Brain Cancer Model Research Articles

Abstract 5756: Apelin-blocking antibodies as potent therapeutics for oncology

Abstract Apelin (APLN) is the endogenous ligand of the G-protein-coupled receptor APJ (APLNR) and triggers a wide range of downstream signaling events, including downregulation of the production of forskolin-induced cAMP and phosphorylation of ERKs, Akt, and p70 S6 kinase. Several lines of evidence suggest that the apelin/APJ axis promotes tumor growth. First, apelin is a potent inducer of angiogenesis and cell proliferation, and its expression is upregulated in hypoxic conditions. Apelin is upregulated in several cancer types and can directly promote tumor growth by inducing cell proliferation through ERK and/or Akt activation. Second, Berta et al. (2010) reported that elevated apelin level correlated with increased microvessel densities and poor survival. Third, blocking APJ signaling reduced proliferation and inhibited tumor growth in human colon adenocarcinoma cell lines expressing high levels of apelin (Picault et al., 2014) and in patient-derived hepatocellular carcinoma cells (Muto et al., 2014). Fourth, although anti-VEGF therapies were developed to block tumor angiogenesis, tumors can acquire drug resistance by bypassing the VEGF pathway and initiating an alternative angiogenic pathway such as the apelin/APJ axis to support growth. Therefore, blocking apelin signaling may be an effective therapeutic strategy for various types of cancer. To block the apelin pathway in cancer therapy, we developed several monoclonal antibodies (mAbs) that bind to the apelin peptide and block apelin-APJ signaling. The antibodies bind to all the physiologically active forms of apelin, including apelin-13, -17, -36, and pyro-apelin-13. These antibodies bind to unique epitopes with an affinity in the picomolar range. The antibodies potently neutralized apelin activity in a cell-based assay with IC50 values in the low nanomolar range. Two antibodies were tested in a mouse oxygen-induced retinopathy (OIR) model to assess their inhibitory effect on angiogenesis. Humanization of the antibodies identified several potent leads that are being characterized for their effect in suppressing the proliferation of tumor cells overexpressing APJ. A search of PDX models found that apelin is overexpressed in specific tumor types including colorectal cancer, kidney cancer, and sarcomas. Remarkably, in PDX models of brain cancer, both apelin and APJ are overexpressed. Indeed, a recent report by Harford-Wright et al. (2017) identified the apelin-APJ system as a central regulator of expansion of patient-derived glioblastoma cells. For recurrent glioblastoma, current anti-VEGF therapies have little to no effect, making the apelin-APJ system an important and exciting target for treating this devastating disease. We will show the efficacy of our lead antibody in PDX models of brain cancer as well as data in support of using apelin-neutralizing mAbs as cancer therapies. Citation Format: Arvin Tam, Ming Wang, Hui Zou. Apelin-blocking antibodies as potent therapeutics for oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5756.

Magnesium Oxide in Nanodimension: Model for MRI and Multimodal Therapy

The prime focus of this investigation is to determine which morphology of magnesium oxide (MgO) is nontoxic and accumulates in sufficient quantity to a human brain cellular/tissue model. Thus, nanostructured MgO was synthesized from a coprecipitation technique involving twin synthetic protocols and the resulting product was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), size distribution histogram, Fourier-transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) analysis and elemental composition was confirmed by EDX analysis. They were tested for selective antigen response in a human brain cancer model through biodistribution, biotoxicity via MTT assay, and tissue morphology. In addition, the MRI compatibility of MgO nanostructures and immunofluorescence studies were investigated on nanoconjugates with different immunoglobulins in the brain section. The results indicated that MgO had some degree of bindings with the antigens. These results led to the empirical modeling of MgO nanomaterials towards toxicity in cancer cells by analyzing the statistical data obtained by experiments. All these results are providing new rational strategy with the concept of MgO for MRI and PTT/PDT.

