In vitro evaluation of (S)-2-amino-3-[3-(2-¹⁸F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid (¹⁸F-FIMP) as a positron emission tomography probe for imaging amino acid transporters

CHO-K1 (RCB0285) Chinese hamster ovary cells, T3M-4 (RCB1021) human pancreatic adenocarcinoma cells, A549 (RCB0098) human lung carcinoma cells, and MCF7 (RCB1904) human breast adenocarcinoma cells were obtained from the RIKEN BioResource Research Center through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Agency for Medical Research and Development (AMED), Japan. CHO-K1 and T3M-4 cells were cultured in Ham’s F12 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Inc., Kerrville, TX), 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Inc.). A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Nacalai Tesque, Inc.) supplemented with 10% FBS (Equitech-Bio, Inc.), 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Inc.). MCF7 cells were cultured in Minimum Essential Medium (MEM; Nacalai Tesque, Inc.) supplemented with 10% FBS (Equitech-Bio, Inc.), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Inc.). NCI-H460 (ATCC HTB-177) human lung cancer cells were obtained from the American Type Culture Collection, Manassas, VA. NCI-H460 cells were cultured in Roswell Park Memorial Institute 1640 medium (Nacalai Tesque, Inc.) supplemented with 10% FBS (Equitech-Bio, Inc.), 100 units/mL penicillin, and 100 μg/mL streptomycin (Nacalai Tesque, Inc.).

The coding regions of LAT1 (SLC7A5; GenBank accession no. NM_003486, nucleotides 78 – 1601), ATB^0,+ (SLC6A14; GenBank accession no. NM_007231, nucleotides 132 – 2060), and xCT (SLC7A11; GenBank accession no. NM_014331, nucleotides 281 – 1786) were amplified by polymerase chain reaction (PCR) with the introduction of SalI and NotI sites, then ligated into the SalI and NotI sites of the pGEM-T Easy Vector system (Promega, Madison, WI). The coding region of ASCT2 (SLC1A5; GenBank accession no. NM_005628, nucleotides 621 – 2246) was amplified by PCR with the introduction of EcoR V and NotI sites, then ligated into the EcoR V and NotI sites of the pGEM-T Easy Vector system (Promega). These inserts were subcloned into the pEBMulti-Hyg vector (Fujifilm Wako Pure Chemical Corporation, Tokyo, Japan). These constructs were referred to as pEBMulti-Hyg-LAT1, pEBMulti-Hyg-ATB^0,+, pEBMulti-Hyg-xCT, and pEBMulti-Hyg-ASCT2, respectively.

For the transfection of these expression vectors, CHO-K1 cells were seeded onto a 100-mm dish at a density of 1 × 10⁶ to 2 × 10⁶ cells/dish and cultured overnight. At 60% to 70% confluence, the cells were transfected with 10 μg of the pEBMulti-Hyg vector (mock) or pEBMulti-Hyg-LAT1 by using TransIT-X2 (Mirus Bio LLC, Madison, WI), pEBMulti-Hyg-ATB^0,+ or pEBMulti-Hyg-ASCT2 by using TurboFectin 8.0 (OriGene Technologies, Inc., Rockville, MD), or pEBMulti-Hyg-xCT by using ViaFect (Promega) according to the manufacturers’ protocols. Subsequently, at 24 to 48 h after transfection, cells were subcultured into a 100-mm dish containing culture medium supplemented with Hygromycin B Gold (0.4 mg/mL; InvivoGen, San Diego, CA) for 2 to 3 weeks. The cells were then seeded onto a 96-well plate with a single cell per well. The culture medium was changed every 2 days for 2–3 weeks until a single colony could be seen. Cells were trypsinized, then transferred into a 24-well plate containing 500 μL of culture medium supplemented with Hygromycin B Gold. The cells were subcultured into a 6-well plate, and subsequently into a 100-mm dish, and maintained in the presence of Hygromycin B Gold.

