MR imaging features of high-grade gliomas in murine models: How they compare with human disease, reflect tumor biology, and play a role in preclinical trials

Murine models are the most commonly used and best investigated among the animal models of HGG. They constitute an important weapon in the development and testing of new anticancer drugs and have long been used in preclinical trials. Neuroimaging methods, particularly MR imaging, offer important advantages for the evaluation of treatment response: shorter and more reliable treatment end points and insight on tumor biology and physiology through the use of functional imaging DWI, PWI, BOLD, and MR spectroscopy. This functional information has been progressively consolidated as a surrogate marker of tumor biology and genetics and may play a pivotal role in the assessment of specifically targeted drugs, both in clinical and preclinical trials. The purpose of this Research Perspectives was to compile, summarize, and critically assess the available information on the neuroimaging features of different murine models of HGGs, and explain how these correlate with human disease and reflect tumor biology.This work was supported by the Programme for Advanced Medical Education from Fundaçâo Champalimaud, Fundaçâo Calouste Gulbenkian, Ministério da Saúde and Fundaçâo para a Ciência e Tecnologia, Portugal, to the first author (A.R.B.), and by grants from the Spanish Ministry of Science and Innovation SAF 2008–01327 and the Community of Madrid S-BIO-2006–0170, to the last author (S.G.C.).Peer Reviewe

Exploring Brain-Derived Progenitor Cells as a therapeutic delivery system to Glioblastoma

Glioblastoma (GBM) is a devastating incurable malignant brain cancer in need of new treatments. We have begun to investigate the feasibility of a primary adult cell type (Brain-Derived Progenitor Cells, BDPCs) as a novel therapeutic delivery system to GBM. Our objective was to track the viability of BDPCs after intratumoral infusion into syngeneic orthotopic rat GBM tumours using non-invasive bioluminescence imaging (BLI). We hypothesize rat BDPCs will survive greater than 1 week following infusion into orthotopic F98 GBM tumors. BDPCs harvested from the cortex of adult Fischer rats were expanded in culture then engineered to co-express firefly Luciferase for BLI as well as the fluorescence protein tdTomato. In vitro assays displayed consistent lentiviral engineering of transgenes as well as statistically significant GBM-homing by BDPCs (p \u3c 0.01). All animals showed in vivo BLI signal until the study’s endpoint, confirming viable BDPCs were still present. Histological examination revealed small numbers of fluorescent BDPCs at the tumours’ invading edges in frozen coronal sections

Characterization of an Orthotopic Rat Model of Glioblastoma Using Multiparametric Magnetic Resonance Imaging and Bioluminescence Imaging

Glioblastoma multiforme (GBM) is a lethal and incurable disease. The C6 rat model of GBM shares several similarities to human GBM and longitudinal non-invasive imaging may allow tumour features to be studied. In this thesis, a multimodality imaging framework, consisting of bioluminescence imaging (BLI) and multiparametric magnetic resonance imaging (mpMRI), was applied to the C6 rat model to characterize the growth of orthotopic tumours. BLI signal, a measure of cell viability, tended to increase and then decrease in the majority of animals, whereas tumour volume (from MRI) continually increased. Cellular viability and tumour volume did not correlate across all days, highlighting the value of using complimentary imaging modalities. Apparent diffusion coefficient maps and immunohistochemistry suggests decreases in BLI signal are in part due to decreased tumour cellularity (i.e. necrosis). This is the first use of BLI and mpMRI to characterize this model, and highlights the inter-subject variability in tumour growth

Brain tumors: preclinical imaging and novel therapies

Vandertop, W.P. [Promotor]Würdinger, T. [Promotor]Noske, D.P. [Copromotor]Hulleman, E. [Copromotor

Preclinical models of glioblastoma:limitations of current models and the promise of new developments

Acknowledgements. The authors thank members of the Speirs group for helpful comments. Financial support. This study was supported by the University of Aberdeen Development Trust and a University of Huddersfield PhD studentship (PV-B).Peer reviewedPostprin

