Chemicals
All reagents were obtained from Sigma-Aldrich (Buchs, Switzerland) unless specified.
Radiochemistry
[18F]FPyPEGCBT-c(RGDfK) was prepared via the reported method (Inkster et al. 2015). In summary, 18F prosthetic group [18F]FPyPEGCBT was prepared by way of nucleophilic aromatic [18F]fluorination of its 2-trimethylammonium pyridine precursor and partially purified by solid-phase extraction on molecularly imprinted polymer sorbent (AffiniMIP®). [18F]FPyPEGCBT was then coupled to cysteine-modified c(RGDfK) in DMF over 30 minutes at 43 °C. [18F]FPyPEGCBT-c(RGDfK) was purified by High Performance Liquid Chromatography (HPLC) and formulated in 10 % EtOH in isotonic saline for preclinical use. The entire synthesis was completed on a single automated synthesis unit (GE Healthcare, Ecublens, Switzerland) over 124–132 min.
Cell lines
The U-87 MG glioblastoma and SKOV-3 ovarian cancer cell lines (ECACC, Salisbury, UK) were cultured at 37 °C and 5 % CO2 in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Zug, Switzerland) supplemented with 10 % (v/v) fetal calf serum (Life Technologies).
siRNA transfection
siRNAs targeting human αV, β1, β3 or β5 integrin subunits were synthesised by MWG (Ebersberg, Germany). siRNA sequences used were: for αV, sense 5′-UCCAUUCAUGUACUUUUCC-3′; β1, sense 5′-AUGUAACCAACCGUAGCA-3′; β3, sense 5′-CAAGCCUGUGUCACCAUAC-3′; β5, sense 5′-GCUCGCAGGUCUCAACAUA-3′. An siRNA targeting luciferase was used as a non-specific control, sense 5′-CGUACGCGGAAUACUUCGA-3′. 48 h prior to an experiment, U-87 MG (4 × 104 cells per well) and SKOV-3 (1 × 104 cells per well) cells were reverse transfected in 96-well plates with a final concentration of 50 nM of indicated siRNAs using RNAiMAX (Life Technologies) according to the manufacturer’s protocol.
Immunoblotting
Protein extracts were prepared in a RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1 % SDS, 0.5 % Na deoxycholate, 1 % Igepal CA630, 2 mM EDTA, 50 mM NaF, pH 8) containing a cocktail of protease inhibitors (Roche, Rotkreuz, Switzerland) and titrated using the DC Protein Assay (Biorad, Cressier, Switzerland). Five to 10 μg of protein extracts were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Amersham, GE Healthcare, Glattbrugg, Switzerland). Membranes were blocked for 1 h at room temperature, incubated overnight at 4 °C with primary antibodies, incubated for 1 h with secondary antibodies, developed using standard ECL protocol and analysed using a Chemidoc MP (Biorad). The primary antibodies raised against αV (611012), β1 (610467) and β3 (611140) integrins were from BD (Allschwil, Switzerland), against β5 integrin (3629S) from Cell Signalling (Berverly, MA, USA) and Actin (A2066) from Sigma-Aldrich. HRP-conjugated secondary antibodies were obtained from Biorad.
Cell binding assay
In vitro integrin-binding of FPyPEGCBT-c(RGDfK) was evaluated using a standard displacement assay of [125I]echistatin. U-87 MG and SKOV-3 cells were seeded in 96-well plates 24 h prior to the experiment (U-87 MG, 6 × 104 cells per well; SKOV-3, 3 × 104 cells per well). On the day of the experiment, the cells were washed twice with binding buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.1 % BSA), then co-incubated for 1 h at 37 °C in binding buffer with 370 Bq/well [125I]echistatin (Perkin Elmer, Schwerzenbach, Switzerland) and a concentration range (10−9 to 10−4 M) of FPyPEGCBT-c(RGDfK) or c(RGDfV) (PeptaNova, Sandhausen, Germany) as a reference peptide. Cells were then washed three times with binding buffer and lysed in 1 M NaOH. The cell-associated radioactivity was measured in a Wizard2 2470 γ-counter (Perkin Elmer). The IC50 (Inhibitory Concentration of 50 %) values were calculated by fitting the data by nonlinear regression using GraphPad Prism (GraphPad software, La Jolla, CA, USA).