For the transfection of gene-specific small interfering ribonucleic acids (siRNAs), each of the overexpressing cell lines were seeded onto a 100-mm dish at a density of 2 × 10⁶ cells/dish and cultured overnight. At 60–70% confluence, cells were transfected with 50 to 100 nM of gene-specific siGENOME human siRNA (Dharmacon, Inc., Lafayette, CO) by using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. The efficacy of knockdown was calculated from the data of immunocytochemical staining as follows: the efficacy of knockdown = number of negative cells/number of total cells.

Plasma membrane fractions were prepared as described previously [23]. The cell pellet was suspended in homogenization buffer containing 10 mM Tris–HCl (pH 7.5), 250 mM sucrose, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid, and protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). The cells were homogenized and centrifuged at 1,000 × g for 5 min at 4 °C. The supernatant was centrifuged at 430,000 × g for 15 min, and the membrane pellet was resuspended in 0.75 mL of 30% iodixanol solution (20 mM Tris–HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid, 30% (w/v) iodixanol (Axis-Shield PoC AS, Oslo, Norway), and 125 mM sucrose). The membrane suspension was overlaid sequentially by 4 mL each of 25%, 17.5%, 10%, and 2.5% iodixanol solutions, then centrifuged at 100,000 × g for 16 h. Each fraction was collected from the top. All fractions were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. After confirmation by western blotting with plasma membrane markers, fractions from the 2.5% to 10% iodixanol interface were pooled as the plasma membrane fraction. The protein concentration of each sample was determined using the bicinchoninic acid (BCA) method. The membrane fractions were dissolved in 1% Fos-Choline-12 (Anatrace, Maumee, OH) mixed with Laemmli sample buffer and subjected to SDS-PAGE.

The protein sample was separated by SDS-PAGE using a 10% to 20% gradient polyacrylamide gel, and the separated proteins were transferred electrophoretically to a Hybond-P polyvinylidene difluoride transfer membrane (GE Healthcare, Chicago, IL). The membrane was pre-blocked in Bullet Blocking One blocking solution (Nacalai Tesque, Inc.) at room temperature for 1 h. The membrane was then incubated with the blocking solution containing a 1:10,000 dilution of rabbit anti-human LAT1 polyclonal antibody (TransGenic Inc., Fukuoka, Japan), 1:2,000 dilution of rabbit anti-human ATB^0,+ polyclonal antibody (Medical & Biological Laboratories Co., Ltd., Nagoya, Japan), 1:1,000 dilution of rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc., Danvers, MA), 1:100 dilution of rabbit anti-mouse xCT polyclonal antibody (TransGenic Inc.), 1:1,000 dilution of rabbit anti-mouse CD98 polyclonal antibody (Sino Biological Inc., Beijing, China), or 1:100,000 dilution of rabbit anti-human sodium potassium ATPase monoclonal antibody (Abcam Inc., Cambridge, UK). The membrane was treated with a 1:5,000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; Sigma-Aldrich Co., LLC, St. Louis, MO) and developed using ECL Select Western Blotting Detection Reagent (GE Healthcare) before visualization under an LAS-3000 luminescent image analyzer (Fujifilm Corporation, Tokyo, Japan).