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

Abstract A124: Discovery of the clinical candidate AZD1390: a high-quality, potent, and selective inhibitor of ATM kinase with the ability to cross the blood-brain barrier

Abstract Glioblastoma multiforme (GBM) is the most common and lethal form of primary brain tumor, and current treatment (surgery followed by fractionated radiotherapy and temozolomide) provides a median survival of just 12-15 months (1,2). The poor prognosis associated with GBM is attributed to an extensive infiltration into surrounding brain tissue (thereby limiting the effectiveness of surgical excision), an intrinsic chemo/radioresistance of the tumor, and the presence of the blood-brain barrier (BBB), which limits the ability of certain chemotherapies to reach the tumor. Ataxia telangiectasia mutant (ATM) is a serine/threonine protein kinase from the phosphatidylinositol 3-kinase-related kinase (PIKK) family of protein kinases and plays a crucial role in the cellular DNA damage response signalling activated by DNA double-strand breaks (DSB). Activated ATM promotes DNA repair and S/G1-cell cycle checkpoints to prevent premature mitosis, maintain genomic integrity, and promote appropriate cell survival or death pathways. DSBs arise intrinsically through the collapse of stalled replication forks, which are induced by a wide range of chemotherapies, or extrinsically through exposure to ionizing radiation. Therefore, ATM inhibition represents an exciting clinical opportunity as a target to hyper-sensitize tumors to chemo/radiotherapy. The optimization of compound properties suitable to allow efficient BBB penetration remains a significant challenge within Medicinal Chemistry, and failure to consider these can severely restrict the utility of an agent for CNS disease. Herein, we describe the identification of AZD1390, a first-in-class orally available and CNS penetrant ATM inhibitor suitable for the treatment of intracranial malignancies. This presentation represents the first disclosure of the Medicinal Chemistry strategies employed to optimize BBB penetration, alongside the SAR for ATM potency, selectivity, and pharmacokinetic properties. AZD1390 is an exceptionally potent inhibitor of ATM in cells (IC50 = 0.78 nM) with >10,000-fold selectivity over closely related members of the PIKK family of enzymes and excellent selectivity across a broad panel of kinases. AZD1390 displays excellent oral bioavailability in preclinical species (66% in rat and 74% in dog), is not a substrate for human efflux transporters, and has been shown to efficiently cross the BBB in non-human primate PET studies. Profound tumor regressions and increased animal survival (>50 days) have been observed in orthotopic xenograft models of brain cancer following just 2 or 4 days combination treatment of AZD1390 with radiotherapy, compared to radiotherapy treatment alone. These data support the potential of CNS-penetrant ATM inhibitors to provide an important new therapeutic agent for the treatment of intracranial malignancies. AZD1390 is currently undergoing early clinical assessment.

PDTM-17. IDENTIFICATION OF HUMAN PEDIATRIC BRAIN TUMORS CELLS IN PATIENT DERIVED ORTHOTOPIC XENOGRAFT MOUSE MODELS BY USING SPECIES-SPECIFIC PTGER2 GENE qPCR

AbstractBACKGROUNDPediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of PDOX models as well as several paired permanent cell lines using and culture techniques. However, an inherent risk of xenograft models is the contamination of isolates of human tumors with host cells. Therefor strategies to identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models.METHODSpecies specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in xenograft and cultured cells. RT-qPCR was carried out using a StepOnePlus™ RT-PCR System. Reaction mix contained SYBR Green master mix, respective primers and 5ng of total gDNA in a total reaction volume of 10μL. The qPCR conditions were as follows: 50°C-2min, 95°C-10min, 40 cycles of (95°C-15sec, 61°C-1min). Primers were additionally controlled by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR.RESULTSVarious models of pediatric brain cancer established in our group were investigated for mouse cell contamination. Xenograft as well as cultured cells were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell in PDOX models. This enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS.CONCLUSIONIn conclusion we have successfully implemented an reliable qRT-PCR approach to determine the human/mouse cell ration in any mixed PDOX cell population.

