¹⁸F-5-Fluoroaminosuberic Acid as a Potential Tracer to Gauge Oxidative Stress in Breast Cancer Models

Radiosynthesis of ¹⁸F-FASu

Radiosyntheses were performed on a TRACERlab FXFN module (GE Healthcare) in a lead-shielded hot cell using our reported method (17). Reverse-phase high-performance liquid (HPLC) chromatography was carried out on an Agilent 1200 series system equipped with a diode array detector and a Raytest GABI Star scintillation detector using a Phenomenex Monolithic C₁₈ 4.6 × 100 mm column. Chemicals and reagents were purchased from Sigma Aldrich, Acros Organics, and Fisher Scientific. Radiofluorination ¹⁸F⁻ was produced in a TR13 cyclotron from bombardment of H₂¹⁸O at 20 μA. The produced H₂¹⁸O/¹⁸F⁻ was delivered to an FXFN automated synthesis module, where the ¹⁸F⁻ was trapped on a QMA resin. The resin was purged with helium, and the fluoride was subsequently released using a mixture of Kryptofix[2.2.2] (10.1 mg in 200 μL of acetonitrile) and K₂CO₃ (2.1 mg in 200 μL of water). The solvent was removed by the repeated volatilization of acetonitrile (1.5 mL) under reduced pressure. The precursor (di-tert-butyl 2-((bis-tert-butoxycarbonyl)amino)-5-(tosyloxy)octanedioate, compound 1, Fig. 1), 1.4 mg, was previously dried in vacuo and dissolved in 500 μL of anhydrous dimethyl sulfoxide in preparation for labeling. The precursor was added to Kryptofix[2.2.2]/¹⁸F⁻, and the reaction was heated at 95°C for 20 min, after which the reaction mixture was cooled to 35°C, diluted by 7 mL of H₂O, and then loaded onto a Waters tC18 Plus Sep-Pak. The Sep-Pak was washed with 17 mL of distilled, deionized H₂O, and di-tert-butyl 2-((bis-tert-butoxycarbonyl)amino)-5-fluorooctanedioate (compound 2, Fig. 1) was eluted using 1.5 mL of acetonitrile. In a separate apparatus, the solvent was removed under the flow of N₂ at 95°C. Trifluoroacetic acid (500 μL) and anisole (8 μL) were added, and the mixture was heated at 95°C for 7 min. After trifluoroacetic acid was removed in vacuo, the residue was dissolved in 0.6 mL of phosphate-buffered saline and then passed through a Waters tC18 light Sep-Pak. The Sep-Pak was washed with an additional 0.6 mL of phosphate-buffered saline. The combined phosphate-buffered saline solution that contained ¹⁸F-FASu was filtered through a 0.22-μm membrane to generate the final product. Decay-corrected radiochemical yield was 18% ± 6% (n = 6), radiochemical purity was more than 98%, and specific activity was 17.5 ± 7 GBq/mmol (n = 6). The identities of compound 2 and ¹⁸F-FASu were confirmed by HPLC comparison with the standards.

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FIGURE 1.

Radiosynthesis of ¹⁸F-FASu. DMSO = dimethyl sulfoxide; TFA = trifluoroacetic acid.

Quality control of the radiosynthesis was performed by ultraviolet and radio-HPLC. The chromatograms are shown in Supplemental Figure 1. The retention time difference between ¹⁸F-FASu and FASu was 0.27 min, in line with the delay between the ultraviolet detector and the γ-detector.

The HPLC method was as follows: Phenomenex Monolithic C18 column, 100 mm × 4.6 mm, 1 mL/min; A: 0.1% trifluoroacetic acid in H₂O, B: 0.1% trifluoroacetic acid in acetonitrile; gradient flow from 100% A at 0 min to 20% A and 80% B in 15 min.

In Vivo and In Vitro Stability Studies

For the serum stability study, 0.74 MBq of ¹⁸F-FASu in 10 μL of phosphate-buffered saline was incubated with 100 μL of Rag2M plasma for 5, 15, 30, 45, 60, 90, and 120 min. After each incubation, the sample was centrifuged through a membrane with a molecular weight cutoff of 10 kDa (Microcon, 145,000 rpm; Millipore) and analyzed by HPLC. No degradation was observed until 60 min. At 90 min, a small shoulder peak (20%) appeared. At 120 min, the same shoulder peak was observed at 27% compound to primary product peak.

For in vivo stability studies, urine and blood samples were collected from two Rag2M mice at 10 and 60 min after injection. The blood samples were centrifuged to collect the serum. Both serum and urine were passed through a 10-kDa membrane to remove protein, followed by HPLC analysis. No metabolites were observed for urine samples at 10 or 60 min. For the blood samples, no degradation was present at 10 min, and 15.5% ± 0.5% degradation was observed at 60 min.

