Small-Animal PET and Biodistribution Studies

Tumor bearing mice (n=5 per treatment arm) received ~300 μCi of ⁸⁹Zr-Tf in 100 μL volume intravenously using a custom mouse tail vein catheter with a 28-gauge needle and a 100–150 mm long polyethylene microtubing (0.28 mm I.D. × 0.64 mm O.D., Scientific Commodities, Inc., Lake Havasu City, AZ). Approximately 32 μg of Tf at a specific activity of 0.4 mCi/nmol was administered per mouse. The mice were imaged on a dedicated small animal PET/CT scanner (Inveon, Siemens Healthcare, Malvern, PA). Mice were imaged at 48 hours post injection. Animals were scanned for 40 minutes for PET, and the CT acquisition was 10 minutes. The coregistration between PET and CT images was obtained using the rigid transformation matrix from the manufacturer-provided scanner calibration procedure since the geometry between PET and CT remained constant for each of PET/CT scans using the combined PET/CT scanner. Animals were anesthetized with gas isoflurane at 2% concentration mixed with medical grade oxygen. PET data were framed dynamically for the first two time points. The durations of the 0.5-h PET data were: 10×10s, 5×40s, 1×300s, and 5×600s. The 3–5 h PET data were also divided to two 1800s frames. The in vivo CT parameters were 120 projections of continuous rotations to cover 220° with an x-ray tube operated at 80 kVp, 0.5 mA, and 175 ms exposure time.

Manufacturer-provided ordered subsets expectation maximization (OS-EM) algorithm was used for PET reconstruction that resulted in 128×128×159 matrices with a voxel size of 0.776×0.776×0.796 mm³. The CT image was created using a conebeam Feldkamp reconstruction algorithm (COBRA) provided by Exxim Computing Corporation (Pleasanton, CA). The matrix size of the reconstructed CT images was 512×512×662 with an isotropic voxel size of 0.191×0.191×0.191 mm³. The photon attenuation correction was performed for PET reconstruction using the coregistered CT-based attenuation map to ensure the quantitative accuracy of the reconstructed PET data.

To evaluate the uptake of ⁸⁹Zr- Tf in human xenografts, biodistribution studies were conducted following imaging. Animals were euthanized by CO₂ asphyxiation after scans were completed. Sixteen tissues, including the tumor, were harvested immediately following sacrifice. The tissues were weighed and counted using a Wizard3 gamma counter (Perkin Elmer) to assess ⁸⁹Zr concentration. Calibration with known amounts of ⁸⁹Zr was performed to determine the amount of activity in each organ. This activity was then decay corrected and the percentage of the injected dose per gram (%ID/g) of tissue was calculated and reported.