Abstract 4874: Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors

Abstract Background: Pediatric brain tumors continue to be among the most difficult to cure and are the leading cause of cancer-related deaths in children. Research investigating pediatric brain cancer is still impaired by the lack of cancer models. We established a large panel of orthotopic xenograft models as well as several paired permanent cell lines using in vivo and in vitro culture techniques. However an inherent risk of xenograft models is the contamination of isolates of human tumors with host (mouse) cells. Therefor strategies to correctly and timely identify the human/mouse ratio are essential. In this study we investigated Prostaglandin Receptor EP2 (PTGER2) as a useful target to identifying mouse cell contamination in PDOX models. Method: Species specific primers based on Alcoster et al., 2011 were used to detect PTGER2 in genomic DNA extracts of xenograft and cultured cells. Real-time qPCR was carried out using a StepOnePlus™ Real-Time PCR System. Reaction mix contained SYBR Green select master mix, respective primers and 5 ng of total genomic DNA in a total reaction volume of 10 μL. The qPCR conditions were as follows: 50°C-2 min, 95°C-10 min, 40 cycles of (95°C-15 sec, 61°C-1 min). Primer specificity was controlled in addition by analysis of amplified products on a 1.2% agarose gel. FACS was used to isolate human cells according to anti-Human HLA-ABC Ab and mouse-specific Ab cocktail surface staining and subjected to qPCR. Results: Various models of pediatric brain cancer (PXA, MB, GBM, EPN, ATRT, DIPG, PNET) established in our group were investigated for mouse cell contamination. Cells from xenograft isolates as well as from cultured xenografts were routinely analyzed. Our study confirms PTGER2 as a useful target for accurately estimate mouse cell contamination in brain cancer xenograft models. This further enabled us to improve detection and characterization of HLA-ABC/mouse-cocktail double negative population as seen in FACS. Conclusion: In conclusion we have successfully implemented an easy qRT-PCR approach to determine the human/mouse cell ration in any mixed cell population. Particular for PDOX models it is paramount to have reliable information on mouse cell contamination. Citation Format: Frank K. Braun, Mari Kogiso, Lin Qi, Huiyuan Zhang, Yuchen Du, Holly B. Lindsay, Sibo Zhao, Sarah G. Injac, Xiao-Nan Li. Application of quantitative analysis of mouse cell contamination and purification of human tumor cells in patient derived orthotopic xenograft mouse models of pediatric brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4874. doi:10.1158/1538-7445.AM2017-4874

Evaluation of [18F]-ATRi as PET tracer for in vivo imaging of ATR in mouse models of brain cancer

RationaleAtaxia telangiectasia and Rad3-related (ATR) threonine serine kinase is one of the key elements in orchestrating the DNA damage response (DDR). As such, inhibition of ATR can amplify the effects of chemo- and radiation-therapy, and several ATR inhibitors (ATRi) have already undergone clinical testing in cancer. For more accurate patient selection, monitoring and staging, real-time in vivo imaging of ATR could be invaluable; the development of appropriate imaging agents has remained a major challenge. Methods3-amino-N-(4-[18F]phenyl)-6-(4-(methylsulfonyl)phenyl)pyrazine-2-carboxamide ([18F]-ATRi), a close analogue of Ve-821, (a clinical ATRi candidate), was readily accomplished similarly to already established synthetic procedures. Structurally, 18F was introduced at the 4-position of the aromatic ring of Ve-821 for generating a labeled ATR inhibitor. In vitro experiments were conducted in U251 MG glioblastoma cell lines and ex vivo biodistribution were performed in subcutaneous U251 MG xenograft bearing athymic nude mice following microPET imaging. Results[18F]-ATRi has a similar pharmacokinetic profile to that of Ve-821. Using an U251 MG glioblastoma mouse model, we evaluated the in vivo binding efficiency of [18F]-ATRi. Blood and tumor showed a statistically significant difference between mice injected with only the probe or following blocking experiment with Ve-821 (1.48±0.40%ID/g vs. 0.46±0.12%ID/g in tumor and 1.85±0.47%ID/g vs. 0.84±0.3%ID/g in blood respectively). Conclusions[18F]-ATRi represents the first 18F positron emission tomography (PET) ATR imaging agent, and is designed on a low nanomolar and clinically relevant ATR inhibitor.

C-terminal domain of p42 Ebp1 is essential for down regulation of p85 subunit of PI3K, inhibiting tumor growth.

Potential tumor suppressor p42, ErbB3-binding protein 1 (EBP1) inhibits phosphoinositide 3-kinase (PI3K) activity reducing the p85 regulatory subunit. In this study, we demonstrated that overexpression of p42 promoted not only a reduction of wild type of p85 subunit but also oncogenic mutant forms of p85 which were identified in human cancers. Moreover, we identified the small fragment of C-terminal domain of p42 is sufficient to exhibit tumor suppressing activity of p42-WT, revealing that this small fragment (280–394) of p42 is required for the binding of both HSP70 and CHIP for a degradation of p85. Furthermore, we showed the small fragment of p42 markedly inhibited the tumor growth in mouse xenograft models of brain and breast cancer, resembling tumor suppressing activity of p42. Through identification of the smallest fragment of p42 that is responsible for its tumor suppressor activity, our findings represent a novel approach for targeted therapy of cancers that overexpress PI3K.

