Orthotopic Brain Tumor Model Research Articles

Metallofullerene-Nanoplatform-Delivered Interstitial Brachytherapy Improved Survival in a Murine Model of Glioblastoma Multiforme

Fullerenes are used across scientific disciplines because of their diverse properties gained by altering encapsulated or surface-bound components. In this study, the recently developed theranostic agent based on a radiolabeled functionalized metallofullerene ((177)Lu-DOTA-f-Gd(3)N@C(80)) was synthesized with high radiochemical yield and purity. The efficacy of this agent was demonstrated in two orthotopic xenograft brain tumor models of glioblastoma multiforme (GBM). A dose-dependent improvement in survival was also shown. The in vivo stability of the agent was verified through dual label measurements of biological elimination from the tumor. Overall, these results provide evidence that nanomaterial platforms can be used to deliver effective interstitial brachytherapy.

CT322, a VEGFR-2 antagonist, demonstrates anti-glioma efficacy in orthotopic brain tumor model as a single agent or in combination with temozolomide and radiation therapy

Glioblastomas are among the most aggressive human cancers, and prognosis remains poor despite presently available therapies. Angiogenesis is a hallmark of glioblastoma, and the resultant vascularity is associated with poor prognosis. The proteins that mediate angiogenesis, including vascular endothelial growth factor (VEGF) signaling proteins, have emerged as attractive targets for therapeutic development. Since VEGF receptor-2 (VEGFR-2) is thought to be the primary receptor mediating angiogenesis, direct inhibition of this receptor may produce an ideal therapeutic effect. In this context, we tested the therapeutic effect of CT322, a selective inhibitor of VEGFR-2. Using an intracranial murine xenograft model (U87-EGFRvIII-luciferase), we demonstrate that CT322 inhibited glioblastoma growth in vivo and prolonged survival. Of note, the anti-neoplastic effect of CT322 is augmented by the incorporation of temozolomide or temozolomide with radiation therapy. Immunohistochemical analysis of CT322 treated tumors revealed decreased CD31 staining, suggesting that the tumoricidal effect is mediated by inhibition of angiogenesis. These pre-clinical results provide the foundation to further understand long term response and tumor escape mechanisms to anti-angiogenic treatments on EGFR over-expressing glioblastomas.

Polo-Like Kinase 1 Inhibition Kills Glioblastoma Multiforme Brain Tumor Cells in Part Through Loss of SOX2 and Delays Tumor Progression in Mice

Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer and given these new therapies are urgently needed. To identify molecular targets, we queried a microarray profiling 467 human GBMs and discovered that polo-like kinase 1 (PLK1) was highly expressed in these tumors and that it clustered with the proliferative subtype. Patients with PLK1-high tumors were more likely to die from their disease suggesting that current therapies are inactive against such tumors. This prompted us to examine its expression in brain tumor initiating cells (BTICs) given their association with treatment failure. BTICs isolated from patients expressed 110-470 times more PLK1 than normal human astrocytes. Moreover, BTICs rely on PLK1 for survival because the PLK1 inhibitor BI2536 inhibited their growth in tumorsphere cultures. PLK1 inhibition suppressed growth, caused G(2) /M arrest, induced apoptosis, and reduced the expression of SOX2, a marker of neural stem cells, in SF188 cells. Consistent with SOX2 inhibition, the loss of PLK1 activity caused the cells to differentiate based on elevated levels of glial fibrillary acidic protein and changes in cellular morphology. We then knocked glial fibrillary acidic protein (GFAP) down SOX2 with siRNA and showed that it too inhibited cell growth and induced cell death. Likewise, in U251 cells, PLK1 inhibition suppressed cell growth, downregulated SOX2, and induced cell death. Furthermore, BI2536 delayed tumor growth of U251 cells in an orthotopic brain tumor model, demonstrating that the drug is active against GBM. In conclusion, PLK1 level is elevated in GBM and its inhibition restricts the growth of brain cancer cells.

