Focus 220

Small animal PET imaging was performed when the tumor was visible on 3 consecutive MRI scans on a FOCUS 220 micro-PET scanner (CTI-Siemens, Germany). The rats were anesthetized with isoflurane in O₂/air 3:7 (induction 5%, maintenance 2%), and a catheter for tracer injection was inserted into the lateral tail vein. After fixation in the animal holder, the emission scan started with intravenous injection of 10 to 64 MBq ⁶⁸Ga-FAPI-46 in 500 µL 0.9% NaCl. The acquisition time was 60 min to 120 min. Emission scans were followed by an 8 min transmission scan with a rotating ⁵⁷Co-point source.
Breathing rate and body temperature were monitored and held at approx. 60 breaths/min and 37 °C, respectively. The emission scans were histogrammed into time frames (2 × 1 min, 2 × 2 min, 6 × 4 min, 18 × 5 min for time–activity curves, and 4 × 30 min/4 × 15 min for display) and fully 3D rebinned (span 3, ring difference 47), followed by OSEM3D/MAP reconstruction. The resulting voxel sizes were 0.38 × 0.38 × 0.80 mm³. For all further processing of the images, including statistics, the software VINCI 5.21 for MacOS X (Max Planck Institute for Metabolism Research, Cologne, Germany) was used. Images were intensity-normalized to injected dose and corrected for body weight (SUV_bw). To this end, every frame was divided by injected dose and multiplied by body weight times 100.

PET imaging was performed on the microPET FOCUS220 (Siemens) under standard anesthesia and monitoring procedures [29 (link)]. Data acquisition started with the IV bolus injection of [¹⁸F]UCB-H (32.9 ± 1.0 MBq/kg, 0.35 ± 0.07 µg/kg). Dynamic PET images were reconstructed using standard OSEM-2D algorithms while correcting for radioactive decay, scatter, attenuation and detectors inhomogeneity, which were measured prior to PET scanning using respectively ⁵⁷Co and ⁶⁸Ge external sources.

A total of seven FAZA-PET/CT imaging sessions were performed on day 0, 2, 4, 7, 10, 16, and 21 following initiation of treatment using a triple mouse imaging bed (Fig. 1). [¹⁸F]FAZA was produced by CanProbe (Ontario, Canada) with a radiochemical purity of 95.7 ± 3.7 % (calculated over seven productions). PET imaging (Focus 220, Siemens) was performed at 2-h post-FAZA administration (0.79 ± 0.06 MBq/g of body weight, Additional file 1: Figure S1). Imaging at 2-h post-FAZA injection was reported to be a desirable imaging time point based on tracer kinetics at the tumor site (i.e., reaching steady state) and on the fact that at this time, post-injection, the tumor tracer uptake levels correlated well with tissue hypoxia [5 (link), 30 (link)]. Each PET acquisition consisted of a 20-min emission scan followed by an 8-min ⁵⁷Co transmission scan for attenuation and scatter correction. Then, a CT scan (GE Locus Ultra, 80 kVp, 50 mA) was performed with animals in the same position in order to provide anatomical data for image registration. Treatment response was quantified based on CT tumor volumes.Fig. 1

Illustration of a the triple mouse PET imaging set-up and b the experimental workflow. Day 0 corresponds to the day of treatment initiation. Note that the fur of the mice was colored for identification purposes

PET imaging was conducted as previously reported (Nagai et al., 2020 (link)). Briefly, PET scans were performed using a microPET Focus 220 scanner (Siemens Medical Solutions). Monkeys were immobilized by ketamine (5-10 mg/kg) and xylazine (0.2-0.5 mg/kg) and then maintained under anesthetized condition with isoflurane (1%-3%) during all PET procedures. Transmission scans were performed for ∼20 min with a Ge-68 source. Emission scans were acquired in 3D list mode with an energy window of 350-750 keV after intravenous bolus injection of [¹¹C]DCZ (324.9-382.3 MBq). Emission data acquisition lasted for 90 min. To estimate the specific binding of [¹¹C]DCZ, regional binding potential relative to nondisplaceable radioligand (BP_ND) was calculated by PMOD with an original multilinear reference tissue model (MRTMo) (Yan et al., 2021 (link)). PET scans were conducted at 45 d after injection of vectors for the two monkeys (#229 and #245) and for one monkey expressing hM4Di in the amygdala (#237) between 496 and 984 d, during which enough hM4Di expression for the occupancy study was observed after its introduction.

PET measurements were performed using the microPET Focus 220 small animal PET scanner (Preclinical Solutions, Siemens Healthineers, Knoxville, TN, USA). To track the motion, the MicronTracker Sx60 (ClaroNav Inc., Toronto, Canada) was used, as in [11 (link)]. The MicronTracker (MT) is a stereo-optical system which tracks preregistered planar markers, deriving the 6 degrees-of-freedom (i.e. the marker pose) from two simultaneously acquired but spatially offset images. It was used to track a small marker (2.2×1.7 cm) attached to the rat’s head, at a frequency of 25–30 Hz. The experimental setup is shown in Fig. 1.
Fig. 1

a The MicronTracker in front of the microPET scanner. The large marker attached to the front of the scanner is the reference marker used to aid in transforming from the MT coordinates to the microPET coordinates. b The head marker used in rat studies. c An unrestrained rat with an attached head marker inside a tube within the microPET. The catheter port can be seen between its shoulders. Figure reproduced with permission of IOP Publishing from [14 (link)]. Ⓒ Institute of Physics and Engineering in Medicine. All rights reserved

10 MBq of [¹⁸F]FDG was injected intravenously (iv) into mice for 1 h. Live mice were then scanned using a microPET scanner (Siemens Focus 220) to detect fluorescent uptake. Mice were also scanned with a microCT scanner (GE Locus Ultra) to collect anatomical information to assist with tumor volume quantification. One minute prior to being euthanized, mice were injected iv with 40mgkg⁻¹ of Hoechst 33,258. Tumors were harvested, frozen in liquid nitrogen and sectioned at 8 μm. Tumor perfusion was measured using ImageJ after visualizing the sections with UV illumination. Specifically, the Hoeschst dye was UV activated at 350 nm, then absorbance measured at 460 nm.

PET scan was performed according to our previous reported methods with modifications 17 (link), 18 (link). Briefly, rats were sedated with isoflurane (3%) in a sedation chamber and kept anesthetized with isoflurane (1.5-2.5%). PET images were acquired using the Siemens FOCUS 220 PET scanner (Siemens Preclinical Solutions, Knoxville, TN) with a reconstructed image resolution of ~2 mm. Following a transmission scan, ¹¹C-glyburide was injected intravenously. List-mode data were acquired and dynamic scan data were reconstructed with a filtered back projection algorithm with corrections for attenuation, normalization, scatter and randoms. The left and right brain regions of interest (ROIs) were manually drawn based on the PET image. Regional time-activity curves (TACs) were generated for the left and right brain hemispheres.