Cell Culture and Uptake Experiments

The HEK-293T cell line was purchased from Clontech Laboratories. The ZR-75-1 cell line was purchased from ATCC. The MCF-7 cell line was obtained as a gift from Dr. C. Kent Osborne (Baylor College of Medicine) and authenticated by DDC Medical. The MDA-MD-231 cell line was obtained as a gift from Dr. Connie Eaves and authenticated by DDC Medical.

The MDA-MB-231 cell line was cultured in complete Dulbecco modified Eagle medium (with 4,500 mg/L glucose + 10% fetal bovine serum + 1% penicillin/streptomycin). ZR-75-1 was cultured in complete RPMI-1640 (with 10% fetal bovine serum + 1% penicillin/streptomycin). MCF-7 was cultured in complete Dulbecco modified Eagle medium with 1% minimum-essential-medium nonessential amino acid solution. Cells were maintained in 10-cm tissue culture dishes in a humidified incubator at 37°C with 5% CO₂ and routinely subcultured at approximately 95% confluency.

For uptake studies, cells were seeded into 24-well plates (1 × 10⁵ cells/mL) such that confluency was achieved the next day. Each plate was washed 3 times with cold HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)–Hanks balanced salt solution, and then 0.148 MBq of ¹⁸F-FASu in 400 μL of HEPES–Hanks balanced salt solution was added. Cell numbers were established from a single well using a Moxi Z Mini Automated Cell Counter (Orflo Technologies). Subsequently, the samples were incubated at 37°C with orbital shaking for the indicated duration. After incubation, supernatants were removed, cells were washed twice with cold HEPES–Hanks balanced salt solution, 400 μL of 1 M NaOH were added to the cells, the NaOH lysate was collected 10 min later, and 25 μL of the lysate was taken to assess the protein concentration using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The activity in the solution and lysate was measured using a PerkinElmer Wizard 2480 γ-counter and normalized to the protein concentration.

For diethyl maleate (DEM)–induced OS experiments, DEM was added at various concentrations approximately 24 h before harvesting. The uptake study was conducted using the method described above.

For amino acid transporter inhibition experiments, all chemicals were purchased from Sigma-Aldrich and used without further modification. l-Glu, d-Asp, 2-amino-2-norbornanecarboxylic acid (BCH), l-Leu, and l-Ser were used at a 2 mM concentration, and sulfasalazine was used at a 1.0 mM concentration. The inhibitors were added to MDA-MB-231 cells and incubated with ¹⁸F-FASu at 37°C with 5% CO₂ for 60 min.

Small Interfering RNA (siRNA) Knockdown Experiments

MDA-MB-231 cells were seeded at 1 × 10⁵ cells/mL in a 24-well plate with complete Dulbecco modified Eagle medium 24 h before transfection with siRNA (xCT siRNA and nontargeting control siRNA, both from Santa Cruz Biotechnologies). Cells were transfected at 80% confluency with a mixture containing 25 nM siRNA, 1.5 μL of TransitIT siQuest transfection reagent (Mirus), and Opti-MEM reduced serum medium (Life Technologies) prepared and added according to the manufacturers’ directions. The cells were left to incubate at 37°C with 5% CO₂ for 24 h. Then, 0.1 mM DEM was added to the cells and incubated an additional 24 h, followed by a tracer uptake study using the method described above.

Glutathione Quantification

Glutathione content was determined, in triplicate, using the Thiol Detection Assay Kit from Cayman Chemical per the manufacturer’s instructions. The fluorescent signal was recorded using a FlexStation 3 spectrophotometer (Molecular Devices). The glutathione content in each sample was estimated from the standard curve and normalized to protein content.

HEK::xCT Cell Line

The lentiviral plasmid Lv205 (pReceiver-ORF-IRES-eGFP-IRES-Puro) containing the open reading frame of human SLC7A11 (GeneCopoeia) was used to generate virus particles using a second-generation packaging system. HEK-293T cells grown in antibiotic-free medium were transduced using 3:1 plasmids (Lv205-xCT and the packaging plasmids) to the transduction reagent ratio following the manufacturer’s instructions (transit-LT1; Mirus) and incubated at 37°C. The cell culture supernatant containing the virus was harvested 48 and 72 h after the transduction, passed through a 0.45-μm low-protein binding filter, and directly incubated on HEK-293T cells grown to 50% confluency. After 48 h of incubation, cells were passaged 3 times with complete medium before those harboring the xCT-plasmid were selected by adding puromycin at 3 μg/mL.

