Inveon software

Titration of dose and longitudinal imaging was performed in CT26 tumor-bearing mice (150-200 mm³) and was only performed for ⁸⁹Zr-DFO-CD8a. Mice were injected with ⁸⁹Zr-DFO-CD8a (1.34 ± 0.1 MBq) without or with 5, 10, 30 or 100 µg unlabelled CD8a-F(ab)'2 intravenously (total volume ~ 200 µL in PBS, N=3/group). Small animal PET/CT imaging was performed 1, 4, 24 and 72 hours after injection on an Inveon Multimodality PET/CT scanner (Siemens, Germany). Mice were anesthetized with sevoflurane (3-4% in 80% N₂, 20% O₂) during PET/CT imaging. Static PET data were acquired in list mode with an acquisition time of 300, 300, 600 and 900 seconds for the 1, 4, 24 and 72 time-point, respectively. Images were reconstructed using a 3D maximum a posteriori algorithm with CT based attenuation correction. Image analysis (Inveon Software, Siemens) was performed by drawing CT based regions of interest (ROIs) over the tumor, whole heart, liver, kidney, muscle, inguinal lymph node (ILN), axillary lymph node (ALN) and cervical lymph node (CLN). ROIs over the spleen were drawn by PET based thresholding. The uptake of ⁸⁹Zr-DFO-CD8a was quantified as % injected dose per gram tissue (%ID/g) assuming a soft tissue density of 1 g/cm³. Blood was withdrawn by cardiac puncture and mice were euthanized after the imaging session, organs resected, weighted and the radioactivity counted in a gamma counter.

PET and CT images were analysed as fused images using the Inveon software (Siemens). Regions of interest (ROIs) were drawn around liver, kidney, spleen, muscle, tumors and the left-ventricle. Uptake in the left ventricle of the heart was taking as a measure of the blood concentration. Percentage of injected dose per gram (%ID/g) was calculated.

The mice were intraperitoneally anesthetized with 1 mL/kg zoletil:rompun solution (4:1, voleme/volume) at 0, 4, and 8 weeks. Hindlimb muscle of the mice was obtained by Micro-CT analysis with a single photon emission tomography system at the research center of Chemon Inc. (Gyeonggi-do, Korea). Total hindlimb muscle volumes were obtained using Siemens Inveon software (Inveon, Washington, DC, USA).

Experiments of micro-computed tomography (micro-CT) were performed with an animal positron emission tomography (PET)/CT/single photon emission computed tomography (SPECT) system (Inveon, Siemens, USA) prior to the sacrifice of animals under 1.5–2% isoflurane in O₂ anesthesia. Computed tomography pictures were further analyzed using Siemens Inveon software to calculate the three-dimensional volume of the fat mass between lumbar vertebrae one to five.

MRI experiments were performed on a Bruker Biospec 7.0 (Bruker Biospin, Ettlingen, Germany). Mice were anaesthetized using 2% isoflurane and a water heating system, combined with a rectal thermometer, was used in order to maintain body temperature at 36.5–37.5°C. A 30-mm surface coil was fixed covering the skull of the mouse. A TurboRare T2w protocol was used for generation of transverse and coronal images. 8 transverse slices and 12 coronal slices with a thickness of 0.5 mm were acquired using a repetition time (TR) of 2500 ms and an echo time (TE) of 33 ms. The total scan time was 5 minutes and 20 seconds for each orientation. A field of view of 20×20 mm was chosen and sampled into a matrix size of 256×256 mm resulting in a spatial resolution of 0.078. The MRI images were then transferred in DICOM format into the Inveon software (Siemens Medical Solutions) for image analysis. ROIs covering the total tumour area were manually drawn on each slice and a tumour volume was obtained by interpolating the ROIs from all transverse images and the coronal images, respectively. The total tumour volume was calculated as the mean of the tumour volume in the transverse and coronal images.

The optimal imaging time-point was assessed by longitudinal small animal PET/CT imaging in HCC827 tumour-bearing mice (N = 8). Mice were injected intravenously immediately after EOS through the tail vein with 1.89 ± 0.02 (range 1.78–2.01) MBq site-specifically labelled ⁸⁹Zr-DFO-6E11 and 30 μg of unlabelled 6E11. Mice were anesthetized with sevoflurane (4% in 65% N₂, 35% O₂) and subjected to PET/CT imaging on an Inveon Multimodality PET/CT scanner (Siemens) 4, 24, 72, and 144 h post-injection (300, 300, 600, and 1200 s PET acquisition time, respectively).
Following optimization of imaging time-point, specificity of ⁸⁹Zr-DFO-6E11 was evaluated in HCC827, H1993, and H1703 xenograft mouse models (N = 7/model) as well as CT26 and B16F10 syngeneic mouse models (N = 6/model). Mice were injected intravenously immediately after EOS with 1.2 ± 0.09 (range 0.65–2.82) MBq. PET/CT imaging was conducted 72 h post-injection (600 s PET acquisition time).
All images were reconstructed using a 3D maximum a posteriori algorithm with CT-based attenuation correction. Image analysis (Inveon Software, Siemens) was performed by drawing CT-based region of interests (ROIs). The uptake of ⁸⁹Zr-DFO-6E11 was quantified as percent injected dose per gram tissue (%ID/g) assuming a tissue density of 1 g/cm³.