Preclinical Positron Emission Tomography with Body Conforming Animal Molds for Cloud-Based Automated Image Analysis in Mice

All procedures performed on animals followed regulations and established guidelines and were reviewed and approved by Pfizer's Institutional Animal Care and Use Committee or through an ethical review process. PET imaging study was performed following all institutional radiation safety protocols. Female C57BL/6 mice, 10-12 weeks old, with body weights ranging from 18-26 g, were used in this study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Prepping and staging of animals

NOTE: This step involves staging the animals for PET imaging with BCAM template-guided tumor implantation.

1. Prior to cell implantation, shave the fur of the mice in the desired region of tumor growth.
2. Set up the anesthesia station and attach a BCAM mold of the appropriate size to the pre-clip with the top flipped up 90 degrees. This will serve as the tumor implantation base (Figure 1A).
3. Anesthetize animals in a suitable induction box with 2.0% isoflurane and 98% oxygen at a flow rate of 0.5-1.5 L/min.
   NOTE: All anesthesia systems are variable.
   1. Use a calibrated system and test before use. Place an anesthetized mouse on the base and extend its limbs (Figure 1B).
4. Gently place a BCAM tumor injection template mold (Figure 1C) on the dorsal side of the mouse (Figure 1D).
5. Carefully create a temporary circular reference mark at the desired cut-out location (e.g., shoulder or flank, left or right) with a marker (Figure 1D). Remove the template mold.
6. Prepare the cells for injection by drawing up the desired volume into a syringe. Attach a 27 G needle to the syringe and fill the dead volume space. A volume of 0.1-0.2 mL per inoculation is recommended.
7. Lift skin with fingers or forceps using the temporary marks from step 1.5 as a reference to create a tent, slowly insert the needle, and inject cells subcutaneously in the desired location. Discard the needle in a biohazard sharps container.
8. Monitor the tumor growth. The average tumor volume at enrollment in this example was 200-300 mm³.
   NOTE: Avoid using metal components such as ear tags or microchips to prevent CT imaging artifacts.

2. Drawing radioactive doses

1. Following all institutional radiation safety protocols, carefully draw up a target dose of [¹⁸F]FDG in the range of approximately 70-80 µCi into a sterile syringe suitable for intravenous injection.
   NOTE: The 70-80 µCi range is optimal to fit the dynamic range of the Sofie G8 PET scanner. Other imagers may have different dynamic ranges that require a different target dose.
2. Carefully measure the radioactive dose in a dose calibrator by placing the syringe into the well chamber and lowering the dipper. Pay attention to selecting the appropriate isotope or calibration number prior to measurement.
3. Take a stable reading. Record date and time. Adjust the dose if needed.
4. (Optional) Use sterile saline to bring the final dose volume to 0.1-0.2 mL.
   NOTE: The desired injection volume may vary.
5. Dose is ready to be administered.

3. Intravenous dose administration

1. Following institutional IACUC and Radiation safety protocols, prepare the mouse tail for IV injection by dilating the tail vein. In this study, the tail vein was dilated by placing the animal cage or induction chamber on a slide warmer set at 42 °C.
   NOTE: It may take up to 10 min of warming for the tail vein to fully dilate.
2. Before injection, record mouse weight. This will determine which weight-specific BCAM to use for PET/CT imaging.
3. Once ready, transfer the mouse into a suitable restrainer (e.g., a Tail vein injection platform was used in the present study) and locate the tail vein.
4. Carefully handle the radioactive syringe and IV inject the dose into a conscious mouse. Record the injection time.
5. Carefully set the syringe aside and clean excess blood drops off the tail with gauze. Remove the mouse from the restrainer and place it back in the appropriate housing.
6. Carefully place the syringe back in the dose calibrator chamber and record any residual readings for proper dose calculations. Record the date and time of the residual activity reading.
7. Once the injections are complete, take caution and place cohorts of mice behind lead enclosures and/or away from personnel, following institution-safe radiation practices. Note all materials used are subject to minor radioactive contamination. Monitor all areas with a calibrated Geiger counter and clean per radiation safety guidelines.

