Molecular Imaging and Biology

Differences in transport mechanisms of trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid in inflammatory, prostate cancer, and glioma cells: comparison with L-[methyl-11C]methionine and [18F]fluoro-D-deoxy glucose

Molecular Imaging and Biology

Shuntaro Oka1,*, Hiroyuki Okudaira1, Masahiro Ono1,2, David M. Schuster3, Mark M. Goodman3, Keiichi Kawai2,4, Yoshifumi Shirakami1

1 Research Center, Nihon Medi-Physics Co., Ltd., Chiba, Japan

2 Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan

3 Division of Nuclear Medicine and Molecular Imaging, Department of Radiology and Imaging Sciences, Emory University, Atlanta, Georgia, USA

4 Biomedical Imaging Research Center, University of Fukui, Fukui, Japan

*Corresponding author: Shuntaro Oka

Tel: +81 438 62 7611

Fax: +81 438 62 5911

E-mail:

Kitasode 3-1, Sodegaura, Chiba 299-0266, Japan

Supplemental Tables and Figures

Materials and Methods

Isolation of Rat Inflammatory Cells

All reagents were purchased from Life Technologies (Carlsbad, CA) and Sigma-Aldrich (St. Louis, MO), unless otherwise stated. T cells were isolated from the mesenteric and iliac lymph nodes of 2 to 4 Copenhagen (COP) rats in every experiment and pooled in Hanks’ balanced salt solution without Mg2+ and Ca2+ (HBSS(-)) including 0.5% bovine serum albumin (BSA, Rockland Immunochemicals, Boyertown, PA) and 0.5 mM ethylenediaminetetraacetic acid (BIO-RAD, Hercules, CA) (reaction buffer). Lymph nodes were minced and a single cell suspension was obtained. Then, the mononuclear cell fraction including T cells was isolated by density gradient centrifugation using Lymphoprep-Rat (Cedarlane, Burlington, ON) according to the manufacture’s protocol, followed by resuspension in AIM-V medium at 1 × 107 cells/mL. The mononuclear cell fraction was seeded in cell culture dishes (100φ mm, BD Biosciences, Franklin Lakes, NJ) and incubated for 2 h in an incubator at 37°C in 5% CO2 to adhere macrophages and monocytes to the dishes. The medium, including non-adherent cells was collected and moved into new culture dishes (100φ mm), then 2-mercaptoethanol (2-ME) and recombinant rat IL-2 were supplemented at 50 mM and 50 U/mL, respectively. To activate T cells, concanavalin A (Con A) was added at 0.25 mg/mL, followed by cultivation in an incubator at 37°C in 5% CO2. After 2 days, cells were collected and T cells were purified by negative selection using magnetic microbeads labeled mouse anti-rat CD45RA monoclonal antibody (mAb) (clone: OX-33, Miltenyi Biotech, Bergisch Gladbach, Germany) and MACS cell separation system (Miltenyi Biotech) according to the manufacture’s protocol, then resuspended in RPMI1640 including 10% COP rat serum, 100 mg/mL streptomycin, and 100 U/mL penicillin (maintenance medium) until used in experiments.

B cells were obtained from the spleens of 2 to 3 COP rats in every experiment and pooled in the reaction buffer. Spleens were minced in the reaction buffer and hemolyzed with VersaLyse solution (Beckman Coulter, Brea, CA). Then, the mononuclear cell fraction including B cells were isolated by using Lymphoprep-Rat as well as T cells preparation, resuspended in HBSS containing Mg2+ and Ca2+ (HBSS(+)) including 3% BSA at 1.5 × 107 cells/mL, and seeded into the culture dishes (100φ mm) at approximately 2×106 cells/cm2. After incubation at room temperature for 1 h, because B cells were weakly adherent to the dishes, cells other than B cells were removed by aspiration (31). Then, the adherent cells were washed gently 3 times with HBSS(+) containing 0.3% BSA and detached from the dishes by flushing with HBSS (+) containing 0.3% BSA with a Pasteur pipette. Next, B cells were resuspended in RPMI1640 containing 10% fetal bovine serum (FBS, American Type Culture Collection, Manassas, VA) inactivated at 56°C for 30 min, 50 mM 2-ME, 100 mg/mL streptomycin, and 100 U/mL penicillin, and seeded into non-treatment type cell culture dishes for suspension cells (90φ mm) (Sumitomo Bakelite, Tokyo, Japan). After cultivation for 2 days in a 5% CO2 incubator under the presence or the absence of lipopolysaccharide (LPS, 5 mg/mL) from Escherichia coli O55:B5 (Wako Pure Chemical Industries, Osaka, Japan), cells were collected and undertaken density-gradient centrifugation with Lymphoprep-Rat to remove dead cells, then resuspended in the maintenance medium until used in experiments.