Detection of human brain cancer infiltration ex vivo and in vivo using quantitative optical coherence tomography.

More complete brain cancer resection can prolong survival and delay recurrence. However, it is challenging to distinguish cancer from noncancer tissues intraoperatively, especially at the transitional, infiltrative zones. This is especially critical in eloquent regions (for example, speech and motor areas). This study tested the feasibility of label-free, quantitative optical coherence tomography (OCT) for differentiating cancer from noncancer in human brain tissues. Fresh ex vivo human brain tissues were obtained from 32 patients with grade II to IV brain cancer and 5 patients with noncancer brain pathologies. On the basis of volumetric OCT imaging data, pathologically confirmed brain cancer tissues (both high- and low-grade) had significantly lower optical attenuation values at both cancer core and infiltrated zones when compared with noncancer white matter, and OCT achieved high sensitivity and specificity at an attenuation threshold of 5.5 mm(-1) for brain cancer patients. We also used this attenuation threshold to confirm the intraoperative feasibility of performing in vivo OCT-guided surgery using a murine model harboring human brain cancer. Our OCT system was capable of processing and displaying a color-coded optical property map in real time at a rate of 110 to 215 frames per second, or 1.2 to 2.4 s for an 8- to 16-mm(3) tissue volume, thus providing direct visual cues for cancer versus noncancer areas. Our study demonstrates the translational and practical potential of OCT in differentiating cancer from noncancer tissue. Its intraoperative use may facilitate safe and extensive resection of infiltrative brain cancers and consequently lead to improved outcomes when compared with current clinical standards.

Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

AT-29 * INTRAVENOUS ADMINISTRATION OF TOCA 511 IN PATIENTS WITH RECURRENT GLIOBLASTOMA

INTRODUCTION: Toca 511 (vocimagene amiretrorepvec) is an investigational retroviral replicating vector (RRV) that encodes the transgene cytosine deaminase (CD). Subsequent oral administration of investigational extended-release 5-FC (Toca FC) results in formation of 5-FU within infected tumor cells expressing CD. A clinical study was initiated to evaluate the delivery of Toca 511 vector to brain tumor via intravenous administration (NCT01985256). METHODS: The primary purpose of this study is to identify the highest safe and well tolerated dose of Toca 511 administered intravenously to subjects with recurrent high grade glioma scheduled for repeat resection. Subjects who have a recurrent high grade glioma who are scheduled for repeat surgical resection will receive an intravenous administration of Toca 511 approximately 11 days prior to surgery. At the time of surgery Toca 511 will be re-administered into the walls of the resection cavity. After 5 weeks, subjects then take oral Toca FC for 7 days every five weeks until study completion or progression. Dose escalation is accelerated for the first three cohorts (single patient cohorts) and the subsequent two cohorts follow a standard 3 + 3 design. Doses are escalated at half-log increases from a total of 4.8x10^8 to 4.8x10^10 TU. RESULTS: Preclinical studies defined the pharmacokinetics and efficacy of intravenous Toca 511 with intraperitoneal 5-FC in a syngeneic murine brain cancer model. In humans, the first 2 subjects were administered Toca 511 intravenously without complication or toxicity. Peak circulating Toca 511 in the patient in the second dose cohort was present at approximately one billion viral particles per mL of plasma. The half-life of the vector was approximately 5 minutes. Preliminary safety and tolerability data, in addition to pharmacokinetics of Toca 511 in the blood and resected tumor will be reported. CONCLUSION: Toca 511 has been safely administered intravenously and further dose escalation is ongoing.

Does Our “Flight and Fight” Response Have a Link With Cancer?

Does Our “Flight and Fight” Response Have a Link With Cancer?

Is copper chelation an effective anti-angiogenic strategy for cancer treatment?

