Pilot project 3: Imaging tumor HIF-1 activity in a rat orthotopic brain tumor model by MRIPI: 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 aggressivesurgical and adjuvant therapies. The distinguishing hallmark of GBM tumors is the presence ofpseudopalisading necrosis and angiogenesis, an abnormal neovasculature that channels the metabolic needsfor tumor growth. Neoplastic cells have a special need for metabolites in order to develop into threedimensionalspheroids and solid tumors. Heterogeneous tumor microenvironment is associated with thedevelopment of abnormal vascularization in GBM, often consisting of distended capillaries with leaky walls andsluggish flow as compared with the regular, ordered vasculature of normal tissues [1, 2]. Despite the constanteffort of tumor cells to recruit new blood vessels, hypoxia occurs in tumor masses 150 ^m away from the bloodsupply and tends to be widespread in solid tumors. Viable hypoxic cells in solid tumors are associated with thefailure of radiation and certain chemotherapies, negatively impacting treatment outcomes. The development ofnew therapies will benefit from small-animal tumor-hypoxia models and imaging techniques that can visualizeand monitor the development or inhibition of hypoxia associated with the tumor growth or treatment. The tumorcells in these hypoxic areas express hypoxia-inducible factor (HIF)-1, a pro-survival transcription factor. Undersuch low oxygen concentrations, HIF-1a is stabilized and dimerizes with HIF-1P to form an active transcriptionfactor. 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, includingglycolytic enzymes and vascular endothelial growth factor (VEGF) [3, 4]. These data suggest that HIF-1 is apromising new target for the treatment of GBM. Thus, non-invasive imaging methods are highly desirable forone to determine HIF-1 activity.to monitor therapeutic response in real-time upcoming anti-HIF-1 therapies andto 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 usingMRI, to measure tumor volume, and bioluminescence imaging (BLI) for HIF-1 activity [5]. To generate 9L cellsreporting 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 whoseexpression is driven by six tandem copies of the hypoxia-responsive element (HRE) derived from the VEGFgene promoter [6]. Single cell G418-resistant clones showing elevated luciferase activity under hypoxicconditions were selected. The 9L-V6R cells were stereotactivally injected into the brains of Fischer 344 rats toestablish an orthotopic brain tumor model. 2ME2 treatment was initiated from 8th day of tumor implantation andlasted 9 days. Tumor growth and drug response were monitored before and after the 2ME2 treatment (days 8and 17) by post-gadolinium T1-weighted MRI [5]. Tumor HIF-1 activity was monitored by BLI using a XenogenSmall Animal Imaging system (IVIS Imaging System) equipped with Living Image software. After MRI andBLI, histological analysis was subsequently performed to elucidate the drug action mechanism. Treatment with2ME2 (60 - 600 mg/kg/day) resulted in a dose-dependent reduction in tumor volume [5]. This effect was alsoassociated with improved tumor oxygenation as assessed by Pimonidazole staining, increased HIF-1 a proteinlevels, and microtubule destabilization as seen with histology. Although the use of V6R-driven luciferaseimaging was able to reveal the HIF-1 activity in vivo (see Fig. 1), the BLI data were not consistent withhistological data due to a lack of deep tissue penetration and 3D tomographic information. Therefore, amethod that combines the ability of reporting genetic processes of tumor activity like BLI and high resolution3D anatomic information will fulfill the urgent need in molecular imaging of cancer. Although MRI providessuperior anatomic details and tissue contrast among all imaging modalities, it suffers from low sensitivity in itsapplication in molecular imaging. Most of contrast agents used currently are exogenous and inappropriate fordirectly monitoring the genetic events in vivo.
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