Granulocytes were isolated from the peripheral blood of 1 to 4 COP rats in every experiment. Blood was collected from the abdominal aorta using a syringe anticoagulated with heparin. Five percent dextran (Nacalai Tesque, Kyoto, Japan) dissolved in HBSS(-) was mixed with blood at a ratio of 3:10 and incubated for 45 to 60 min at room temperature to sediment erythrocytes. The leukocyte-rich plasma was removed from above the aggregated erythrocyte pellet and neutrophils in the leukocyte-rich plasma were isolated by density-gradient centrifugation using OptiPrep (AXIS-SHIELD PoC AS, Dundee, Scotland) according to the manufacturer’s protocol. Isolated granulocytes were suspended in maintenance medium and incubated with or without 100 nM phorbol 12-myristate 13-acetate (PMA) (Enzo Life Sciences International, Farmingdale, NY) for 1 h in an incubator at 37°C in 5% CO2, then used in experiments.

Macrophages were obtained from the peritoneal fluid of 3 to 4 COP rats in every experiment. Briefly, 25 mL of ice-cold HBSS(-) including 2 mM EDTA, was injected into the peritoneal cavity and the abdomen was massaged gently for 5 min, and the HBSS including resident peritoneal macrophages was then collected. This procedure was repeated twice. After the intraperitoneal cells were centrifuged, cells were resuspended in RPMI1640 and the number of macrophages was counted under a phase-contrast microscope (Nikon Corporation, Tokyo, Japan). Then, the macrophages were resuspended in RPMI1640 medium at 1 × 106 cells/mL, and 0.3 mL of the cell suspension was seeded in 48-well tissue culture plates (BD Biosciences). After incubation for approximately 2 h in an incubator at 37°C in 5% CO2, the medium was aspirated and each well was washed twice with HBSS(+) to remove non-adherent cells, followed by the replacement of the maintenance medium. After overnight incubation in an incubator (37°C, 5% CO2), media were substituted with fresh warmed maintenance medium. Then macrophages were cultivated for 6 h with or without 5 mg/mL LPS at 37°C in a 5% CO2 incubator and used in experiments.

Validation of Activation Status of Isolated Inflammatory cells

All mAbs were purchased from Biolegend (San Diego, CA), eBioscence (San Diego, CA), BD Biosciences, and Santa Cruz Biotechnology (Santa Cruz, CA). To confirm the activation status, isolated T cell, B cells, and granulocytes were stained with the following fluorescent-dye conjugated mouse anti-rat mAbs: fluorescein isothiocyanate (FITC)-anti-TCRab (clone: R73) and phycoerythrin (PE)-anti-CD25 (clone: OX-39) for T cells; FITC-anti-CD45RA (clone: OX-33), PE-anti-IgM (clone: HIS40), PE-anti-CD86 (clone: 24F), and allophycocyanin (APC)-anti-MHC class II (clone: HIS19) for B cells; PE-anti-granulocyte (clone: RP-1) and APC-anti-CD11b/c (clone: OX-42) for granulocytes. As a negative control, FITC-, PE-, or APC-conjugated mouse IgG1 or IgG2a isotype mAbs were used. Cells were stained with these mAbs for 15 min in a refrigerator and washed twice with cold reaction buffer. For granulocytes, cells were loaded with 5 mM of 2¢,7¢- dichlorodihydrofluorescein diacetate (DCFH-DA) for 15 min during PMA stimulation prior to mAb staining. Finally, the inflammatory cells were resuspended in the cold reaction buffer including 1 mg/mL propidium iodide and applied to flow cytometry. Data were acquired from 20,000 cells in each sample using a FACSCalibur flow cytometer (BD Biosciences) and the positive rates of markers on each inflammatory cell and their mean fluorescence intensity (MFI) were analyzed with WinMDI software (ver. 2.8) (n = 10–12).

The cells adhering to the tissue culture plate were thought to be macrophages. The activation status of macrophages stimulated with LPS was monitored based on morphological changes under a phase-contrast microscope (Nikon Corporation) after staining with Giemsa solution. Cells were observed at ×200 magnification and 100–500 cells per field were counted on 3 randomly selected fields; then, the percentage of spherical (non-stimulated macrophages) and elongated (activated macrophages) cells were calculated. Experiments were repeated 3 times and data were represented as mean ± standard error of the mean (SEM). In addition, the nitrite concentration in the culture supernatants was determined by the standard Griess reaction as a marker of activated macrophages. Briefly, 100 mL of supernatants of macrophage culture was placed in a 96-well flat-bottomed plate (BD Biosciences) and the equivalent volume of Griess reagent was added, followed by incubation for 10 min at room temperature (n = 6–12). The absorbance of each well was measured with a microplate reader (VersaMax; Nihon Molecular Devices, Tokyo, Japan) at 550 nm and the nitrite concentration was determined from a standard curve of sodium nitrite.

Statistics

Experiments were repeated at least twice. All results were expressed as mean ± SD unless otherwise stated. All statistical analyses were performed using SAS for Windows (Ver. 5, SAS Institute, Cary, NC). For datasets with normal distributions, homogeneity of variance was analyzed by the F-test, and homogeneous data were then analyzed using the two-tailed unpaired Student’s t-test, whereas non-homogeneous data were analyzed using the Welch’s t-test. The Wilcoxon rank sum test was used for non-normal datasets. In all cases, P 0.05 was considered to be significant.

