Plasmids
Lentiviral constructs were generated using standard molecular biology techniques (Figure 1). The YFP/GLuc fusion protein was cloned into a lentiviral backbone (LV.YFP/GLuc). The pDisplay™ vector (Life Technologies, Invitrogen, Carlsbad, CA, USA), a vector that anchors any protein to the cell membrane, was cloned into this YFP/GLuc lentiviral backbone, yielding the LV.MT-YFP/GLuc vector. A control vector was prepared by cloning the GLuc gene into a lentiviral plasmid containing a red fluorescent fluorochrome (Red/GLuc).
Cell lines
Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's modified Eagle medium (DMEM), 10% fetal bovine serum (FBS) and penicillin-streptomycin and passaged every 3 days. Cells were grown at 37°C and in a 5% CO2 atmosphere. A total of 100,000 cells were transduced with all three of the aforementioned lentiviral vectors, and 2 days upon transduction, cells were sorted for fluorescence expression and subcloned cells were cultured according to the above-mentioned protocol. A GFP and firefly luciferase (FLuc)-expressing SKOV3 cell line was purchased (Bio-Connect®, Huissen, the Netherlands).
Cell line validation in vitro: microscopy and in vitro BLI
Transduction efficiency was scored by fluorescence microscopy. Further localization of the fluorescent signal was obtained by confocal microscopy (Zeiss LSM710 NLO TiSa multiphoton confocal microscope using Zeiss Zen2011 software, Carl Zeiss NV-SA, Zaventem, Belgium).
Luciferase functionality was determined using in vitro bioluminescence imaging (BLI). HEK293T and SKOV3 cells were plated in six-well plates. For GLuc-expressing cells, a coelenterazine solution (50 μl per well of a 1 mg/ml solution) was added to approximately 2E6 cells per well. In parallel, 2E6 FLuc-expressing SKOV3 cells were imaged upon adding 150 μl of d-luciferin (30 mg/ml) per well. Images were obtained with a Photo Imager camera (Biospace, Paris, France) that allows list-mode acquisition.
Xenograft model
All animal experiments were performed with the approval of the ethical committee for animal research of the Vrije Universiteit Brussel. During transplantation, all mice were anaesthetized with a mixture of oxygen and 5% isoflurane and maintained with a mixture of oxygen and 2.5% isoflurane. Twenty immune-deficient athymic (nu/nu) mice were purchased (Charles River, Chatillon-sur-Chalaronne, France). Cells growing exponentially in culture were suspended in 150 μl of phosphate-buffered saline (PBS) and 150 μl Matrigel (BD Biosciences, San Jose, CA, USA) and transplanted subcutaneously in the hind leg. Mice were inoculated with either MT-YFP/GLuc-expressing cells (n = 4), YFP/GLuc-expressing cells (n = 6) or cells that expressed Red/GLuc (n = 4). Also, six mice were transplanted with the SKOV3 cells. All transplants consisted of 2E6 cells.
In vivo BLI
BLI was performed immediately after SPECT/CT while the animals were still anaesthetized. Coelenterazine (1 mg/ml) or d-luciferin (30 mg/ml) was injected intravenously via the tail vein. A solution of 30 μl substrate and 120 μl NaCl was administered. Immediately after substrate injection, mice were imaged using a Photo Imager camera (Biospace, France). Light emission was measured using the large field-of-view setting and registered using the photon counting technology during 1 min. A grey-scale photographic image of the mice was fused with the bioluminescent images. The most intense luciferase signal is shown in red, the weakest signal in blue. To analyse the images, an elliptical region of interest (ROI) was drawn over the transplant location, using a constant surface area.
Nanobody labelling
Nanobodies were labelled with 99mTc at their hexahistidine tail. For the labelling, [99mTc(H2O)3(CO)3]+ was synthesized by adding 1 ml of 99mTcO4− (0.84 to 3.7 GBq) to an Isolink kit (Mallinckrodt Medical BV, Petten, the Netherlands) containing 4.5 mg of sodium boranocarbonate, 2.85 mg of sodium tetraborate, 10 mg H2O, 8.5 mg of sodium tartrate and 7.15 mg of sodium carbonate at pH 10.5. The vial was incubated at 100°C in a boiling bath for 20 min. The freshly prepared [99mTc(H2O)3(CO)3]+ was allowed to cool at room temperature for 5 min and neutralized with 125 μl of 1 M HCl to pH 7 to 8. A total of 500 μl of the tricarbonyl solution was added to 50 μl of carbonate buffer at pH 8. The mixture was incubated for 90 min at 60°C in a water bath. The 99mTc-nanobody solution was purified on a NAP-5 column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) pre-equilibrated with PBS. The labelling efficiency was determined by instant thin layer chromatography.
Pinhole SPECT/micro-CT
One week after transplantation, anaesthesia was induced with isoflurane gas 3% in an air/oxygen mixture. For the induction of anaesthesia, ketamine/xylazine was given intraperitoneally. The animal was placed in supine position during acquisition, and images were acquired 60 min after tracer injection. Micro-CT imaging was followed by pinhole SPECT on separate systems.
Micro-CT was performed using a dual-source CT scanner (Skyscan 1178 micro-CT, Skyscan, Kontich, Belgium) with a 60-kV voltage and 615-mA tube current at a resolution of 83 μm. The total body scan time was 2 min. Images were reconstructed using filtered back projection (NRecon; Skyscan).
The SPECT acquisitions were performed using a dual-headed gamma camera (e.cam, Siemens Medical Solutions, Hoffman Estates, IL, USA) equipped with a triple pinhole collimator. The collimator has a focal length of 250 mm. Sixty-four projections of each 30 s were acquired over 360° of rotation into a 64 × 64 matrix. The total imaging time was approximately 15 min.
To guide image fusion of the SPECT with the micro-CT, two acrylic circular disks containing 3 3.7 MBq 57Co point sources incorporated in organic ion exchange beads of 1-mm diameter (Canberra, Zellik, Belgium) were firmly fixed to the scanner bed. The disks measured 25 mm in diameter and 3 mm in thickness. The six beads provided reference points in both imaging modalities and were used as markers to generate a spatial transformation matrix. The spatial position of the six fiducial markers was determined manually using A Medical Image Data Examiner (AMIDE).
phSPECT images were analysed using AMIDE [[17]]. 3D elliptical regions of interest were drawn around the transplants on SPECT/micro-CT images. Transplant to muscle ratios were calculated as the mean activity within these ROIs divided by the mean activity within ROIs drawn in muscle tissue.
In vitro biodistribution
After imaging, all animals were killed. Blood, liver, kidneys, muscle and transplants were dissected and weighed. The amount of activity was determined using a gamma counter (Canberra, Zellik, Belgium). Tracer uptake was expressed as the percentage injected dose per gram of tissue (%ID/g).
Statistical analysis
Average transplant-to-muscle ratios were compared between groups using the Kruskall-Wallis test. Post-hoc comparisons were made by Mann-Whitney tests using a Bonferroni correction for multiple testing.