Pharmacokinetic study of neural stem cell-based cell carrier for oncolytic virotherapy: targeted delivery of the therapeutic payload in an orthotopic brain tumor model

Oncolytic virotherapy is a promising novel therapy for glioblastoma that needs to be optimized before introduced to clinic. The targeting of conditionally replicating adenoviruses (CRAds) can be improved by relying on the tumor tropic properties of neural stem cells (NSCs). Here, we report the characterization of an FDA approved NSC, HB1.F3-CD, as a cell carrier for CRAd-S-pk7, a glioma-tropic oncolytic adenovirus. We show that NSCs replicate and release infectious CRAd-S-pk7 progeny capable of lysing glioma cell lines. Moreover, ex-vivo loaded NSCs, injected intracranially in nude mice bearing human glioma xenografts (i) retained their tumor-tropism, (ii) continued to replicate CRAd-S-pk7 for more than a week after reaching the tumor site and (iii) successfully handed-off CRAd-S-pk7 to glioma cells in vivo. Delivery via carrier cells reduced non-specific adenovirus distribution in the mouse brain. Moreover, we assessed biodistribution of loaded NSCs after intracranial injection in animal models semi-permissive to adenovirus replication, the Syrian hamster and cotton rat. NSCs did not migrate to distant organs and high levels of CRAd-S-pk7 DNA were observed only in the injected hemisphere. In conclusion, this optimized carrier system, with high efficiency of adenovirus delivery and minimal systemic toxicity, poses considerable advantages for anti-glioma oncolytic virotherapy.

Abstract 1024: Mural cell transdifferentiation of glioblastoma stem-like cells drives vasculogenic mimicry in glioblastoma development

Abstract Glioblastomas (GBM) are extremely aggressive and have the poorest prognosis among brain tumors. These tumors display extensive microvasculature that presumably originates from endothelial cell angiogenesis. Indeed, many anti-angiogenic therapies have been accepted for glioblastoma treatment based on this premise. Recent evidence has shown that glioblastoma stem-like cells (GSCs) can differentiate into endothelial cells that participate in angiogenesis (Ricci-Vitiani L et al., Nature 2010;468:824-29, Wang et al., Nature 2010;468:829-33, & Soda et al., PNAS 2011; 108:4274-80). However, it remains to be clarified if these GSC-derived endothelial cells make a significant contribution to tumor vasculature. Here, we surveyed eleven GBM cases and found that glioblastomas consisted of 34.1% of endothelial cell-associated vasculature, while 59.7% of microvasculature arose from mural-like tumor cells that co-express mural cell markers smooth muscle alpha actin and platelet-derived growth factor receptor, and VEGF receptor 2 (Flk-1), but not CD31, CD34 or VE-cadherin. This pattern of vascularization indicates an alternative microvascular circulation known as vasculogenic mimicry (VM). GSCs derived from patients with GBM displayed the multipotency of neuronal stem cells capable of transdifferentiation into mural-like tumor cells that promoted VM. GSCs developed vigorous mural cell-associated vascular channels but few endothelial cell vessels in orthotopic brain tumor models. Furthermore, the development of these tumor cell-associated channels was dependent on expression of VEGF receptor 2 (Flk-1). Suppression of Flk-1 activity and gene expression by a variety of different inhibitory approaches abrogated GSC transdifferentiation and vascularization in vitro, and inhibited VM in xenografted tumor models in vivo. Collectively, this study establishes a pivotal role for transdifferentiation of GSCs in the development of VM, which contributes to an aggressive phenotype of GBM, and points to Flk-1 as a potential target for therapeutic intervention. 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 1024. doi:1538-7445.AM2012-1024

REST Regulates Oncogenic Properties of Glioblastoma Stem Cells

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. Although many GBM tumors are believed to be caused by self-renewing, glioblastoma-derived stem-like cells (GSCs), the mechanisms that regulate self-renewal and other oncogenic properties of GSCs are only now being unraveled. Here we showed that GSCs derived from GBM patient specimens express varying levels of the transcriptional repressor repressor element 1 silencing transcription factor (REST), suggesting heterogeneity across different GSC lines. Loss- and gain-of-function experiments indicated that REST maintains self-renewal of GSCs. High REST-expressing GSCs (HR-GSCs) produced tumors histopathologically distinct from those generated by low REST-expressing GSCs (LR-GSCs) in orthotopic mouse brain tumor models. Knockdown of REST in HR-GSCs resulted in increased survival in GSC-transplanted mice and produced tumors with higher apoptotic and lower invasive properties. Conversely, forced expression of exogenous REST in LR-GSCs produced decreased survival in mice and produced tumors with lower apoptotic and higher invasive properties, similar to HR-GSCs. Thus, based on our results, we propose that a novel function of REST is to maintain self-renewal and other oncogenic properties of GSCs and that REST can play a major role in mediating tumorigenicity in GBM.