Cell uptake assay
U-87 MG (6 × 104 cells per well) and SKOV-3 (3 × 104 cells per well) cells were seeded in 96-well plates 24 h prior to the experiment. On the day of the experiment, cells were washed twice with binding buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.1 % BSA) then incubated for 15 to 120 min with 100 kBq/mL [18F]FPyPEGCBT-c(RGDfK) in binding buffer at 37 or 4 °C. Cells were also co-treated with 10−4 M c(RGDfV) for 1 h. [18F]FPyPEGCBT-c(RGDfK) uptake in cells with siRNA-mediated depletions of specific integrin subunits was also assessed. After indicated uptake times, cells were washed three times with binding buffer and lysed in 1 M NaOH. The cell-associated radioactivity was measured in a Wizard2 2470 γ-counter (Perkin Elmer).
Animal models
All animal experiments were performed in compliance with the current Swiss animal protecting laws and protocols of the local authorities (approval G36/3528). Six to eight weeks old female BALB/c nude mice (Harlan, Horst, The Netherlands) were injected subcutaneously into the right flank with 1 × 106 U-87 MG or SKOV-3 cancer cells in 200 μL of a matrix containing 30 % of DMEM and 70 % of HyStem-C hydrogel (ESI BIO, Alameda, CA, USA). After two weeks, when tumour volumes were approximately 500 mm3, the animals were used for microPET/CT and/or biodistribution studies.
Biodistribution study
Mice were anesthetised with 2 % isoflurane and were injected retro-orbitally in the venous sinus with 3–5 MBq of [18F]FPyPEGCBT-c(RGDfK). Mice were then left awake during the uptake times of 30, 60 or 120 min. For the blocking experiments, mice were injected intravenously with 20 mg/kg of c(RGDfV) 5 min prior to the tracer. Mice were then anesthetised, blood was collected by intracardiac puncture and the animals were sacrificed. Indicated organs and tumours were collected, weighed, and the radioactivity quantified in a Wizard2 2470 γ-counter. Data were expressed as percentages of the injected dose per gram (%ID/g).
MicroPET/CT imaging
Injections of [18F]FPyPEGCBT-c(RGDfK) to anesthetised mice were conducted as described above. Mice were injected intraperitoneally with 700 μL of 132 mg/mL meglumine ioxitalamate (Telebrix, 6 % m/v iodide, Guerbet AG, Zürich, Switzerland) to allow abdominal organs discrimination and subjected to CT scans in a Triumph microPET/SPECT/CT system (Trifoil, Chatsworth, CA, USA). Images were obtained at 80 kVp, 160 μA, and 1024 projections were acquired during the 360° rotation with a field of view of 71.3 mm (1.7× magnification). After 20 min of uptake, PET scans were started for a total duration of 110 min allowing the reconstructions of PET frames of 30 ± 10 min, 60 ± 10 min and 120 ± 10 min of uptake. PET scans were reconstructed with the LabPET software using an OSEM3D (20 iterations) algorithm and images were calibrated in Bq/mL by scanning a phantom cylinder. The Triumph XO software, which uses a back-projection engine, was used to reconstruct the CT scans with a reconstruction matrix of 512 and a voxel size of 0.135 mm. Reconstructed CTs were then co-registered with the PET scans using the plugin Vivid (Trifoil) for Amira (FEI, Hillsboro, OR, USA) and exported as dicom files for mice anatomy visualisation. The software Osirix (Pixmeo, Geneva, Switzerland) was used to quantitatively analyse the datasets and generate pictures. PET series were converted to display Standardised Uptake Values (SUV) adjusted to the body weight of the animals and merged with CT sets. Regions of interest (ROI) were drawn on contiguous slides according to CT scans. ROIs were subsequently computed to 3D volumes allowing the quantification of the [18F]FPyPEGCBT-c(RGDfK) uptake in the different organs.
Metabolic assay
Fractions of blood collected during biodistribution studies were used to assess metabolic stability of [18F]FPyPEGCBT-c(RGDfK). Metabolic activity was quenched (v/v) with a solution of 20 % MeCN in PBS (100 mM, pH 7.4). Blood samples were centrifuged at 4 °C (2000 × g, 8 min) and the serum was removed. Serum proteins were precipitated with MeCN (1:1) and centrifuged at 4 °C again (21380 × g, 8 min). Supernatants were removed from pellets, and the activity associated with both was measured. In this fashion, the extraction efficiency of this step was estimated to be 91.0 % ± 4.8 (n = 18). For each sample, an aliquot of supernatant (2 μL) was spotted onto silica gel plates and eluted with 4:6 MeOH-10 % (w/w) aqueous ammonium acetate. Phosphor imaging screens were exposed to dried TLC plates for 10–25 min and analysed by autoradiography using a Cyclone plus and the built-in Optiquant software (Perkin Elmer).
Figures and statistics
Statistical analyses and regressions were conducted using Graphpad prism. Differences between samples were assessed using one-way ANOVA statistical test. Figures were generated using Microsoft Excel, Graphpad Prism, Adobe Photoshop and Adobe Illustrator.