Concorde microsystems focus 220

3-N-Boc-5′-O-dimethoxytrityl-3′-O-nosyl-thymidine, 2,3′-anhydro-5′-O-benzoyl-2′-deoxythymidine, 3′-deoxy-3′-fluorothymidine, and tetrabutylammonium hydrogen carbonate (TBAHCO₃, 75 mM in ethanol) were purchased from ABX (Advanced Biochemical Compounds, Radeberg, Germany). All trapping and purification cartridges were purchased from Waters (Milford, MA) or Grace Davison Discovery Sciences (Deerfield, OR). Anhydrous acetonitrile, potassium carbonate (K₂CO₃), dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), hydrochloric acid (HCl), and Kryptofix-222 were purchased from Sigma-Aldrich (St. Louis, MO), and absolute ethanol (200 proof) from Pharmaco Aaper (Brookfield, CT). Membrane filters (0.2-μm) were purchased from Pall (Port Washington, NY). Manifold kits for FDG were purchased from Rotem Industries, Inc. (Arava, Israel). All radiosyntheses were performed using either a Bioscan Coincidence GE FDG reaction module (Fairfield, CT) or a GE TRACERlab FX_F-N reaction module. Analytical HPLC was performed using a Hitachi HPLC system (LaChrom Elite) equipped with in-line Hitachi UV (LaChrom Elite model L-2400) and radiometric (Carroll-Ramsey Associates, Berkeley, CA) detectors and a Hitachi LaChrom reversed-phase analytical column (150 mm × 4.6 mm). MicroPET scanning was accomplished using a Concorde Microsystems Focus 220 (Siemens, Berlin, Germany).

Animal handling methods for PET imaging studies were conducted as reported^{5 ,42 (link)}. Prior to imaging, animals were allowed to acclimate to facility environment for at least 1 h in a warmed chamber at 31.5 °C. Animals were administered 10.4–11.8 MBq 4-[¹⁸F]fluoroglutamine via intravenous injection and imaged using a Concorde Microsystems Focus 220 microPET scanner (Siemens Preclinical Solutions). During imaging, animals were maintained under 2% isoflurane anesthesia in oxygen at 2 L/min and kept warm for the duration of the PET scan. PET images in xenograft-bearing mice were acquired as 60-minute dynamic data sets. Imaging was initiated three hours post-treatment following vehicle or V-9302 (75 mg/kg) administration. PET data were reconstructed using a three-dimensional (3D) ordered subset expectation maximization/maximum a posteriori (OSEM3D/MAP) algorithm. The resulting three-dimensional reconstructions had an x-y voxel size of 0.474 mm and inter-slice distance of 0.796 mm. ASIPro software (Siemens Preclinical Solutions) was used to manually draw 3D regions of interest (ROIs) surrounding the entire tumor volume. 4-[¹⁸F]fluoroglutamine uptake was quantified as the percentage of the injected dose per gram of tissue (%ID/g). Significance was calculated using a t-test in Graphpad Prism. Error is reported as standard deviation (SD).