All chemicals were purchased from Sigma-Aldrich (St. Gallen, Switzerland), unless otherwise specified. CLINDE precursor was provided by the Australian Nuclear Science and Technology Organization (ANSTO).
GL26 mouse glioma cells were kindly provided by Prof. L. Zitvogel (Institut G. Roussy, Paris, France). They were routinely cultured in DMEM, supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate (Life technologies, Zug, Switzerland). A stable GL26 cell line expressing enhanced green fluorescent protein (EGFP) was obtained by a lentiviral vector transfection according to the manufacturer instructions (ViraPower Lentiviral Expression System, Life technologies, Zug, Switzerland). Blasticidin (from 2 to 5 μg/ml of culture medium during 2 weeks) was used for the selection of clone of GL26 cells expressing EGFP. GL26-EGFP cells (50.000 cells in 2.5 μl of culture medium/injection) were stereotactically implanted in the striatum of adult male C57/Bl6 mice (Janvier Laboratories, Saint Berthevin, France, n = 10), weighing 28.2 ± 3.2 g, under isoflurane (4% induction and 1.5% to 2% maintenance) anesthesia. Three of these animals received a concurrent second injection of culture medium only in the contralateral striatum. All animal experiments were approved by the Animal Ethics Committee of the Canton of Geneva and were in accordance with the European Union regulations on animal research.
[123I]CLINDE were labeled as previously described . At 4 weeks post-injection, mice were injected with 62.24 ± 18.90 MBq of radiotracer and underwent a dynamic SPECT session with the U-SPECT-II scanner (MILABS, Utrecht, Netherlands), using a list mode acquisition protocol consisting of 60 frames of 84 s. For the displacement study, four out of ten mice were injected with unlabeled CLINDE (10 mg/kg) at the 82th minute post-injection of the labeled tracer. Total scan duration was 165 min.
SPECT tomograms were reconstructed with an ordered subsets expectation maximization (OSEM) algorithm (using four subsets and six iterations). SPECT data were corrected for radioactive decay, but no correction of attenuation and scatter was applied. A 0.4-mm FWHM filter was applied on images. The dynamic SPECT images were first averaged over time frames between 50 and 80 min in order to enhance visualization of the different structures. These images were manually co-registered to a mouse magnetic resonance imaging (MRI) and volume of interest (VOI) template  that was implemented in PMOD software (version 3.6, 2014, PMOD Technologies Ltd, Zurich, Switzerland), and the co-registration parameters were employed for dynamic image co-registration. Standardized uptake values (SUV) of tumor and contralateral striatum radioactivity were extracted from averaged frames between 50 and 80 min, using PMOD and compared by means of t-test (Microsoft Excel, 14.4.7).
Mice were sacrificed after the end of the scan session at 4 weeks post-implantation of GL26 cells. Brains were harvested and frozen in isopentane (−20°C) and serial coronal sections were taken. Brain sections were used for ex vivo autoradiography, fluorescence microscopy, Nissl staining, immunohistochemistry, and in situ hybridization. Two digoxigenin-labeled riboprobes complementary to the Mus musculus translocator protein (TSPO) mRNA sequences (NM-00977.4: coding sequences 102-380 and 322-577, respectively) were used. The cDNA amplicons from total RNA extracted from a mouse brain cortex were inserted in a TOPO-pCR4 vector (Invitrogen, Carlsbad, CA, USA) followed by linearization and in vitro transcription in presence of dUTP-digoxigenin (Roche, Basel, Switzerland) according to the manufacturer instructions (Invitrogen, Carlsbad, CA, USA). The in situ hybridization histochemistry procedures were previously reported in detail . The use of sense probes, in serial tissue sections, resulted in the absence of any hybridizing signal whereas the two antisense independent probes mentioned above gave the same patterns of labeling. Thus, these data were in favor of the specificity of the hybridizing signal for TSPO mRNA visualized in the tumor tissue sections. TSPO-immunoreactivity expressed by glioma cells was further suggested by immunohistochemistry and the TSPO-immunoreactive band seen in a Western blot using an anti-TSPO antibody (goat anti-mouse TSPO antibody LS-B5755, LifeSpan Biosciences, Inc., Seattle, WA, USA; working dilution 1:1,000). The Western blotting procedure was published with detail elsewhere .