*4.14. Animal Injection and Imaging*

Tumors were allowed to grow for about five weeks until they reached 0.5–1.0 cm of size in one dimension [35,36] (i.e., around 50–250 mm<sup>3</sup> volume), and then mice were divided in two groups. The experimental group (*n* = 5) was intravenously injected (retro-orbital venous sinus) [37,38] with 150 µL of a [111]In-IP-001 solution (2.06 nmol, 7.4MBq), while the blocked group (*n* = 5) was injected with 150 µL of a [111]In-IP-001 solution added to a 50-fold molar excess of cold ligand. All injections were performed with the animals under isoflurane anesthesia (1–3%), using 30G ultra-fine insulin syringes. Real-time, live, and fast dynamic screening studies were performed right after injection and on selected time points post-injection, on a dedicated desktop, mouse-sized, planar scintigraphic system (γ-eyeTM by BIOEMTECH, Athens, Greece). The system supports fusion with a digital mouse photograph for anatomical co-registration. The main detector is based on two position-sensitive photomultiplier tubes coupled to a CsI(Na) pixelated scintillator and a medium-energy lead collimator with parallel hexagonal holes that supports a range of SPECT isotopes. The system's field of view is 5 <sup>×</sup> 10 cm<sup>2</sup> , with a spatial resolution of ~2 mm. For the planar imaging, mice were kept under isoflurane anesthesia and under a constant temperature of 37 ◦C, and scans had a duration of 10 min. This allowed for a fast test, right after injection, to check whether the injection was successful, and on later time points to quickly evaluate the bio-kinetics of the tracer. Tomographic SPECT/CT imaging was performed with y-CUBETM and x-CUBETM (Molecubes, Belgium), respectively, at the same selected time points post-injection. The SPECT system provides a spatial resolution of 0.6 mm for mouse imaging and of 1.5 mm for rat imaging. The CT system performs a spiral scan, it can provide images with 100 um resolution, and images were acquired with 50 kVp. Mouse imaging was performed by keeping the mice anaesthetized under isoflurane and under a constant temperature of 37 ◦C. SPECT scans were acquired with a 45 min duration, and each SPECT scan was followed by a high-resolution CT scan for coregistration purposes. The SPECT data were reconstructed through a maximum-likelihood expectation-maximization (MLEM) algorithm, with a 500 µm voxel size and 100 µm iterations. CT data were reconstructed through an iterative image space reconstruction

algorithm (ISRA) algorithm with a 100 µm voxel size. Tomographic images are presented through maximum intensity projection (MIP) view). The experimental group was imaged at 2, 4, 8, and 24 h post-injection with both imaging systems, while the blocked group was imaged at 4 and 24 h.