Multifunctional Theranostic Nanoparticles for Brain Tumors

Antiangiogenic approaches have been extensively exploited to provide a rationally designed therapy for the treatment of brain cancer. The brain tumor endothelium, with characteristics of high proliferation, high permeability, and high expression of proangiogenic factors (such as vascular endothelial growth factor, VEGF), is a particularly appealing therapeutic target for this strategy. Many antibody drugs, which primarily target the interaction between VEGF and its receptors, have been investigated in clinical trials but have shown only modest effects. Recent research published by Agemy et al.1 has alternatively harnessed a tumor-homing peptide (CGKRK) to specifically deliver multifunctional theranostic nanoparticles composed of iron oxide nanoworm (NW) and mitochondria-targeted cytotoxic peptide D[KLAKLAK]2 to glioblastoma multiforme (GBM) endothelium. Moreover, with the help of a tumor-penetrating peptide (iRGD), the NWs were capable of infiltrating the tumor tissue after extravasation for tumor cell eradication. This strategy was evaluated in lentivirus-induced, transplantable, and orthotopic brain tumor models. Complete tumor ablation was achieved in the first model and significant prolongation of survival was observed in the latter two models, suggesting promise for eventual clinical application.

Metallofullerene-based Nanoplatform for Brain Tumor Brachytherapy and Longitudinal Imaging in a Murine Orthotopic Xenograft Model

To demonstrate in an orthotopic xenograft brain tumor model that a functionalized metallofullerene (f-Gd₃N@C₈₀) can enable longitudinal tumor imaging and, when radiolabeled with lutetium 177 (¹⁷⁷Lu) and tetraazacyclododecane tetraacetic acid (DOTA) (¹⁷⁷Lu-DOTA-f-Gd₃N@C₈₀), provide an anchor to deliver effective brachytherapy. All experiments involving the use of mice were carried out in accordance with protocols approved by the institutional animal care and use committee. Human glioblastoma U87MG cells were implanted by using stereotactic procedures into the brains of 37 female athymic nude-Foxn1nu mice and allowed to develop into a tumor for 8 days. T1- and T2-weighted magnetic resonance (MR) imaging was performed in five mice. Biodistribution studies were performed in 12 mice at four time points over 7 days to evaluate gadolinium content. Survival studies involved 20 mice that received infusion of a nanoplatform by means of convection-enhanced delivery (CED) 8 days after tumor implantation. Mice in survival studies were divided into two groups: one comprised untreated mice that received f-Gd₃N@CC₈₀ alone and the other comprised mice treated with brachytherapy that received 1.11 MBq of ¹⁷⁷Lu-DOTA-f-Gd₃N@CC₈₀. Survival data were evaluated by using Kaplan-Meier statistical methods. MR imaging showed extended tumor retention (25.6% ± 1.2 of the infused dose at 52 days, confirmed with biodistribution studies) of the f-Gd₃N@CC₈₀ nanoplatform, which enabled longitudinal imaging. Successful coupling of ¹⁷⁷Lu to the f-Gd₃N@CC₈₀ surface was achieved by using a bifunctional macrocyclic chelator. The extended tumor retention allowed for effective brachytherapy, as indicated by extended survival time (> 2.5 times that of the untreated group) and histologic signs of radiation-induced tumor damage. The authors have developed a multimodal nanoplatform and have demonstrated longitudinal tumor imaging, prolonged intratumoral probe retention, biodistribution, and extended survival in an orthotopic xenograft brain tumor model.