Results

Isolation and Validation of Activated Inflammatory Cells

The activation status of isolated T cells, B cells, and granulocytes stimulated with Con A, LPS, and PMA, respectively, were determined by flow cytometry (Fig. S1; all data are summarized in Table S2). It is known that T and B cells increase in size upon activation, and their sizes (FSC) were increased 1.9- and 1.4-fold, respectively, compared to non-stimulated (NS) cells (Fig. S1a, b). As for T cells stimulated with Con A, the positive rate and the expression intensity (MFI) of CD25, an activation marker of T cells, were increased 3.4- and 150.9-fold, respectively, in comparison with NS T cells (Fig. S1a). In B cells stimulated with LPS, the expression of CD86, a B cell activation marker, was higher in terms of percentage (2.7-fold) and MFI (27.6-fold) than NS B cells (Fig. S1b). Furthermore, the MFI of MHC II, another activation marker of B cells, was also increased 2.5-times of NS B cells, although the percentage of positive cells for MHC II was not changed (>99.0%, Fig. S1b). These results demonstrated that both T and B cells were activated by stimulation with Con A and LPS, respectively.

Because granulocytes stimulated with activators produce oxidants in cells, we measured oxidant levels utilizing the conversion of nonfluorescent 2¢,7¢-dichlorodihydrofluorescein diacetate (DCFH-DA) to the fluorescent compound 2¢,7¢-dichlorofluorescein (DCF) when DCFH-DA is hydrolyzed and oxidized. The results showed that 90.4% of the PMA-stimulated granulocytes were positive for DCF (NS: 17.6%) and the MFI from DCF in stimulated cells was 9.4-fold higher than that in NS granulocytes (Fig. S1c). In addition, the expression of CD11b/c, an activation marker of granulocytes, was also enhanced in granulocytes stimulated with PMA (3.4-fold vs. NS, Fig. S1c). Thus, it was thought that granulocytes were activated adequately by PMA.

For LPS-stimulated macrophages, the morphological changes of cells were observed by microscopy after Giemsa staining. Although macrophages with an elongated shape were approximately half of the total cells (NS: 12.6%), almost all the cells showed cytoplasmic foaming (Fig. S1d). Furthermore, the concentration of nitrite in the culture medium of LPS-stimulated macrophages was increased 2.3-fold in comparison with NS macrophages (Fig. S1d). These results suggest that most macrophages were activated or in the process of being activated.

Fig. S1 Validation studies of the activation status of T cells (a), B cells (b), granulocytes (c), and macrophages (d). Non-stimulated (NS) and activated rat inflammatory cells (T cells, B cells, and granulocytes were stimulated with Con A, LPS, and PMA, respectively) were stained with fluorochrome-labeled monoclonal antibodies indicated in each panel, and then analyzed with a flow cytometer. Forward scattering (FSC) correlates with the cell volume. The X- and Y-axes of each panel show the fluorescence intensity and number of cells, respectively. The morphological changes of macrophages stimulated with LPS were observed under phase-contrast microscopy with Giemsa-stained specimens (original magnification: 200), and the numbers of spherical (non-stimulated macrophages) and elongated (activated macrophages) cells were counted. The nitrite concentration in the supernatants from macrophage cultures was measured using the Griess reagent and a microplate reader. Data were acquired from 8 experiments and are represented as mean ± SEM. Detailed data are shown in Table S2.

Competitive Inhibition Tracer Uptake Experiments

Figure S2 Competitive inhibition of anti-[14C]FACBC (FACBC) and [14C]Met (Met) transport in rat T cells by naturally occurring and synthetic amino acids. Cells were stimulated with Con A, and then 10 mM anti-[14C]FACBC and [14C]Met were incubated with or without 2 mM naturally occurring and synthetic amino acids in sodium (a), choline (b), and lithium (c) buffer. The control transport of tracers in each buffer was normalized to 100%. Each bar represents the mean ± SD of 2–3 independent experiments (n = 5–11). * P 0.05, ** P 0.01.

Figure S3 Competitive inhibition of anti-[14C]FACBC (FACBC) and [14C]Met (Met) transport in rat B cells by naturally occurring and synthetic amino acids. Cells were stimulated with LPS, and then 10 mM anti-[14C]FACBC and [14C]Met were incubated with or without 2 mM naturally occurring and synthetic amino acids in sodium (a), choline (b), and lithium (c) buffer. The control transport of tracers in each buffer was normalized to 100%. Each bar represents the mean ± SD of 2–3 independent experiments (n = 5–6). * P 0.05, ** P 0.01.

Figure S4 Competitive inhibition of anti-[14C]FACBC (FACBC) and [14C]Met (Met) transport in a rat prostate cancer cell line (MLLB2) by naturally occurring and synthetic amino acids. Ten micromolar anti-[14C]FACBC and [14C]Met were incubated with or without 2 mM naturally occurring and synthetic amino acids in sodium (a), choline (b), and lithium (c) buffer. The control transport of tracers in each buffer was normalized to 100%. Each bar represents the mean ± SD of 2–3 independent experiments (n = 6–9). * P 0.05, ** P 0.01.