereotactic technique, rat C6 gliomas resembled histopathological features of human glioma. This kind of model was a more reliant and reproducible one, with 96. 67% yield of intracranial tumor as well as no extracranial growth extension. The difference between cortex temperature conducted from center target and deep tissue temperature around target had no statistical significance (P>0.05). Conclusion A rat C6 brain glioma model resembles histopathological features of human glioma, as a perfect model to study LITT of glioma. Infrared thermograph technique to measure temperature conveniently, effectually, non invasive and the data could be treated by software in LITT research. Combining thermocouple to measure deep tissue temperature, it would have a better effect. Key words: Glioma; Stereotaxic techniques; Thermocoagulatian,laser; Rats

Delivery of temozolomide to the tumor bed via biodegradable gel matrices in a novel model of intracranial glioma with resection

We have completed in vivo safety and efficacy studies of the use of a novel drug delivery system, a gel matrix-temozolomide formulation that is injected intracranially into the post-resection cavity, as a candidate for glioma therapy. A rat intracranial resection model of C6-GFP intracranial glioma was used for safety and toxicity studies. Biodistribution studies were performed using gel matrix-gallocyanine formulations and were evaluated at various time intervals using real-time analysis of dye distribution. Additionally, the resection model was used to determine the efficacy of gel matrix-temozolomide as compared to blank gel matrix. A subcutaneous human xenograft glioma model was used to further assess the efficacy of gel matrix-temozolomide in reducing the overall tumor load. Gel matrix-temozolomide exhibited minimal cytotoxicity toward normal brain tissue while displaying high levels of oncolytic activity toward glioma cells. In the intracranial glioma resection and subcutaneous glioma model, administration of gel matrix-temozolomide directly to the tumor bed was well tolerated and effective at reducing the tumor load. A significant reduction of tumor load was observed (P < 0.0001) in the 30% temozolomide group (approximately 95%) as compared to blank control. There was little morbidity and no mortality associated with gel matrix treatment. Gel matrix-temozolomide appears to be safe and effective when used in vivo to treat intracranial glioma and warrants further development as a potential adjuvant therapy.

High‐resolution longitudinal assessment of flow and permeability in mouse glioma vasculature: Sequential small molecule and SPIO dynamic contrast agent MRI

The poor prognosis associated with malignant glioma is largely attributable to its invasiveness and robust angiogenesis. Angiogenesis involves host-tumor interaction and requires in vivo evaluation. Despite their versatility, few studies have used mouse glioma models with perfusion MRI approaches, and generally lack longitudinal study design. Using a micro-MRI system (8.5 Tesla), a novel dual bolus-tracking perfusion MRI strategy was implemented. Using the small molecule contrast agent Magnevist, dynamic contrast enhanced MRI was implemented in the intracranial 4C8 mouse glioma model to determine K(trans) and v(e), indices of tumor vascular permeability and cellularity, respectively. Dynamic susceptibility contrast MRI was subsequently implemented to assess both cerebral blood flow and volume, using the macromolecular superparamagnetic iron oxide, Feridex, which circumvented tumor bolus susceptibility curve distortions from first-pass extravasation. The high-resolution parametric maps obtained over 4 weeks, indicated a progression of tumor vascularization, permeability, and decreased cellularity with tumor growth. In conclusion, a comprehensive array of key parameters were reliably quantified in a longitudinal mouse glioma study. The syngeneic 4C8 intracerebral mouse tumor model has excellent characteristics for studies of glioma angiogenesis. This approach provides a useful platform for noninvasive and highly diagnostic longitudinal investigations of anti-angiogenesis strategies in a relevant orthotopic animal model.