Aditya Bansal

Choline phosphorylation and regulation of transcription of choline kinase α in hypoxia

Choline kinase catalyzes the phosphorylation of choline, the first step of phospholipid synthesis. Increased phosphorylation of choline is a hallmark characteristic of the malignant phenotype in a variety of neoplasms. However, in hypoxic cancer cells, choline phosphorylation is decreased. To understand the mechanism behind this altered metabolic state, we examined the expression and regulation of the major choline kinase isoform, choline kinase α (ChKα), in hypoxic PC-3 human prostate cancer cells. Hypoxia decreased choline phosphorylation, choline kinase activity, and ChKα mRNA and protein levels. Promoter analysis studies revealed a region upstream of the ChKα gene bearing a conserved DNA consensus binding motif, hypoxia response element-7 (HRE7), at position −222 relative to +1 translation start site, for binding the hypoxia dependent master regulator transcription factor, hypoxia-inducible factor 1α (HIF-1α). Electrophoretic mobility shift competition/supershift assay and chromatin immunoprecipitation assay confirmed binding of HIF-1α to HRE7. A putative promoter of ChKα was isolated from PC-3 genomic DNA and cloned into a luciferase-based reporter vector system. In PC-3 cells, hypoxia decreased the expression of luciferase under the control of the ChKα promoter. Mutation of HRE7 abrogated this hypoxia effect, further demonstrating the involvement of HRE7 in hypoxia-sensitive regulation of ChKα. The results strongly suggest that transcriptional control of choline phosphorylation is largely mediated via HIF-1α binding to the newly identified HRE7.

Synthesis of 18F-Tetrafluoroborate via Radiofluorination of Boron Trifluoride and Evaluation in a Murine C6-Glioma Tumor Model

The sodium/iodide symporter (NIS) is under investigation as a reporter for noninvasive imaging of gene expression. Although 18F-tetrafluoroborate (18F-TFB, 18F-BF4−) has shown promise as a PET imaging probe for NIS, the current synthesis method using isotopic exchange gives suboptimal radiochemical yield and specific activity. The aim of this study was to synthesize 18F-TFB via direct radiofluorination on boron trifluoride (BF3) to enhance both labeling yield and specific activity and evaluation of specific activity influence on tumor uptake. Methods: An automated synthesis of 18F-TFB was developed whereby cyclotron-produced 18F-fluoride was trapped on a quaternary methyl ammonium anion exchange cartridge, then allowed to react with BF3 freshly preformulated in petroleum ether/tetrahydrofuran (50:1). The resultant 18F-TFB product was retained on the quaternary methyl ammonium anion exchange cartridge. After the cartridge was rinsed with tetrahydrofuran and water, 18F-TFB was eluted from the cartridge with isotonic saline, passing through 3 neutral alumina cartridges and a sterilizing filter. Preclinical imaging studies with 18F-TFB were performed in athymic mice bearing NIS-expressing C6-glioma subcutaneous xenografted tumors to determine the influence of specific activity on tumor uptake. Results: Under optimized conditions, 18F-TFB was synthesized in a radiochemical yield of 20.0% ± 0.7% (n = 3, uncorrected for decay) and greater than 98% radiochemical purity in a synthesis time of 10 min. Specific activities of 8.84 ± 0.56 GBq/μmol (n = 3) were achieved from starting 18F-fluoride radioactivities of 40–44 GBq. An avid uptake of 18F-TFB was observed in human NIS (hNIS)–expressing C6-glioma xenografts as well as expected NIS-mediated uptake in the thyroid and stomach. There was a positive correlation between the uptake of 18F-TFB in hNIS-expressing tumor and specific activity. Conclusion: A rapid, practical, and high-specific-activity synthesis of the NIS reporter probe 18F-TFB was achieved via direct radiofluorination on BF3 using an automated synthesis system. The synthesis of high-specific-activity 18F-TFB should enable future clinical studies with hNIS gene reporter viral constructs.

Tracking and Therapeutic Value of Human Adipose Tissue–derived Mesenchymal Stem Cell Transplantation in Reducing Venous Neointimal Hyperplasia Associated with Arteriovenous Fistula