Cells grown on 8-well chamber slides (Matsunami Glass Ind., Ltd., Osaka, Japan) were briefly washed with Dulbecco’s phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature. After washing once with PBS for 5 min, the cells were treated with 0.1% Triton X-100 in PBS for 10 min at room temperature for permeabilization. The cells were washed once with PBS for 5 min, followed by blocking for 60 min at room temperature in PBS containing 4% FBS for LAT1 and ATB^0,+, PBS containing 5% normal goat serum (NGS) and 0.3% Triton X-100 for ASCT2, and PBS containing 1% NGS for xCT. The cells were then incubated with primary antibodies diluted in PBS containing 4% FBS for LAT1 and ATB^0,+, PBS containing 1% bovine serum albumin and 0.3% Triton X-100 for ASCT2, and PBS containing 1% NGS for xCT. Rabbit anti-human LAT1 polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ATB^0,+ polyclonal antibody (Sigma-Aldrich Co., LLC) was diluted at 1:50, rabbit anti-human ASCT2 polyclonal antibody (Cell Signaling Technology, Inc.) was diluted at 1:50, and rat anti-human xCT monoclonal antibody (Cosmo Bio Co., Ltd., Tokyo, Japan) was diluted at 1:100. After an overnight incubation at 4 °C, the cells were washed with PBS, then incubated with Alexa488-conjugated goat anti-rabbit IgG (1:500, Thermo Fisher Scientific) in PBS containing 4% FBS and Hoechest33258 (1:1000, Dojindo Laboratories, Kumamoto, Japan) for 90 min at room temperature for LAT1 and ATB^0,+, Alexa488-conjugated goat anti-rabbit IgG (1:500, Thermo Fisher Scientific) in PBS containing 1% bovine serum albumin, 0.3% Triton X-100, and Hoechest33258 (1:1000, Dojindo Laboratories) for 120 min at room temperature for ASCT2, and Alexa488-conjugated donkey anti-rat IgG (1:2000, Thermo Fisher Scientific) in PBS containing 4% FBS and Hoechest33258 (1:1000, Dojindo Laboratories) for 60 min at room temperature for xCT. The cells were washed with PBS, then mounted on slides with PBS containing 0.1 M dithiothreitol and 50% glycerol. Immunofluorescent staining was observed under the C1 confocal microscope system (Nikon Instech Co., Ltd., Tokyo, Japan).

A ¹⁴C-labeled ligand uptake assay was performed using each of the overexpressing and mock cell lines grown to 90% to 100% confluence on collagen-coated 24-well plates. The combinations of ligand and inhibitor used were: L-¹⁴C-leucine (Moravek, Inc., Brea, CA) and 2-amino-2-norbornanecarboxylic acid (BCH; Sigma-Aldrich Co., LLC) for LAT1; ¹⁴C-glycine (Moravek, Inc.) and α-methyl-DL-tryptophan (AMT; Sigma-Aldrich Co., LLC) for ATB^0,+; L-¹⁴C-glutamine (PerkinElmer, Waltham, MA) and L-γ-glutamyl-p-nitroanilide (GPNA; Fujifilm Wako Pure Chemical Corporation) for ASCT2; and L-¹⁴C-glutamic acid (Moravek, Inc.) and sulfasalazine (SF; Fujifilm Wako Pure Chemical Corporation) for xCT. The incubation and washing solutions used were Na-free Hank’s Balanced Salt Solution (pH 7.4) for LAT1 and xCT, and Hank’s Balanced Salt Solution (pH 7.4) for ATB^0,+ and ASCT2. First, the cells were washed three times with wash solution, then incubated in the same solution for 10 min at 37 °C. Next, the ¹⁴C-labeled ligands (1 μM) were added, then incubated for 3 min at 37 °C with or without the corresponding inhibitor (1 mM). The reaction was stopped by washing the cells three times with ice-cold wash solution. The cells were then lysed in 500 μL of 0.1 N NaOH, followed by incubation for 15 min at room temperature. The lysates of each well were collected in scintillation vials and mixed with Clear-sol Ι (Nacalai Tesque, Inc.). The protein concentration of a portion of the lysates was determined using the BCA method. The radioactivity was measured by a Tri-Carb 2800TR scintillation counter (PerkinElmer, Waltham, MA).

An ¹⁸F-FIMP uptake assay was also performed using each of the overexpressing and mock cell lines grown to 90% to 100% confluence on collagen-coated 12-well plates. The inhibitors and solutions used were the same as described for the ¹⁴C-labeled ligand uptake assay. First, the cells were washed three times with wash solution, then incubated in the same solution for 10 min at 37 °C. Next, ¹⁸F-FIMP (370 kBq) was added, then the cells were incubated for 3 min at 37 °C with or without the corresponding inhibitor (1 mM). The reaction was stopped by washing the cells three times with ice-cold wash solution. The cells were then lysed in 1 mL of 0.1 N NaOH, followed by incubation for 15 min at room temperature. The lysates of each well were collected in microtubes. The protein concentration of a portion of the lysates was determined using the BCA method. The radioactivity was measured by a 2480 WIZARD² Auto Gamma Counter (PerkinElmer).