Angiogenesis and the acquisition of an angiogenic phenotype is important for cancer cell proliferation. Copper in an essential trace element that participates in many enzymatic complexes like the cytochrome c, superoxide dismutase and lysyl oxidase and it is involved in processes, like embryogenesis, growth, angiogenesis and carcinogenesis. In particular, its involvement in carcinogenesis was described for the first time in oral submucous fibrosis, where fibroblasts produce large amounts of collagen in the presence of copper. Copper’s action in carcinogenesis is two-fold: (1) it participates in reactions with an increased redox potential that result in the production of oxidative products and oxidative stress. Through this mechanism, copper may cause DNA mutations in the nucleus and mitochondria or alterations to membrane phospholipids, (2) it participates in angiogenesis even in the absence of angiogenic molecules, as it was reported for the first time in rabbit cornea models with copolymer pellets charged with PGE1.Copper chelation regimens like penicillamine and tetrathiomolybdate are being described in the literature as having anti-angiogenic, anti-fibrotic and anti-inflammatory actions. Animal models of brain cancer that evaluated the anti-angiogenic properties of copper, have proven evidence of the reduction of tumor’s microvascular supply, tumor volume and vascular permeability after plasma copper levels reduction. Interestingly, plasma copper levels reduction was shown to suppress micrometastases generation in mice models of breast cancer.We hypothesize that copper chelation therapy: increases oxidative stress in cancer cells to a level that does not allow survival because of the reduction of anti-oxidative enzymes production. It may also result in inhibition of angiogenesis and of the initiation of the angiogenic switch, because copper normally enhances endothelial cell migration and proliferation, improves binding of growth factors to endothelial cells and enhances the expression of angiogenic molecules. Copper chelation may also reduce extracellular matrix degradation and cancer spread, through reduction of MMP-9 production and probably of other collagenases and may inhibit propagation of micrometastases. However, copper chelation therapy may enhance angiogenesis through reduction of thrombospondin-1, that results into an increase in VEGF–VEGFR2 complexes and a high level of active MMP-9.These hypotheses help in understanding of the anti-angiogenic action of copper chelation therapies and of the complex network of interactions between copper and other molecules involved in angiogenesis. It may also stimulate further research regarding differences in copper metabolism, the effects of anti-copper regimens on organs, the development of resistance, and their possible angiogenic action through thrombospondin expression reduction.

Systemic Delivery of SapC-DOPS Has Antiangiogenic and Antitumor Effects Against Glioblastoma

Saposin C-dioleoylphosphatidylserine (SapC-DOPS) nanovesicles are a nanotherapeutic which effectively target and destroy cancer cells. Here, we explore the systemic use of SapC-DOPS in several models of brain cancer, including glioblastoma multiforme (GBM), and the molecular mechanism behind its tumor-selective targeting specificity. Using two validated spontaneous brain tumor models, we demonstrate the ability of SapC-DOPS to selectively and effectively cross the blood-brain tumor barrier (BBTB) to target brain tumors in vivo and reveal the targeting to be contingent on the exposure of the anionic phospholipid phosphatidylserine (PtdSer). Increased cell surface expression of PtdSer levels was found to correlate with SapC-DOPS-induced killing efficacy, and tumor targeting in vivo was inhibited by blocking PtdSer exposed on cells. Apart from cancer cell killing, SapC-DOPS also exerted a strong antiangiogenic activity in vitro and in vivo. Interestingly, unlike traditional chemotherapy, hypoxic cells were sensitized to SapC-DOPS-mediated killing. This study emphasizes the importance of PtdSer exposure for SapC-DOPS targeting and supports the further development of SapC-DOPS as a novel antitumor and antiangiogenic agent for brain tumors.

Gene Set Based Integrated Data Analysis Reveals Phenotypic Differences in a Brain Cancer Model

A key challenge in the data analysis of biological high-throughput experiments is to handle the often low number of samples in the experiments compared to the number of biomolecules that are simultaneously measured. Combining experimental data using independent technologies to illuminate the same biological trends, as well as complementing each other in a larger perspective, is one natural way to overcome this challenge. In this work we investigated if integrating proteomics and transcriptomics data from a brain cancer animal model using gene set based analysis methodology, could enhance the biological interpretation of the data relative to more traditional analysis of the two datasets individually. The brain cancer model used is based on serial passaging of transplanted human brain tumor material (glioblastoma - GBM) through several generations in rats. These serial transplantations lead over time to genotypic and phenotypic changes in the tumors and represent a medically relevant model with a rare access to samples and where consequent analyses of individual datasets have revealed relatively few significant findings on their own. We found that the integrated analysis both performed better in terms of significance measure of its findings compared to individual analyses, as well as providing independent verification of the individual results. Thus a better context for overall biological interpretation of the data can be achieved.

