Dev Proj 3: Imaging Tumor HIF-1 activity in a Rat Orthotopic Brain Tumor Model...

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Pilot project 3: Imaging tumor HIF-1 activity in a rat orthotopic brain tumor model by MRI PI: Xiaoping Hu, Ph.D. Co-Pis: Hyunsuk Shim, Ph.D. and Anthony Chan, Ph.D. Background and Specific Aims: Glioblastoma multiforme (GBM) is a fatal disease despite aggressive surgical and adjuvant therapies. The distinguishing hallmark of GBM tumors is the presence of pseudopalisading necrosis and angiogenesis, an abnormal neovasculature that channels the metabolic needs for tumor growth. Neoplastic cells have a special need for metabolites in order to develop into threedimensional spheroids and solid tumors. Heterogeneous tumor microenvironment is associated with the development of abnormal vascularization in GBM, often consisting of distended capillaries with leaky walls and sluggish flow as compared with the regular, ordered vasculature of normal tissues [1, 2]. Despite the constant effort of tumor cells to recruit new blood vessels, hypoxia occurs in tumor masses 150 ^m away from the blood supply and tends to be widespread in solid tumors. Viable hypoxic cells in solid tumors are associated with the failure of radiation and certain chemotherapies, negatively impacting treatment outcomes. The development of new therapies will benefit from small-animal tumor-hypoxia models and imaging techniques that can visualize and monitor the development or inhibition of hypoxia associated with the tumor growth or treatment. The tumor cells in these hypoxic areas express hypoxia-inducible factor (HIF)-1, a pro-survival transcription factor. Under such low oxygen concentrations, HIF-1a is stabilized and dimerizes with HIF-1P to form an active transcription factor. The dimer binds to the DNA sequence 5'-RCGTG-3' (HRE), located in the promoter of target genes. This subsequently leads to up-regulation of factors that promote tumor growth and angiogenesis, including glycolytic enzymes and vascular endothelial growth factor (VEGF) [3, 4]. These data suggest that HIF-1 is a promising new target for the treatment of GBM. Thus, non-invasive imaging methods are highly desirable for one to determine HIF-1 activity.to monitor therapeutic response in real-time upcoming anti-HIF-1 therapies and to determine if anti-HIF-1 therapy is suitable for patients. Previously, we evaluated whether 2-methoxyestradiol (2ME2), an HIF-1 inhibitor, has therapeutic potential for tumor in a 9L rat orthotopic gliosarcoma model using MRI, to measure tumor volume, and bioluminescence imaging (BLI) for HIF-1 activity [5]. To generate 9L cells reporting HIF-1 activity, we stably co-transfected 9L rat glioma cells with a luciferase expression vector (V6R) to monitor HIF-1 activity and pCDNA3.1 for drug selection (mixture ratio of luciferase vector: pCDNA 3.1 = 5:1) using Lipofectamine 2000 (Invitrogen). The V6R HIF-1 reporter plasmid contains a luciferase gene whose expression is driven by six tandem copies of the hypoxia-responsive element (HRE) derived from the VEGF gene promoter [6]. Single cell G418-resistant clones showing elevated luciferase activity under hypoxic conditions were selected. The 9L-V6R cells were stereotactivally injected into the brains of Fischer 344 rats to establish an orthotopic brain tumor model. 2ME2 treatment was initiated from 8th day of tumor implantation and lasted 9 days. Tumor growth and drug response were monitored before and after the 2ME2 treatment (days 8 and 17) by post-gadolinium T1-weighted MRI [5]. Tumor HIF-1 activity was monitored by BLI using a Xenogen Small Animal Imaging system (IVIS¿ Imaging System) equipped with Living Image software. After MRI and BLI, histological analysis was subsequently performed to elucidate the drug action mechanism. Treatment with 2ME2 (60 - 600 mg/kg/day) resulted in a dose-dependent reduction in tumor volume [5]. This effect was also associated with improved tumor oxygenation as assessed by Pimonidazole staining, increased HIF-1 a protein levels, and microtubule destabilization as seen with histology. Although the use of V6R-driven luciferase imaging was able to reveal the HIF-1 activity in vivo (see Fig. 1), the BLI data were not consistent with histological data due to a lack of deep tissue penetration and 3D tomographic information. Therefore, a method that combines the ability of reporting genetic processes of tumor activity like BLI and high resolution 3D anatomic information will fulfill the urgent need in molecular imaging of cancer. Although MRI provides superior anatomic details and tissue contrast among all imaging modalities, it suffers from low sensitivity in its application in molecular imaging. Most of contrast agents used currently are exogenous and inappropriate for directly monitoring the genetic events in vivo.