Small Interfering RNA Imaging Probes for Neurological Applications

Small interfering RNAs (siRNAs) have emerged as a potent new class of therapeutics that regulate gene expression through sequence-specific inhibition of mRNA translation. Clinical trials of siRNAs have highlighted the need for robust delivery and detection techniques that would enable the application of these therapeutics to increasingly complex diseases and organ systems. Here we detail the generation and evaluation of siRNA-based optical and magnetic resonance imaging (MRI) contrast agents for the treatment of neurological diseases, including ischemic stroke and glioblastoma multiforme brain cancer. First, we designed and tested a fluorescent probe for neuroprotection in the setting of stroke that consists of siRNA complexed with myristoylated poly-arginine peptide (MPAP). MPAP, a peptide shown to cross cell membranes and the blood brain barrier, promoted robust internalization of siRNA by neurons in vitro and in mouse brain after intracerebral injection. Cellular uptake of MPAP-siRNA probes directed against a protein implicated in stroke pathology, c-Src, led to statistically-significant reductions of endogenous mRNA expression. The neuroprotective potential of probes was tested in a mouse model of ischemic stroke. Second, superparamagnetic iron oxide nanoparticles were investigated as vectors for siRNA delivery to glioblastoma multiforme brain tumors. Nanoparticles were designed to enhance chemotherapeutic treatment of tumors through siRNA-mediated knockdown of O6-methylguanine–DNA methyltransferase (MGMT), a protein implicated in glioblastoma chemotherapy resistance. The iron oxide core of nanoparticles rendered them detectable by MRI while fluorescent labeling was used for optical imaging. Functionalizing nanoparticles with the peptide chlorotoxin enabled tumor targeting and cellular accumulation of probe. Probe uptake was accompanied by reductions in MGMT activity and enhanced cellular responses to the chemotherapeutic temozolomide. Nanoparticles were tested in an orthotopic glioblastoma mouse model, where intratumoral administration proved effective in suppressing MGMT expression and tumor volume. These studies serve as proof-of-principle that siRNA-based imaging agents can be used as therapeutic tools for diseases of the central nervous system.Engineering and Applied Science

Cancer and glioma : an integrated approach of gene therapy and bioluminescence imaging

Glioblastoma multiforme (GBM) is the most malignant variant of glioma. This tumor does not only display an extremely aggressive, invasive growth pattern, but is also very difficult to treat. With a two-year survival rate of 40% and a median survival of 12-18 months after treatment, prognosis is poor. Current treatment options are not successful in halting tumor progression and GBM tumors are highly heterogeneous, display all kinds of anti-apoptotic escape routes, suppress the immune system, invade the surrounding parenchyma with unmatched aggressiveness and possess a whole array of tools to rearrange the extra tumoral environment to their advantage. The aim of this thesis is to combine the strengths of gene-therapy and bioluminescence Imaging (BLI) for the development of novel reporter systems in order to study glioma tumor biology and its response to therapeutic compounds. We optimized the currently available BLI luciferases (Gaussia luciferase, Vargula hilgendorfi) and assays (Gluc blood assay, Mycoplasma detection assay). We explored a new multimodal targeted liposome formulation with increased relaxivity for the treatment and imaging of cancer. Finally we combined the newly developed and enhanced reporters to test a new therapeutic combination for the treatment of Glioma (TRAIL, Lanatoside C).UBL - phd migration 201