HMSC-mediated Concurrent Delivery of Endostatin and Carboxylesterase to Mouse Xenografts Suppresses Glioma Initiation and Recurrence

Glioma stem cells (GSCs) are known to be maintained within a "vascular niche"; thereby, disruption of this microenvironment using antiangiogenesis agents is a promising therapeutic modality. However, this regimen leads to treatment failure and tumor recurrence in patients with glioblastoma multiforme (GBM). Therefore, more effective therapeutic approaches that can eradicate GSCs and the bulk tumors are needed. Toward this goal, we examined the antitumor effects of an antiangiogenesis approach combined with conventional chemotherapy on suppressing glioma xenograft growth. We established three genetically engineered mesenchymal stem cell (MSC) lines (GE-AF-MSCs) by stably transducing the gene encoding endostatin (an antiangiogenesis factor), the gene encoding secretable form of carboxylesterase 2 (sCE2, a prodrug-activating enzyme), or a mixture of both genes. Among the three GE-AF-MSC cell lines, injection of amniotic fluid (AF)-MSCs-endostatin-sCE2 cells into U87MG-EGFRvIII-driven orthotopic brain tumor and postsurgery tumor recurrence models, and subsequent CPT11 treatment yielded the strongest antitumor responses, including diminished angiogenesis, increased cell death, and a reduced Nestin-positive GSC population. Therefore, our antitumor strategy provides a novel basis for designing stem cell-mediated therapeutic approaches to target and eradicate GSCs and the bulk tumors.

The expression of BST2 in human and experimental mouse brain tumors

Glioblastoma multiforme (grade IV astrocytoma) is a highly malignant brain tumor with poor treatment options and an average lifespan of 15 months after diagnosis. Previous work has demonstrated that BST2 (bone marrow stromal cell antigen 2; also known as PDCA-1, CD137 and HM1.24) is expressed by multiple myeloma, endometrial cancer and primary lung cancer cells. BST2 is expressed on the plasma membrane, which makes it an ideal target for immunotherapy. Accordingly, several groups have shown BST2 mAb to be effective for targeting tumor cells. In this report, we hypothesized that BST2 is expressed in human and mouse brain tumors and plays a critical role in brain tumor progression. We show that BST2 expression is upregulated at both the mRNA and protein level in high grade when compared to low grade human astrocytoma ( p < 0.05). To test the relevance of BST2, we utilized the intracranially (IC)-injected GL261 cell-based malignant brain tumor mouse model. We show that BST2 mRNA expression is increased in mouse brain IC-injected with GL261 cells, when compared to mouse brain IC-injected with saline at 3 weeks post-operative ( p < 0.05). Furthermore, BST2 immunofluorescence predominantly localized to mouse brain tumor cells. Finally, mice IC-injected with GL261 cells transduced with shRNA for BST2 ± preincubated with BST2 mAb show no difference in overall lifespan when compared to mice IC-injected with GL261 cells transduced with a scrambled shRNA ± preincubated with BST2 mAb. Collectively, these data show that while BST2 expression increases during brain tumor progression in both human and mouse brain tumors, it has no apparent consequences to overall lifespan in an orthotopic mouse brain tumor model.

Abstract 2355: Full-length wild-type CD99 increases the migration and invasiveness of human malignant glioma cells

Abstract The malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed CD99, which is a transmembrane glycoprotein, is more expressed in malignant glioma than in normal brain. It has been implicated in cell adhesion and migration in various cells, even if the function of this gene is not understood clearly. This gene has two isoforms of wild-type and splicing variant. We revealed that only wild-type CD99 is expressed in human glioma cells and tissues. Through MTS, colony-forming, and anoikis assays, we found that wild-type CD99 in glioma cells has enough resistance for anoikis and ability to survive and grow in anchorage independence, as is opposed to previous reports in other tumors. Using tissue microarray, we validated that CD99 demonstrates higher expression in gliomas compared to non-neoplastic brain. We inhibited CD99 expression by siRNA and demonstrated decreased glioma cell migration and invasion. In contrast, when CD99 was over-expressed in glioma cells, we observed enhancement of cell migration and invasiveness. Orthotopic brain tumor model with nude mice harboring CD99 overexpressing glioma cells results in significant decreased survival rate, compared to control mice. Moreover, higher invasiveness is observed in overexpressing mice by pathological review. Taken together, our findings suggest that CD99 play an important role in the migration and invasion of human gliomas. CD99 could be a target to inhibit migration and invasion especially in CD99-expressing gliomas. 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 2355. doi:10.1158/1538-7445.AM2011-2355