The green fluorescent protein tag of HEK::xCT allows confirmation of the expression of xCT using confocal microscopy. The green fluorescent protein and normal image overlay are shown in Supplemental Figure 2 (supplemental materials are available at http://jnm.snmjournals.org).

Quantitative Polymerase Chain Reaction (PCR)

Transcriptional expression of xCT (SLC7A11) in MDA-MB-231, MCF-7, and ZR-75 cells was determined relative to hypoxanthine phosphoribosyltransferase 1 (HPRT1). Total RNA was purified using the GenElute Mammalian Total RNA Miniprep Kit (Sigma), treated with amplification grade DNase I (Sigma), and measured using a NanoDrop spectrophotometer. A 50-ng quantity of total RNA from each sample was reverse-transcribed in a 20-μL reaction using a SuperScript VILO cDNA synthesis kit (Invitrogen). Quantitative PCR was set up in 384-well plates and performed on a QuantStudio 6K Flex Real-Time PCR system. Each 10-μL reaction contains 1 μL of template cDNA, 500 μM forward and reverse primers, 250 μM probe (IDT), and 1× SsoAdvanced Universal Probes Supermix (Bio Rad). MDA-MB-231 was assayed in triplicate; MCF-7 and ZR-75 were assayed in 2× triplicate. The concentration of each target was determined by interpolating the threshold cycle value from respective standard curves of known concentrations.

The IDT PrimeTime quantitative PCR primers were Hs.PT.58.38930943 for SLC7A11 and Hs.PT.58v.45621572 for HPRT1.

To construct the SLC7A11 and HPRT1 standard curves, total RNA from MDA-MB-231 cells was purified and reverse-transcribed, and SLC7A11 and HPRT1 genes were amplified using the same primers as for the quantitative PCR reaction and Q5 High-Fidelity DNA polymerase (New England BioLabs) per the manufacturer’s instructions. PCR products were separated on a 2% agarose gel, and target bands were extracted. The DNA was purified using the Monarch DNA Gel Extraction Kit (New England BioLabs) and quantified using the Qubit dsDNA HS assay kit (Thermo Fisher). Standard curves were constructed using 10-fold serial dilutions and assayed by quantitative PCR in triplicate (Supplemental Fig. 3).

Small-Animal PET and Biodistribution Studies

All animal experiments were conducted in accordance with the guidelines established by the Canadian Council on Animal Care and approved by the Animal Care Committee of the University of British Columbia. Female immunodeficient RAG2M mice (12–14 wk, 19–26 g) were obtained from an in-house breeding colony at the Animal Resource Centre of the British Columbia Cancer Research Centre.

MDA-MB-231, ZR-75-1, MCF-7, and HEK-293T tumors were inoculated subcutaneously with 5–10 × 10⁶ cells in Matrigel (Corning) on the dorsal flank of the mice. MCF-7 and ZR-75-1 required implantation of a 17β-estradiol releasing pellet (0.36 mg/60 d, Innovative Research of America) 7 d before inoculation.

Once palpable tumors measuring about 7–10 mm were obtained (∼2–4 wk), the mice were anesthetized by 2% isoflurane inhalation and intravenously injected with about 3.7 MBq of ¹⁸F-FASu. The activity in the syringe was counted before and after injection to determine the exact injected dose.

For blocking experiments, the radiotracer was coinjected with a 100 mg/kg dose of nonradioactive aminosuberic acid. After the injections, the mice were allowed to recover and roam freely in their cages for 60 min. The animals were then anesthetized by 2% isoflurane inhalation and euthanized by CO₂ at various time points. Blood was promptly withdrawn via a cardiac puncture, and the tissues of interest were harvested, rinsed in phosphate-buffered saline, blotted dry, and weighed. The radioactivity of the collected mouse tissues was counted on a γ-counter and expressed as the percentage injected dose per gram of tissue (%ID/g).

Mice destined for a static emission scan were anesthetized with 2% isoflurane inhalation 1 or 2 h after intravenous injection of the tracer and placed on the scanner. A baseline CT scan was obtained for localization and attenuation correction. During the acquisition, anesthesia was maintained and the mice were kept warm with a heating pad. A single static emission scan was acquired for 15 min. Afterward, the mice were euthanized and the organs harvested for biodistribution as described above.

Statistical Analysis

Test statistics and P values were calculated using Minitab Express analysis software. The descriptive statistics are reported as mean ± SD when applicable. Means were compared using a 2-sample t test, assuming equal variance. A P value of less than 0.05 was considered significant.