PURPOSE To determine if adventitial transplantation of human adipose tissue-derived mesenchymal stem cells (MSCs) to the outflow vein of B6.Cg-Foxn1(nu)/J mice with arteriovenous fistula (AVF) at the time of creation would reduce monocyte chemoattractant protein-1 (Mcp-1) gene expression and venous neointimal hyperplasia. The second aim was to track transplanted zirconium 89 ((89)Zr)-labeled MSCs serially with positron emission tomography (PET) for 21 days. MATERIALS AND METHODS All animal experiments were performed according to protocols approved by the institutional animal care and use committee. Fifty B6.Cg-Foxn1(nu)/J mice were used to accomplish the study aims. Green fluorescent protein was used to stably label 2.5 × 10(5) MSCs, which were injected into the adventitia of the outflow vein at the time of AVF creation in the MSC group. Eleven mice died after AVF placement. Animals were sacrificed on day 7 after AVF placement for real-time polymerase chain reaction (n = 6 for MSC and control groups) and histomorphometric (n = 6 for MSC and control groups) analyses and on day 21 for histomorphometric analysis only (n = 6 for MSC and control groups). In a separate group of experiments (n = 3), animals with transplanted (89)Zr-labeled MSCs were serially imaged with PET for 3 weeks. Multiple comparisons were performed with two-way analysis of variance, followed by the Student t test with post hoc Bonferroni correction. RESULTS In vessels with transplanted MSCs compared with control vessels, there was a significant decrease in Mcp-1 gene expression (day 7: mean reduction, 62%; P = .029), with a significant increase in the mean lumen vessel area (day 7: mean increase, 176% [P = .013]; day 21: mean increase, 415% [P = .011]). Moreover, this was accompanied by a significant decrease in Ki-67 index (proliferation on day 7: mean reduction, 81% [P = .0003]; proliferation on day 21: mean reduction, 60%, [P = .016]). Prolonged retention of MSCs at the adventitia was evidenced by serial PET images of (89)Zr-labeled cells. CONCLUSION Adventitial transplantation of MSCs decreases Mcp-1 gene expression, accompanied by a reduction in venous neointimal hyperplasia.

Improved production and processing of 89Zr using a solution target

OBJECTIVE The objectives of the present work were to improve the cyclotron production yield of (89)Zr using a solution target, develop a practical synthesis of the hydroxamate resin used to process the target, and develop a biocompatible medium for (89)Zr elution from the hydroxamate resin. METHODS A new solution target (BMLT-2) with enhanced heat dissipation capabilities was designed by using helium-cooled dual foils (0.2 mm Al and 25 μ Havar) and an enhanced water-cooled, elongated solution cavity in the target insert. Irradiations were performed with 14 MeV protons on a 2M solution of yttrium nitrate in 1.25 M nitric acid at 40-μA beam current for 2 h in a closed system. Zirconium-89 was separated from Y by use of a hydroxamate resin. A one-pot synthesis of hydroxamate resin was accomplished by activating the carboxylate groups on a carboxymethyl cation exchange resin using methyl chloroformate followed by reaction with hydroxylamine hydrochloride. After trapping of (89)Zr on hydroxamate resin and rinsing the resin with HCl and water to release Y, (89)Zr was eluted with 1.2 M K2HPO4/KH2PO4 buffer (pH3.5). ICP-MS was used to measure metal contaminants in the final (89)Zr solution. RESULTS The BMLT-2 target produced 349±49 MBq (9.4±1.2 mCi) of (89)Zr at the end of irradiation with a specific activity of 1.18±0.79 GBq/μg. The hydroxamate resin prepared using the new synthesis method showed a trapping efficiency of 93% with a 75 mg resin bed and 96-97% with a 100-120 mg resin bed. The elution efficiency of (89)Zr with 1.2M K2HPO4/KH2PO4 solution was found to be 91.7±3.7%, compared to >95% for 1 M oxalic acid. Elution with phosphate buffer gave very small levels of metal contaminants: Al=0.40-0.86 μg (n=2), Fe=1.22±0.71 μg (n=3), Y=0.29 μg (n=1). CONCLUSIONS The BMLT-2 target allowed doubling of the beam current for production of (89)Zr, resulting in a greater than 2-fold increase in production yield in comparison with a conventional liquid target. The new one-pot synthesis of hydroxamate resin provides a simpler synthesis method for the (89)Zr trapping resin. Finally, phosphate buffer elutes the (89)Zrfrom the hydroxamate resin in high efficiency while at the same time providing a more biocompatible medium for subsequent use of (89)Zr.

Synthesis and evaluation of 18F-hexafluorophosphate as a novel PET probe for imaging of sodium/iodide symporter in a murine C6-glioma tumor model

Noninvasive imaging of iodide uptake via the sodium/iodide symporter (NIS) has received great interest for evaluation of thyroid cancer and reporter imaging of NIS-expressing viral therapies. In this study, we investigate 18F-labeled hexafluorophosphate (HFP or PF6-) as a high-affinity iodide analog for NIS imaging. 18F-HFP was synthesized by radiofluorination of phosphorus pentafluoride·N-methylpyrrolidine complex and evaluated in human NIS (hNIS)-expressing C6 glioma cells and a C6 glioma xenograft mouse model. 18F-HFP was obtained in radiochemical yield of 10 ± 5%, radiochemical purity of >96% and specific radioactivity of 604 ± 18 MBq/µmol. Specific uptake of 18F-HFP and high affinity of 19F-HFP were observed in hNIS+ C6-glioma cells. PET imaging showed robust uptake of 18F-HFP in NIS-expressing tissues (thyroid, stomach, and hNIS+ C6 glioma xenografts), and the uptake of 18F-HFP was blocked by NaClO4 pretreatment. Specific accumulation in hNIS-expressing xenograft (hNIS+) was observed relative to isogenic control tumor (hNIS-). Clearance of 18F-HFP was predominantly through renal excretion. The biodistribution showed consistent results with PET imaging. Minimal bone uptake was observed over 2 h period post-injection, indicating excellent in vivo stability of 18F-HFP. Although improvement in specific radioactivity is desirable, the results indicate that 18F-HFP is a promising candidate radiotracer for further evaluation for NIS imaging.