Recapitulation of Tumor Heterogeneity and Molecular Signatures in a 3D Brain Cancer Model with Decreased Sensitivity to Histone Deacetylase Inhibition

IntroductionPhysiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS).MethodsCNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat.ResultsMacroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches.ConclusionsOur comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system.

Imaging the efficacy of UVC irradiation on superficial brain tumors and metastasis in live mice at the subcellular level

The effect of UVC irradiation was investigated on a model of brain cancer and a model of experimental brain metastasis. For the brain cancer model, brain cancer cells were injected stereotactically into the brain. For the brain metastasis model, lung cancer cells were injected intra-carotidally or stereotactically. The U87 human glioma cell line was used for the brain cancer model, and the Lewis lung carcinoma (LLC) was used for the experimental brain metastasis model. Both cancer cell types were labeled with GFP in the nucleus and RFP in the cytoplasm. A craniotomy open window was used to image single cancer cells in the brain. This double labeling of the cancer cells with GFP and RFP enabled apoptosis of single cells to be imaged at the subcellular level through the craniotomy open window. UVC irradiation, beamed through the craniotomy open window, induced apoptosis in the cancer cells. UVC irradiation was effective on LLC and significantly extended survival of the mice with experimental brain metastasis. In contrast, the U87 glioma was relatively resistant to UVC irradiation. The results of this study suggest the use of UVC for treatment of superficial brain cancer or metastasis.

Gene therapy-mediated reprogramming tumor infiltrating T cells using IL-2 and inhibiting NF-κB signaling improves the efficacy of immunotherapy in a brain cancer model.

Immune-mediated gene therapy using adenovirus expressing Flt3 ligand and thymidine kinase followed by ganciclovir administration (Flt3/TK) effectively elicits tumor regression in preclinical glioma models. Herein, we assessed new strategies to optimize Flt3L/TK therapeutic efficacy in a refractory RG2 orthotopic glioblastoma model. Specifically, we aimed to optimize the therapeutic efficacy of Flt3L/TK treatment in the RG2 model by overexpressing the following genes within the brain tumor microenvironment: 1) a TK mutant with enhanced cytotoxicity (SR39 mutant TK), 2) Flt3L-IgG fusion protein that has a longer half-life, 3) CD40L to stimulate DC maturation, 4) T helper cell type 1 polarizing dendritic cell cytokines interleukin-12 or C-X-C motif ligand 10 chemokine (CXCL)-10, 5) C-C motif ligand 2 chemokine (CCL2) or C-C motif ligand 3 chemokine (CCL3) to enhance dendritic cell recruitment into the tumor microenvironment, 6) T helper cell type 1 cytokines interferon-γ or interleukin-2 to enhance effector T-cell functions, and 7) IκBα or p65RHD (nuclear factor kappa-B [NF-κB] inhibitors) to suppress the function of Foxp3+ Tregs and enhanced effector T-cell functions. Anti-tumor immunity and tumor specific effector T-cell functions were assessed by cytotoxic T lymphocyte assay and intracellular IFN-γ staining. Our data showed that overexpression of interferon-γ or interleukin-2, or inhibition of the nuclear factor kappa-B within the tumor microenvironment, enhanced cytotoxic T lymphocyte-mediated immune responses and successfully extended the median survival of rats bearing intracranial RG2 when combined with Flt3L/TK. These findings indicate that enhancement of T-cell functions constitutes a critical therapeutic target to overcome immune evasion and enhance therapeutic efficacy for brain cancer. In addition, our study provides novel targets to be used in combination with immune-therapeutic strategies for glioblastoma, which are currently being tested in the clinic.

