Vivoquant software

All animal procedures were approved by the University of Alabama at Birmingham IACUC, animal protocol number IACUC-22618. Male athymic nude mice (Charles River, Wilmington, MA, USA) were allowed to acclimate for at least 72 h before any procedures were performed. For tumor implantation, mice were subcutaneously injected with 300,000 HT29 cells 3 weeks before imaging. For imaging, mice were injected with 1 μg of radiolabeled NOTA-NT-20.3 or 1 μg of radiolabeled NOTA-NT-20.3 and 200 μg non-radiolabeled NOTA-NT-20.3 (n = 4 per group) and imaged at 1 h (gallium-68, copper-64, cobalt-55), 4 h, and 24 h (copper-64 and cobalt-55). Mice were imaged on a GNEXT microPET/CT (Sophie, Springfield, VA, USA). After the last imaging timepoint, mice were euthanized and organs were taken for biodistribution. Organ weights and radioactive counting were collected on a Hidex gamma counter (Lablogic, Clair-Mel City, FL, USA). Images were processed and SUVs were calculated using VivoQuant software (Invicro, Boston, MA, USA).

Preclinical PET/CT images were acquired using a NanoScan^® PET/CT (Mediso Medical Imaging Systems, Budapest, Hungary) scanner with mice under isoflurane (2% in oxygen) anesthesia. The KB tumor xenograft-bearing CD1 nude mice (n=3 for each liposome) each received approximately 5.0 MBq of either ⁸⁹Zr-FA-DFO-liposome or ⁸⁹Zr-DFO-liposome in 100 µL PBS via i.v. injection. PET scanning was performed for 30 min at 6, 24, and 48 h postinjection followed by a CT scan. All PET/CT data were reconstructed with the Monte Carlo-based full-three-dimensional iterative algorithm Tera-Tomo (Mediso Medical Imaging Systems). Raw PET data were reconstructed into 30-min bins using reconstruction settings (four iterations, six subsets, 0.4×0.4×0.4 mm³ voxel size) as well as intercrystal scatter correction. All reconstructed data were analyzed with VivoQuant software (v2.5; inviCRO, LLC, Boston, MA, USA).

Mice were fasted overnight and ¹⁸FDG (Cardinal Health, Omaha, NE) was injected intravenously to fasted mice, and brain glucose uptake was evaluated by PET scan. Briefly, mice were anesthetized by 2% isoflurane along with oxygen. ¹⁸FDG with an activity of 70 µCI in a total volume of 0.1 ml PBS was intravenously injected into the lateral tail vein and allowed for 10 min of uptake. At 30 min post-injection, 10 min PET acquisitions were carried out using Molecube beta-CUBE (MOLECUBES NV, Gent, Belgium). CT scans were acquired using TriFoil imaging Triumph (Tri-foil imaging Northridge, CA). The X-ray tube was used at 150 μA and 75 kV. Each run obtained 512 projections with an exposure time of 230 ms. VIVOQUANT software (inviCRO, Boston, MA) was used to overlay the CT and PET reconstructed images for glucose uptake measurements.

To perform quantitative analysis of the 3D images, the lung region of interest (ROI) was manually selected based on the anatomical structure in each slice of the CT image. The ROIs were applied to the co‐registered MPI‐CT images and the MPI signal intensity of each pixel in the lung ROIs was extracted. We defined the SPIO extravasation index (SEI) as the pixel intensities (PXLI) summed over all the pixels in the lung divided by PXLI summed over the entire body and normalized by the volume of the lung (Vlung, cm³): $$\mathrm{SEI}=\frac{\sum _{\text{lung}}{\text{PXLI}}_{i,j,k}}{\sum _{\text{whole body}}{\text{PXLI}}_{i,j,k}*V_{\text{lung}}},$$ where i,j,k denote the coordinates that identify the location of each pixel. 3D image segmentation and quantification were performed using the CT & MPI Visualization Tools in 3Dmed software. 2D image quantification and processing were performed using VivoQuant™ software (VivoQuant 4.0, Invicro, Boston, MA, USA).

Imaging studies were performed using a small-animal SPECT/CT camera (NanoSPECT/CT^TM, Mediso Medical Imaging Systems, Budapest, Hungary). [¹⁷⁷Lu]folate was injected into the lateral tail vein of tumor-bearing mice (25 MBq, ~1 nmol per mouse). SPECT scans of 38 min duration were performed 4 h and 24 h after injection of the [¹⁷⁷Lu]folate after CT scans of 7.30 min duration. The images were acquired using Nucline Software (version 1.02, Mediso Ltd., Budapest, Hungary). The reconstruction was performed using HiSPECT software, version 1.4.3049 (Scivis GmbH, Göttingen, Germany). Images were analyzed using VivoQuant software (version 3.0, inviCRO Imaging Services and Software, Boston, US). Gauss post-reconstruction filter (FWHM = 1 mm) was applied twice to the SPECT images, and the scale of radioactivity was set as indicated on the images (minimum value = 3 Bq/voxel to maximum value = 30 Bq/voxel).

Animal imaging studies were ethically reviewed and carried out in accordance with the Animals (Scientific Procedures) Act 1986 (ASPA) UK Home Office regulations governing animal experimentation. SPECT/CT imaging was accomplished using a pre-clinical nanoScan SPECT/CT Silver Upgrade instrument (Mediso) calibrated for technetium-99m. All scans were acquired by helical SPECT (4-head scanner with 4 × 9 [1.4 mm] pinhole collimators), and helical CT with 1.4 mm aperture collimators. All acquired images were reconstructed using a full 3D Monte Carlo-based iterative algorithm (Tera-Tomo; Mediso) and further processed and analysed using VivoQuant software (inviCRO, USA).