2.2.2. GBM-Specific Pharmacokinetics of (*S*)-(−)-[18F]Fluspidine in the Orthotopic U87-MG Mouse Model

Encouraged by the in vitro and in vivo findings on the specific expression of sig1R in orthotopically grown U87-MG tumours and the sig1R-specific binding of (*S*)-(−)-[18F]fluspidine, we proceeded with PET studies with (*S*)-(−)-[18F]fluspidine performed under baseline conditions in nude mice-bearing orthotopic U87-MG (n = 3). Mean injected activity was 9.1 MBq with a molar activity of 92.5 GBq/μmol at the time of injection, resulting in a mean chemical concentration of 4.2 nmol/kg. The tumour growth was assessed by MRI with a T2-weighted sequence, and a 60 min dynamic PET scan, followed by T1 and T2-weighted sequences, performed when the tumour size was 28 <sup>±</sup> 8 mm3 (i.e., 23 to 30 days after implantation). The regions-of-interest (ROIs) were delineated on the T2-weighted MR images and then applied on the PET data to generate the regional TACs.

As reflected by the TACs presented in Figure 4, although not statistically significant, the uptake of (*S*)-(−)-[18F]fluspidine in the tumour is lower and more slowly than the activity uptake in the control region with maximal SUV values of 0.82 at 3 min p.i. and 1.24 at 1 min p.i., respectively. However, because the washout from the tumour was slower, the tumour and CL TACs intersected at about 30 min p.i., demonstrating with SUVs of 0.38 and 0.28 at 60 min p.i. a higher retention of activity in the tumour region compared to the CL, respectively. The retarded washout of the sig1R-specific (*S*)-(−)-[18F]fluspidine from the orthotopic tumour is in accordance with the autoradiographic data, indicating a higher availability of sig1R in the U87-MG tumour tissue in comparison to CL.

**Figure 4.** PET/MR imaging of sig1R in mice with orthotopic xenograft of human GBM cells (U87-MG). Average time-activity curves after i.v. administration of (S)-(−)-[18F]fluspidine of the tumour (red dots) and the contralateral (black squares) regions of interest (*n* = 3). Statistical test: Student *t*-test, \* *p* < 0.05.

The intratumoral heterogeneity of sig1R expression already discovered by the radioligand and antibody investigations in vitro was detectable also by the in vivo imaging study. The early PET images between 2 and 9 min after injection show an heterogeneous uptake of (*S*)-(−)-[18F]fluspidine into the tumour (Figure 5D, upper panel). According to the histological analyses of the explanted tumour tissue, performed immediately after the PET scans, the tumour inner part is characterised by a lower cell density compared to the periphery along with extra-cellular oedema area highlighting presumably areas of necrosis (Figure 5A–C). Therefore, the heterogeneous uptake of (*S*)-(−)-[18F]fluspidine may also (or additionally) be caused by reduced blood supply to the tumour centre. The PET image at later time points (45 to -60 min p.i.; Figure 5D, lower panel) pictures a more homogenous uptake of the tracer, along with a low slope, reflecting an accumulation.

**Figure 5.** Hematoxylin-eosin staining of U87-MG tumour: (**A**) tumour bulk in the right striatum of a mouse brain (×2, scale bar: 1000 μm); (**B**) tumour periphery presents area of high density of cell nuclei; (**C**) tumour centre presents area of lower cell density accompanied by oedema. (×40, BAT: brain adjacent to tumour; T: tumour, E: oedema. Scale bar: 100 μm. (**D**) Representatives coronal PET/MR images of U87-MG tumour-bearing mouse after i.v. administration of (S)-(−)-[18F]fluspidine. The upper panel exhibits the distribution of (*S*)-(−)-[18F]fluspidine at early times p.i. (averaged time frames from 2 to 9 min), and the lower panel exhibits the distribution of (*S*)-(−)-[18F]fluspidine at later times (averaged time frames from 45 to 60 min).