¹⁸F-FIMP (Fig. 1) was synthesized as described previously [20]. Radiochemical purities of > 99.5% were determined by high-performance liquid chromatography.

Data are presented as the mean ± standard deviation. All statistical analyses were performed using Student’s t test on Microsoft Excel 2010 version 14.0 (Microsoft, Redmond, WA). P-values less than 0.05 were considered to be significant.

The protein expression levels in the membrane protein extracts from the positive-control cells, negative-control cells (CHO-K1 cells), control vector-transfected cells (mock), expression vector-transfected cells, and gene-specific siRNA-transfected cells were evaluated by western blot analysis using anti-human LAT1 (Fig. 2A, Suppl. Fig. 1), ATB^0,+ (Fig. 2B, Suppl. Fig. 2), ASCT2 (Fig. 2C, Suppl. Fig. 3), and xCT (Fig. 2D, Suppl. Fig. 4) antibodies. As positive-control cells, MCF-7 (Fig. 2A, Suppl. Fig. 1), T3M-4 (Fig. 2B, Suppl. Fig. 2), NCI-H460 (Fig. 2C, Suppl. Fig. 3), and A549 (Fig. 2D, Suppl. Fig. 4) cells were used. No signals were observed for the control and mock cells on all blots, whereas intense signals were observed for all of the expression vector-transfected cells. These signals were weak, or strongly reduced, by gene-specific siRNA treatment. The signals of the anti-Na + /K + -ATPase antibody, which was used as a loading control, were similar in intensity to the other signals on the same membrane. Full-length blots images are included in an Additional file 1.

The protein expression levels in transfected cells were also evaluated by immunofluorescent analysis using the same specific antibodies used in the western blot analysis (Fig. 3). No signals were observed for the control and mock cells, whereas intense signals were observed for the expression vector-transfected cells. These signals were strongly reduced by gene-specific siRNA treatment (Table 2).

To evaluate the transport activity of the overexpressing cell lines, transporter-mediated ¹⁴C-labeled substrate uptake was measured in the presence or absence of specific inhibitors, i.e., BCH, AMT, GPNA, or SF, in the control vector (mock)-transfected cells and each of the expression vector-transfected cell lines (Fig. 4). The uptake values for all of the ¹⁴C-labeled substrates were significantly higher in the expression vector-transfected cells than in the control (mock) cells. These uptake values were significantly inhibited by each of the corresponding specific inhibitors. In the presence of the inhibitors, the uptake levels were significantly lower in the expression vector-transfected cells than in the control (mock) cells. The uptake values in the control (mock) cells for LAT1, ASCT2, and xCT were significantly inhibited by the corresponding specific inhibitors.

To estimate the affinity of ¹⁸F-FIMP in each transporter-overexpressing cell line, transporter-mediated ¹⁸F-FIMP uptake was measured in the presence or absence of specific inhibitors in the control vector (mock)-transfected cells and each of the expression vector-transfected cell lines (Fig. 5). ¹⁸F-FIMP uptake values were significantly higher in the LAT1- and ATB^0,+-overexpressing cell lines than in the control (mock) cells. These uptake values were significantly decreased by the specific inhibitors BCH and AMT (Fig. 5A and B). In contrast, the ¹⁸F-FIMP uptake values in the ASCT2- and xCT-overexpressing cell lines were not significantly higher than those in the control (mock) cells (Fig. 5C and D). The ¹⁸F-FIMP uptake values in the control (mock) cells were significantly inhibited by each of the specific inhibitors, i.e., BCH, AMT, GPNA, and SF.