CNS Penetration of Tyrosine Kinase Inhibitors in Mouse Models

For the past three decades, advances in the treatment of central nervous system (CNS) tumors such as malignant glioma have only been modest. One particular challenge facing treatment of brain tumors is the delivery of therapeutically effective concentrations of anti-cancer agents to the target site in the brain. The sanctuary of the brain is protected by several barrier systems such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). These barriers restrict the passage of anti-cancer drugs into the brain via several protective mechanisms. In the present study, we used cerebral microdialysis sampling, a technique for sampling unbound molecules in brain extracellular fluid (ECF) via semi-permeable probe, to assess the role of murine ATP Binding Cassette (ABC) transporters Bcrp1, P-gp, and Mrp4 in CNS penetration of molecularly targeted agents under investigation for treatment of malignant glioma. We choose the specific inhibitor of epidermal growth factor receptor (EGFR), erlotinib (TarcevaTM), and the specific inhibitor of platelet-derived growth factor receptor (PDGFR), crenolanib, as examples of tyrosine kinase inhibitors currently tested for treatment of malignant glioma. Given the poor microdialysis probe recovery of these lipophilic molecules, we enhanced their recovery by including an affinity-based trapping agent, 10% hydroxypropylbetacyclodextrin (HPBCD), in the perfusate. Using this technique, we studied erlotinib and its major metabolite, OSI-420, penetration in control and transporter-deficient mice. We showed that Bcrp1 is the main efflux transporter preventing erlotinib and OSI-420 penetration in mouse brain. Intracellular accumulation studies confirmed the role of BCRP in erlotinib and OSI-420 transport. We also characterized the role of solute carrier transporters in erlotinib and OSI-420 brain accumulation. Our data show that erlotinib and OSI-420 are substrates for members of the SLC22A family of uptake transporters, OAT3 and OCT2. We then sought to characterize the disposition of tyrosine kinase inhibitors in malignant glioma using cerebral microdialysis. We decided to use a transgenic mouse model that highly recapitulates several features of the human glioma including tumor histology and genetic profiles. However, the bregma commonly used as a reference point to place microdialysis cannula does not appear on images derived by magnetic resonance imaging (MRI), the imaging method used to identify the size and location of the spontaneously arising tumors. Thus, we realized that a new technique to implant the microdialysis guide cannula would be necessary. Using angiography studies of mouse brain vasculature and T2-weighted MRI, we identified the intersection of the midline suture and the rostral rhinal vein on the mouse brain surface as a reference point for implanting the microdialysis cannula. This point correlated with the intersection between the midline and the olfactory bulb/frontal lobe border visualized on T2- weighted MRI. Our method allowed for accurate placement of microdialysis cannula in tumors developing in several regions of the mouse brain. While cerebral microdialysis is commonly used to monitor CNS disposition of single anti-cancer drug at a time, the feasibility of simultaneous sampling of multiple anti-cancer agents via cerebral microdialysis has not been reported. However, combining anti-cancer drugs represents a promising strategy for treatment of resistant CNS tumors, as malignant glioma. Given the role played by EGFR and PDGFR in providing multiple inputs for sustaining glioma cell survival and proliferation, combining inhibitors of EGFR (erlotinib) and PDGFR (crenolanib) represents a promising treatment strategy for these tumors. The goal of our last set of studies was to optimize microdialysis conditions to sample crenolanib and erlotinib as single agents or in combination from tumor ECF in a xenograft mouse model of glioma. By including 10% HPBCD in the perfusate, probe recovery of both erlotinib and crenolanib was significantly increased. To estimate probe recovery we used the zero-flow rate (ZFR) which estimated stock concentrations with 15% accuracy. The enhanced recovery achieved by including HPBCD coupled with sensitive analytical techniques allowed us to determine crenolanib and erlotinib penetration in tumor ECF under steady state conditions. No significant differences were observed in drug penetration between groups treated with single agent or those treated with both drugs. In conclusion, we developed techniques to improve microdialysis probe recovery of lipophilic agents administered as single agents or in combination. We also developed an MRIguided method to implant microdialysis cannula in a spontaneous glioma murine model. These techniques enhance our ability to perform microdialysis studies on a large spectrum of anticancer agents in clinically relevant murine models. Using the developed techniques, we identified efflux and uptake transporters that regulate erlotinib CNS disposition. We evaluated the extent of erlotinib and crenolanib penetration in malignant glioma models. Our results shed more light on the extent of tumor penetration of two tyrosine kinase inhibitors currently being tested for treatment of malignant glioma

Molecular Mechanisms of Therapeutic Resistance in Cancer.

Development of therapeutic resistance limits the efficacy of current cancer treatment. Understanding the molecular basis for therapeutic resistance should facilitate the identification of actionable targets and development of new combination therapies for cancer patients. Yet the understanding of therapeutic resistance still remains incomplete. In this thesis, clinically relevant mouse models coupled with systematic genomic and imaging technologies are used to identify mechanisms driving resistance, which also formulate novel therapeutic paradigms for patients with drug-resistant tumors. In the first study, a genetically engineered mouse model of ovarian endometrioid adenocarcinoma (OEA) was utilized in combination with molecular imaging to understand mechanisms of chemoresistance in OEA. It was demonstrated that AKT signaling pathway was activated upon chemotherapy (cisplatin) administration, which protected cells from apoptosis and thereby leading to the development of resistance. In support of this observation, inhibition of AKT activity improved the efficacy of chemotherapy by enhanced induction of apoptosis. A second study was undertaken to develop a new understanding of the mechanistic basis for therapeutic resistance in glioblastoma using a patient derived xenograft model. An integrated transcriptome analysis revealed that chemoradioresistance was associated with an increased expression of genes involved in the mesenchymal and stem cell phenotype as well as a decreased expression of genes involved in cell death. TGF-β signaling was identified to be central to each of the mesenchymal/stem phenotype and therefore a critical player in modulating therapeutic resistance. In support, treatment with a TGF-β inhibitor partially restored the sensitivity to therapy in TMZ/IR resistant tumors. Overall, this thesis demonstrated the importance of the AKT and TGF-β signaling pathways in therapeutic resistance in a subset of ovarian cancer and glioblastoma patients, which provides clinical guidance for applying new combination therapies. It also demonstrates the concept that the combination of clinically relevant mouse models, molecular imaging and systematic genomic analysis can be used to derive novel insights into the dynamic signaling processes involved with gain of resistance. Future studies are needed to investigate if targeting these resistance mechanisms delays or prevents the development of resistance in treatment-naïve patients.PHDCellular and Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111378/1/hanxiaow_1.pd