PET Imaging of Gliomas

Noninvasive imaging methods, including positron emission tomography (PET), have become essential for diagnosis and staging of gliomas, and monitoring of treatment response. The utility of these techniques have been found to be highly dependent on tumor grade. According to the World Health Organization (WHO) classification of tumors (Kleihues and Sobin 2000), gliomas are classified into 3 main histological types: astrocytoma, oligodendroglioma and glioblastoma. These histological types are further classified on the basis of anaplasia and degree of malignancy as: grade I, noninvasive glioma (pilocytic astrocytoma); grade II, less-invasive glioma (astrocytomas and oligodendrogliomas); grade III, invasive glioma (analplastic astrocytoma/oligodendrogliomas); and grade IV, highly invasive glioma (glioblastoma, or GBM). Low-grade gliomas (grade I and II) typically affect younger patients. Grade I glioma is the most common form of glioma in children and is less frequent in adults (Burkhard et al. 2003) while grade II gliomas are common in adults (mean age of onset is 40 years) (Hagerstrand et al. 2008). Median survival for low-grade glioma is varied but prognosis and treatment require regular follow ups. Low-grade gliomas grow slowly or stabilize spontaneously and with surgical resection, median survival can be 20 years or more (Burkhard et al. 2003). For high-grade gliomas, the mean age of onset is 40 years for grade III glioma and 61 years for GBM (Ohgaki and Kleihues 2005). GBM is the most malignant and most common glioma, accounting for 45% 50% of all adult gliomas. Median survival for grade III glioma is 2-3 years and for GBM is 1 year (Chen 2007). For optimal disease prognosis, treatment and follow up, one should be able to delineate the tumor lesion and most importantly, differentiate benign lesions from neoplastic lesions, low-grade from high grade tumors, and tumor progression from therapy induced necrosis. As will be discussed later, efforts are also being directed toward defining early imaging predictors of response to therapy. Conventional imaging with magnetic resonance imaging (MRI) provides excellent anatomical definition of brain tumors. MRI is highly sensitive in identifying lesions, mass effect, edema, hemorrhage, necrosis and signs of increased intracranial pressure (Chen 2007). Pathologic changes are characterized on MRI by increased water content (edema) and blood-brain barrier (BBB) disruption, visualized as contrast enhancement (Grosu et al. 2002). Most tumors (low-grade or high-grade) have prolonged T1 and T2 relaxation times and thus

Hepatocyte spheroids as an alternative to single cells for transplantation after ex vivo gene therapy in mice and pig models

Background: Autologous hepatocyte transplantation after ex vivo gene therapy is an alternative to liver transplantation for metabolic liver disease. Here we evaluate ex vivo gene therapy followed by transplantation of single‐cell or spheroid hepatocytes. Methods: Pig and mouse hepatocytes were isolated, labeled with zirconium‐89 and returned to the liver as single cells or spheroids. Biodistribution was evaluated through positron emission tomography–computed tomography. Fumarylacetoacetate hydrolase–deficient pig hepatocytes were isolated and transduced with a lentiviral vector containing the Fah gene. Animals received portal vein infusion of single‐cell or spheroid autologous hepatocytes after ex vivo gene delivery. Portal pressures were measured and ultrasound was used to evaluate for thrombus. Differences in engraftment and expansion of ex vivo corrected single‐cell or spheroid hepatocytes were followed through histologic analysis and animals’ ability to thrive off 2‐(2‐nitro‐4‐trifluoromethylbenzoyl)‐1,3‐cyclohexanedione. Results: Positron emission tomography–computed tomography imaging showed spheroid hepatocytes with increased heterogeneity in biodistribution as compared with single cells, which spread more uniformly throughout the liver. Animals receiving spheroids experienced higher mean changes in portal pressure than animals receiving single cells (P < .01). Additionally, two animals from the spheroid group developed portal vein thrombi that required systemic anticoagulation. Immunohistochemical analysis of spheroid‐ and single‐cell–transplanted animals showed similar engraftment and expansion rates of fumarylacetoacetate hydrolase–positive hepatocytes in the liver, correlating with similar weight stabilization curves. Conclusion: Ex vivo gene correction of autologous hepatocytes in fumarylacetoacetate hydrolase–deficient pigs can be performed using hepatocyte spheroids or single‐cell hepatocytes, with spheroids showing a more heterogeneous distribution within the liver and higher risks for portal vein thrombosis and increased portal pressures.