4. Anesthetizing and positioning the animals for PET imaging

1. At an appropriate time (e.g., 10-15 min before the end of the tracer uptake period), turn on all anesthesia equipment and animal warming instruments required for both anesthesia induction and PET imaging. Anesthesia can be set at 2.0%-2.5% isoflurane in 98% oxygen (settings may vary with mouse strain and disease model).
2. Confirm all adapters and valves are switched on and the gas flow rate is 0.5-1.0 L/min. Specific flow rates may vary among anesthesia systems and imaging systems.
3. Place appropriate animal(s) in an induction box and wait until the subject reaches the correct depth of anesthesia.
4. Apply eye lubricant ointment to each eye.
5. Transfer mouse into an appropriately sized BCAM based on body weight.
6. Gently pick up the mouse by the nape of the neck, insert the tail into the tail slot, and wrap it under the BCAM. Place the tail on the tail platform and tape to secure the position. Check that the spine is straight. Gently close the BCAM (Figure 2A,B).
7. Carefully position all four limbs to rest on each of the BCAM paw platforms. Use tape as needed to secure paws to each platform (Figure 2B).
8. Gently snap the BCAM into the imaging bed shuttle by inserting the front end first (Figure 2C). The shuttle should already be connected to the G8 loading dock with anesthesia flowing (Figure 2D). Push down on the back of the BCAM, and a click will indicate that it is secured to the G8 shuttle.

5. Image acquisition

1. Open the G8 PET acquisition software. Study folders, scan parameters, and subject IDs can be created beforehand.
   1. Enter study details into the software: mouse weight, date/time and amount of the initial dose drawn, date/time of injection, and date/time and amount of residual activity reading.
2. Insert the shuttle with the mouse into the scanner opening. A blue indicator light will indicate proper docking to the system.
3. Select the appropriate PET/CT parameters. Acquire imaging data.
   NOTE: In this study, a 7-min static ¹⁸F PET scan was run, followed by a 70-s default CT scan, and a maximum-likelihood expectation-maximization 3D reconstruction algorithm (default and only option in the G8 scanner) was applied to reconstruct the data¹².
4. Upon acquisition completion, the shuttle will return to its initial starting position. Remove the shuttle with the mouse and insert it into the dock. Press on the back of the BCAM vertical tab to remove it from the G8 shuttle, and gently pull up and remove the BCAM with the mouse.
5. Press on the two retaining tabs to flip up and open the top of the BCAM.
6. Gently remove the mouse. Ensure the mouse has fully recovered from anesthesia and is bright, alert, and responsive (BAR) before returning to its home cage.
7. Continue the procedure at set time intervals to scan the entire cohort/group of mice.

6. Uploading images to the SaaS

1. Login to app.invivo.ax and create a project.
2. Click on the red upload tab at the top right of the window. Select an imaging system and reporter used in the imaging study (i.e., [¹⁸F]FDG). Select the folder with imaging data.
3. Navigate to the annotation tab within the project folder. Click on Annotate to add relevant scan information (subject name, sex, BCAM size, subject weight, cohort name, timepoint, and injected dose value). Most metadata will auto-populate.

7. Automated data analysis

1. Within the project folder, all scans should populate. Click on scans to navigate from project, group, and individual subject levels. Select Analyze at the top right corner.
2. At the top right of the analysis ribbon window, under Project, select the plus symbol and select Organ Probability Map ROI. The Organ Probability Map (OPM) is a statistic mouse organ atlas encompassing multiple pre-defined organs and tissues. Select individual organs of interest based on research needs.
   NOTE: In this example, manual segmentation and analysis were done using VivoQuant, using the spline tool, followed by interpolation within the 3D ROI operator. Automated analysis results were compared to the manual segmentation results as a benchmark.