Abstract 3612: A novel and selective c-Met inhibitor against subcutaneous xenograft and othotopic brain tumor models

Abstract HM5016504 is a novel, highly potent and selective c-Met inhibitor under development by HMPL. In preclinical studies, it demonstrated strong in vitro activity against c-Met and its downstream signaling targets, thus blocking the related cellular functions, including proliferation, migration, invasion, scattering and the secretion of VEGF that plays a pivotal role in tumor angiogenesis. The cross-talk between c-Met and EGFR was investigated, and their contribution to downstream signaling cascade was identified in certain tumor cell lines with dysregulation of both c-Met and EGFR. HM5016504 could effectively inhibit the in vivo growth of a number of human tumor xenografts. Particularly, the tumor cells or xengorafts with high fold of c-Met gene amplification showed high sensitivity to HM5016504, such as gastric cancer SNU-5, Hs746T and lung cancer H1993 and EBC-1 cells or their respective xenografts. The compound not only showed activity in subcutaneous xenograft models, but also demonstrated efficacy in an orthotopic brain tumor model induced by U87MG, indicating it could pass blood brain barrier. In vitro and in vivo activities of HM5016504 were found to be closely correlated with the c-Met expression level and activation status. In addition, HM5016504 exhibited a reasonable pharmacokinetic profile and safety window based on the toxicology study results in rats, dogs, and monkeys. The pre-clinical study results suggest that HM5016504 could be a promising c-Met targeting anti-cancer agent. 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 3612. doi:10.1158/1538-7445.AM2011-3612

Decreased CCAAT/enhancer binding protein β expression inhibits the growth of glioblastoma cells

C/EBPβ is a leucine-zipper transcription factor implicated in the control of metabolism, development, cell differentiation, and proliferation. However, it remains unclear its role in tumor development. Here, we show that down-regulation of C/EBPβ by RNA interference inhibits proliferation in the GL261 murine glioblastoma cell line, induces an arrest of the cell cycle at the G0/G1 boundary, and diminishes their transformation capacity and migration. In addition, we show that C/EBPβ regulates the expression of several DNA damage response- and invasion-related genes. Lastly, C/EBPβ depletion significantly retards tumor onset and prolongs survival in a murine orthotopic brain tumor model. Immunohistochemical analysis revealed a significant diminution of proliferating cell nuclear antigen (PCNA) labeling in tumors derived from C/EBPβ-depleted GL261 cells compared with that in controls. These results show, for the first time, the dependence of glioma cells on C/EBPβ and suggest a potential role of this transcription factor in glioma development.

Nucleic Acid Therapeutics: Concepts for Targeted Delivery to Solid Tumors

Solid tumors form a heterogeneous group of diseases, although common features such as hyperproliferation, overexpression of certain growth factor receptors and deregulated vessel formation including leaky vasculature give the opportunity to target macromolecular drug and nucleic acid carriers to tumor tissue. Similar to other macromolecular drugs, nucleic acid carriers have to be designed to enable tumor targeting after systemic injection. Chemical modification of nucleic acids makes them resistant towards enzymatic degradation. Cationic lipids or polycations condense nucleic acids into small, virus-like structures and the surface modification with hydrophilic polymers allows passive accumulation in tumor tissue; tumor cell binding ligands allow cellular targeting. To avoid toxic side effects, biodegradable and biocompatible carriers were designed. The design of thermoresponsive gene carriers allowed their selective tumor accumulation by locoregional hyperthermia. As a therapeutic concept, tumor-specific delivery of antitumoral RNA was realized in an orthotopic brain tumor model. The combination of gene- and radio-therapy enabled selective accumulation of radionuclides in tumors and boosted antitumoral effects. Hence, combining a smart delivery concept for nucleic acids with a suitable therapeutic strategy will allow successful treatment of otherwise incurable malignant diseases.

Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with its functional ability to inhibit angiogenesis and tumor growth in vivo. We have shown that the soluble anti-angiogenic domain of BAI1 (termed Vstat120) requires CD36, a cell surface glycoprotein expressed on microvascular endothelial cells (MVECs), for it to elicit an anti-angiogenic response. We now report that Vstat120 binding to CD36 on MVECs activates a caspase-mediated pro-apoptotic pathway, and this effect is abrogated by histidine-rich glycoprotein (HRGP). HRGP is a circulating glycoprotein previously shown to function as a CD36 decoy to promote angiogenesis in the presence of thrombospondin-1 or -2. Data here show that Vstat120 specifically binds HRGP. Under favorable MVEC growth conditions this interaction allows chemotactic-directed migration as well as endothelial tube formation to persist in in vitro cellular systems, and increased tumor growth in vivo as demonstrated in both subcutaneous and orthotopic brain tumor models, concomitant with an increase in tumor vascularity. Finally, we show that HRGP expression is increased in human brain cancers, with the protein heavily localized to the basement membrane of the tumors. These data help define a novel angiogenic axis that could be exploited for the treatment of human cancers and other diseases where excess angiogenesis occurs.

Astrocyte Elevated Gene-1: A Novel Target for Human Glioma Therapy

Malignant gliomas including glioblastoma multiforme (GBM) and anaplastic astrocytomas are the most common primary brain tumors. Despite multimodal treatment including surgery, chemotherapy, and radiation, median survival for patients with GBMs is only 12 to 15 months. Identifying molecules critical for glioma progression is crucial for devising effective targeted therapy. In the present study, we investigated the potential contribution of astrocyte elevated gene-1 (AEG-1) in gliomagenesis and explored the possibility of AEG-1 as a therapeutic target for malignant glioma. We analyzed the expression levels of AEG-1 in 9 normal brain tissues and 98 brain tumor patient samples by Western blot analysis and immunohistochemistry. AEG-1 expression was significantly elevated in >90% of diverse human brain tumor samples including GBMs and astrocytic tumors, and also in human glioma cell lines compared with normal brain tissues and normal astrocytes. Knockdown of AEG-1 by small interfering RNA inhibited cell viability, cloning efficiency, and invasive ability of U87 human glioma cells and 9L rat gliosarcoma cells. We also found that matrix metalloproteases (MMP-2 and MMP-9) are involved in AEG-1-mediated invasion of glioma cells. In an orthotopic nude mouse brain tumor model using primary human GBM12 tumor cells, AEG-1 small interfering RNA significantly suppressed glioma cell growth in vivo. Taken together, these provocative results indicate that AEG-1 may play a crucial role in the pathogenesis of glioma and that AEG-1 could represent a viable potential target for malignant glioma therapy.

Abstract C98: Blocking glycolysis with 2-deoxy-D-glucose prodrugs to target brain tumors: A novel concept

Abstract Glycolysis is the major energy producing pathway for fast growing, glycolytically dependent tumors, such as gliomas and ependymomas. Blocking glycolysis is, therefore, an important therapeutic strategy when used alone or as a combination therapy to enhance the effects of chemotherapy in energy-starved tumors. In our previous studies, we examined D-glucose-based antimetabolites, including 2-deoxy-D-glucose (2-DG), and confirmed their ability to block glycolysis, discovered that they can induce autophagic cell death in vitro, and established their antitumor activity in vivo in an orthotopic glioma model. However, during our evaluation of 2-DG, we determined that these compounds do not possess sufficient drug-like properties (reasonable biostability, pharmacokinetic characteristics, or distribution to the target) to warrant further investigations into their clinical effectiveness. In an attempt to improve the pharmacokinetic profile of 2-DG, we have designed synthesized, and tested a series of ester-type prodrugs that enhance biostability, biodistribution, and delivery of 2-DG to its CNS target. We will present the synthesis and preliminary evaluation of these compounds, leading to the selection of WP1122 as our lead compound for further drug development. We will also present the subsequent evaluation of WP1122 in vivo in orthotopic brain tumor models and demonstrate its superior pharmacokinetic profile and enhanced CNS uptake. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C98.