Abstract SY11-02: Microenvironment matters: TGFβ regulates the response to DNA damage

Abstract Transforming growth factor β1 (TGFβ) is a central regulator of the microenvironment in both normal tissues and tumors. TGFβ resides in the extracellular matrix as a latent complex that requires activation prior to binding to ubiquitous cell surface receptors to initiate a signaling cascade culminating in nuclear Smad activation of transcription. Most cancer cells are no longer responsive to TGFβ growth suppression, yet maintain a partially intact signaling pathway. Why? Surprisingly, we found that either Tgfb1 genetic deletion in mouse cells or TGFβ inhibition in human cells compromises the execution of the molecular cascade in response to DNA damage. Irradiated epithelial cells exhibit decreased ATM kinase activity and autophosphorylation, reduced phosphorylation of critical DNA damage transducers, and abrogation of cell cycle blockade and apoptosis, which together result in increased radiosensitivity. The ability of exogenous TGFβ to restore these responses indicates that the effect is both cell intrinsic and distal to TGFβ signaling. Recent studies suggest that TGFβ regulates multiple components of the DNA damage response, supporting the conclusion that extrinsic TGFβ is necessary for efficient execution of the intrinsic DNA damage response. TGFβ may thus provide a survival advantage in tumors, which are a source of exuberant TGFβ activity, provides an unexpected avenue for in cancer treatment. Both ionizing radiation and chemotherapy actually elicit TGFβ activity via redox- and integrin-mediated activation. We tested the translational potential of the requirement for TGFβ in the genotoxic stress program during radiotherapy using TGFβ neutralizing antibodies and small molecule inhibitors of the TGFβ type I receptor kinase in experimental models of breast, lung and brain cancer in vivo and in vitro. TGFβ inhibition prior to radiation increased the radiosensitivity of 10 of 12 human cancer cell lines as measured by clonogenic assay and decreased γH2AX phosphorylation. Notably, glioma stem cells were preferentially protected from DNA damage by high levels of TGFβ, which also promoted self-renewal, both of which were compromised by TGFβ inhibition. Administration of TGFβ neutralizing antibody in vivo increased tumor growth delay following single or multiple radiation fractions and also reduced metastasis. We anticipate that use of TGFβ inhibitors in the context of radiotherapy will increase radiosensitivity and impair the protective tumor microenvironment, providing both local and systemic benefit. 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 SY11-02. doi:1538-7445.AM2012-SY11-02

Abstract 2441: Modeling glioblastoma in the mouse: Insights from noninvasive imaging

Abstract What can noninvasive imaging reveal about mouse models of brain cancer and how closely they resemble their human counterparts? Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer. Genetically engineered mouse models provide an appealing experimental framework in which to address the critical need for improved diagnostic and therapeutic strategies for GBM in a preclinical setting. An important step in the validation of such models is to determine how well they recapitulate salient features of human disease, most commonly from a molecular or genetic perspective. While molecular characterization is certainly imperative, we contend that it is also essential to validate mouse models of GBM from an in vivo imaging perspective. In vivo imaging can reveal not only tumor location and size, but also important biological and physiological processes including vascularity, blood brain barrier (BBB) permeability and necrosis. In addition, given the important role of magnetic resonance imaging (MRI) in the management of patients with GBM, there is a wealth of human imaging data that can be readily compared with analogous mouse data. Thus, our objective was to develop a clinically relevant framework for performing (i) image-based characterization of mouse models of GBM and (ii) comparisons with human disease. We have utilized novel mouse models wherein genetic alterations in the pRb, PTEN and Ras signaling pathways yield GBM. Clinically motivated 3T MRI acquisitions were performed including pre-contrast T1 and T2 datasets, dynamic contrast enhanced MRI (DCEMRI) and a post-contrast T1 acquisition. Image analysis was also guided by clinical relevance. T1 and T2 images were qualitatively analyzed using the VASARI lexicon developed for human GBM in the TCGA project. DCEMRI data was mathematically analyzed using a two compartment model yielding Ktrans parametric maps that are proportional to the BBB permeability. Human GBM MRI data and VASARI features from the TCGA study were downloaded from caIntegrator and compared with murine features using the χ2 test. We found that MRI has high sensitivity for GBM (97%) and accurately reflects histologic extent (≥ =0.88) in mice. We observed marked inter and intra lesion heterogeneity in BBB permeability, with some tumors showing over 10 times higher Ktrans in the rim compared to the center. Interestingly, many VASARI features exhibited similar distributions in both mouse and human GBM, however one feature was rarely found in mice (p< 0.001): a thick enhancing margin which is a salient feature of human GBM and reflects a central region of necrosis and a proliferative, vascular rim. In summary, we have developed a new standardized framework for performing clinically relevant MRI comparisons of mouse and human GBM. Remaining challenges include further investigation on the molecular mechanisms for the observed heterogeneous patterns of BBB permeability and the lack of central necrosis in mice. 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 2441. doi:1538-7445.AM2012-2441