Thirty-eight male rats (Charles River Laboratories, Sulzfeld, Germany) were included in this study. An overview of the experiments, the rat strains, glioma models, tracers, pharmacological interventions and number of animals in each group is given in Table 1. All animals were handled in accordance with the Animal Research Committee of the Forschungszentrum Jülich GmbH, the German Animal Welfare Act and the European Community Council directives regarding the protection of animals used for experimental and scientific purposes (2010/63/EU), and with approval by the local ethics committee (Landesamt für Natur, Umwelt und Verbraucherschutz, North-Rhine-Westphalia, Germany, Az: 84-02.04.2016.248). All rats weighted between 230 and 330 g and were housed in groups of two under standard conditions. Food and water was provided ad libitum.
Cell culture and tumor inoculation
U87 and 9L tumor cells (U87: ATCC® HTB-14™, LGC Standards GmbH; 9L: ECACC GS-9L, Salisbury, UK) were cultured in Minimum Essential Medium Eagle (MEM) supplemented with 10% fetal calf serum, 1% glutamine, 1% penicillin/streptomycin and 1% non-essential amino acids. F98 tumor cells were cultured similar to U87 and 9L, but in Dulbecco's Modified Eagle's Medium (DMEM) instead of MEM and without non-essential amino acids. After reaching about 95% of confluency, cells were prepared for inoculation into rat brains. Therefore, cells were washed with PBS and detached by incubation with a trypsin/EDTA solution for 5 min. Afterwards cells were resuspended in cell medium generating a cell concentration of 500.000 cells/5 µl for the U87 cells, 65.000/5 µl for the 9L cells and 30.000/5 µl for the F98 cells.
Cell suspensions were then stereotactically inoculated into the left anterior striatum under anesthesia as described previously . The tumor was allowed to grow for 13 days.
Autoradiography and micro-PET
18F-DCFPyL and 68Ga-PSMA were synthesized as described previously . On day 13 post surgery, ex vivo autoradiography (AR) was performed on 38 rats 40 min after i.v. injection of approximately 40 MBq 68Ga-PSMA or 18F-DCFPyL, respectively (Table 1). Five animals for each tracer and each tumor model were additionally investigated by micro-PET using a small animal Siemens INVEON scanner as described previously . The PET measurement was performed during accumulation phase of the tracers from 20-40 min postinjection. For the AR, animals were sacrificed, rat brains removed and immediately frozen in liquid isopentane (-50 C). Every tenth slice of the 20 μm cryosections of the tumor bearing brain and freshly prepared 20 μm 68Ga and 18F standards, to generate a tracer calibration curve, were exposed to an imaging plate (Fuji Imaging Plate, Raytest) overnight , scanned (Fuji BAS Reader 5000, Raytest) and quantitatively evaluated with a pixel size of 25 μm (AIDA Version 4.50, Raytest). Tracer uptake in the tissue was expressed as standardized uptake value (SUV) by dividing the radioactivity (kBq/ml) in the tissue by the radioactivity injected per gram of body weight.
Cryosections of the brains were stained with DAPI (4′,6-diamidino-2-phenylindole) and evaluated by fluorescence microscopy. Disturbance of the BBB was visually evaluated using Evans blue dye (EBD). Therefore, 500 µl/kg 2% EBD was injected i.v. 30 min before decapitation of the rat for AR. EBD extravasation in brain slices was examined by fluorescence microscopy (LMD6000, Leica Microsystems CMS GmbH) and processed by AIDA software (AIDA Version 4.50, Raytest).
Blood vessels were stained with anti-rat von Willebrand factor antibody (ab6994, Abcam). To visualize activated microglia, anti-rat CD11b (Integrin alpha M) antibody (ab133357, Abcam) was used. Reactive astrocytes were visualized by staining with anti-rat GFAP (glial fibrillary acidic protein) antibody (ab53554, Abcam). Immunofluorescence PSMA staining was applied to tumor bearing brain slices using three different anti-PSMA antibodies (NBP1-45057 and NBP1-89822, Novus Biologicals; ab58779, Abcam) to display PSMA expression. To verify the functionality and specificity of the used PSMA antibodies, rat prostate and kidney tissue were used as positive controls. Immunofluorescence stainings were performed according to standard histology protocols and as described before .
Competition tests were performed with four animals to evaluate binding specificity of 68Ga-PSMA (n=2) and 18F-DCFPyL (n=2). As competitor 30 mg/kg BW 2-(Phosphonomethyl)pentane-1,5-dioic acid (PMPA; Tocris) was co-injected i.v. with the tracer as described in the literature . After 40 min of incubation autoradiography was performed as described above.
In order to evaluate the influence of BBB permeability on tracer uptake, four rats with U87 gliomas received glucocorticoid treatment with Dexamethasone (Jenapharm®, Mibe GmbH). The rats received 8 mg/kg intraperitoneally on day 11 post surgery and 4 mg/kg each on days 12 and 13. Autoradiography was performed after injection of 68Ga-PSMA as described above.
The autoradiograms were co-registered to the DAPI stained sections of adjacent slices to compare tracer binding to morphological data as described previously . In short, a circumference region-of-interest (ROI) was drawn along the borders of the coronal brain slices in the DAPI stained sections. After adapting the size of the corresponding autoradiogram to that ROI, tracer binding in the tumor region was evaluated by three different ROIs: 1) along the outer tumor margin reflecting the total tumor volume, 2) in the central part of the tumor and 3) around the peritumoral area. The ROI in the center of the tumor reflects tracer binding in the tumor tissue, excluding the prominent spherical tracer binding in the peritumoral area. The ROI on the peritumoral area contained only the prominent spherical tracer binding, without the binding in the center. Furthermore, a reference ROI was placed in the normal brain tissue and tumor-to-background ratios (TBR) were calculated by dividing the mean SUV of the different tumor ROIs by the mean SUV of the background ROI. Data evaluation included the tumor volume (mm3), SUV in the ROIs and TBRs.
The evaluation of PET data was performed as described previously . Tumor VOI and contralateral background VOI (110 mm³) were placed for each animal in summed PET images from 20 to 40 min p.i.. The tumor VOI on PET scans was determined by a 3D autocontouring process using a cutoff for the TBR that yielded a tumor size similar to that calculated in subsequent autoradiography. Afterwards, SUV and TBR were calculated as described above.
Statistical evaluation was performed using Sigma Plot 12.5 (Systat Software GmbH). Two-Way repeated measures ANOVAs with Holm-Sidak post-hoc test were performed 1) to compare the volume of total tracer binding in the AR to the histological tumor volume derived from the nuclear staining, 2) to compare tracer uptake in the different tumor models for the AR data; and 3) for the comparison of control animals versus Dex treated animals. For analysis of astrocytes, comparison of staining intensity between the two treatment groups was performed by t-test.