Abstract A148: Palomid 529, a dual mTor1/2 inhibitor, efficiently penetrates the blood-brain barrier and may be an attractive agent for treatment of glioblastoma

Abstract High-grade gliomas (WHO grade III anaplastic astrocytoma and grade IV glioblastoma multiforme) are the most common primary tumors in the central nervous system in adults and uniformly lethal. The current standard treatment (radiotherapy and temozolomide chemotherapy) has only a modest efficacy and better treatments are urgently needed. Activation of the EGFR/PI3K/Akt/mTor signaling cascade is a common event, especially in de novo glioblastoma (GBM) and, therefore, an attractive target for intervention by inhibitors. However, the blood-brain barrier (BBB) is an impediment for the delivery of many agents into the brain. Although GBM has the capacity to disrupt the blood-brain barrier, it is also a very invasive tumor with many tumor cells residing within areas of the brain where the BBB is still intact. Consequently it is important to consider the BBB penetration of novel candidate drugs for treatment of gliomas. We here present a pre-clinical pharmacokinetic study of Palomid 529, which is a novel small molecule inhibitor of mTor. This agent is attractive because it inhibits both the Torc1 and Torc2 complexes, thereby preventing upstream Akt activation through feedback loops. We have used in vitro transwell assays and found that Palomid 529 has negligible affinities for P-glycoprotein (Pgp) and Bcrp1, the two most dominant drug transporters that are expressed at the BBB. Subsequent studies in transporter knockout mouse models receiving i.v. P529 as a micronized drug formulation at a high dose (54 mg/kg) or low dose (5.4 mg/kg) confirmed these results and demonstrated that Palomid 529 penetrates the BBB efficiently with brain-to-plasma ratios exceeding 1. Only at the lowest dose level a minor effect of Pgp and Bcrp1 was observed. Based on these results we are now executing pre-clinical testing of Palomid 529 against orthotopic brain tumor models. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A148.

Targeting energy metabolism in brain cancer through calorie restriction and the ketogenic diet

Malignant brain tumors are a significant health problem in children and adults and are largely unmanageable. As a metabolic disorder involving the dysregulation of glycolysis and respiration (the Warburg effect), malignant brain cancer can be managed through changes in metabolic environment. In contrast to malignant brain tumors that are mostly dependent on glycolysis for energy, normal neurons and glia readily transition to ketone bodies (beta-hydroxybutyrate) for energy in vivo when glucose levels are reduced. The transition from glucose to ketone bodies as a major energy source is an evolutionary conserved adaptation to food deprivation that permits the survival of normal cells during extreme shifts in nutritional environment. Only those cells with a flexible genome, honed through millions of years of environmental forcing and variability selection, can transition from one energy state to another. We propose a different approach to brain cancer management that exploits the metabolic flexibility of normal cells at the expense of the genetically defective and less metabolically flexible tumor cells. This approach to brain cancer management is supported from recent studies in orthotopic mouse brain tumor models and in human pediatric astrocytoma treated with calorie restriction and the ketogenic diet. Issues of implementation and use protocols are discussed.

Antitumor activity of ZD6474 in a metastatic orthotopic brain tumor model

The objective of this study was to examine the antitumor effect of ZD6474, an orally available inhibitor of the vascular endothelial growth factor receptor-2 (VEGFR-2) and the epidermal growth factor receptor (EGFR), on tumor growth in an orthotopic metastatic brain tumor model. In order to determine the antitumor mechanism of ZD6474 treatment, in vitro and in vivo studies were performed. Human breast carcinoma cells (MDA-MB-435) were injected using direct intracranial (IC) inoculation (5x105 cells/100 µl) and internal carotid artery (ICA) injection (5x104 cells/100 µl) in Balb/c-nu female mice. Daily oral treatment with ZD6474 (50 mg/kg) was initiated on day 14 after the establishment of micrometastasis. Mice (n=12 per group) were sacrificed on day 28. Western blot analysis revealed that the autophosphorylation of EGFR and Akt was increasingly decreased with ZD6474 treatment in lung and brain endothelial cells and the MDA-MB-435 cell line. MTT assay also showed that the in vitro antitumor activity of ZD6474 was dependent on EGFR tyrosine kinase inhibition at a higher dose. Daily oral treatment with ZD6474 led to marked inhibition of metastatic tumor growth in the ICA injection and the direct IC inoculation models (median size 3.5 mm3, range 1.6-13.9 mm3) as compared to the control group (median size 62.4 mm3, range 11.5-206.9 mm3). These results suggest that simultaneous inhibition of both the EGFR and VEGFR-2 signaling pathways has a valuable therapeutic effect through its inhibition of the growth of metastatic brain tumors.