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: shuntaro_oka@nmp.co.jp
Kitasode 3-1, Sodegaura, Chiba 299-0266, Japan
1
Supplemental Tables and Figures
Table S1. Characteristics of amino acid transporters
+
Na -dependency
natural AAs
system
AAT name
SNAT1
SLC38A1
✔
✔
✔
SNAT2
SLC38A2
✔
✔
✔
SNAT4
SLC38A4
✔
✔
ASCT1
SLC1A4
ASCT2
SLC1A5
ASC
G-like
neutral AATs
pH-sensitivity
Gln Ser Phe Met Pro Gly Glu Arg
A
dependent
synthetic AAs
SLC family
0
B
PROT
SLC6A7
B0 AT1
SLC6A19
0
✔
✔
✔
✔
✔
SLC6A15
SIT1
SLC6A20
SNAT3(SN1)
SLC38A3
✔
SNAT5(SN2)
SLC38A5
✔
N
+
y LAT1/4F2hc
SLC7A7
✔
✔
✔
✔
✔
1, 2
✔
✔
✔
✔
✔
✔
cotransport with Na
neutral AAs uptake is relatively insensitive;
more active for acidic AAs at acidic pH
cotransport with Na +
amino acid exchanger
―
✔
SLC7A6
SLC7A5
more active at alkaline pH
―
insensitive
―
dependent
✔
✔
✔
more active at alkaline pH
✔
✔
✔
✔
✔
✔
✔
✔
SLC7A8
✔
✔
✔
✔
✔
✔
✔
✔
LAT4
SLC43A2
✔
✔
✔
✔
T
TAT1
SLC16A10
✔
asc
asc-1/4F2hc
SLC7A10
basic AATs
y+L
acidic AATs
SLC6A14
b (BAT1)/rBAT
SLC7A9
+
SLC7A7
y LAT1/4F2hc
+
independent
y+
independent
others
ATB0.+
0,+
independent
X-A.G
-
XC
✔
✔
✔
1, 14
tolerance of Li+ in place of Na +
1, 15
amino acid exchanger; associated with 4F2hc;
16, 17
✔
✔
✔
✔
handles neutral AAs with Na +; tolerance of Li+
16, 18
16, 19
amino acid exchanger; associated with 4F2hc
more active for acidic AAs at low pH
insensitive
―
insensitive
―
insensitive
✔
✔
✔
✔
✔
✔
16, 20
amino acid exchanger; associated with 4F2hc
―
✔
✔
SLC7A1
✔
✔
CAT2
SLC7A2
✔
✔
CAT3
SLC7A3
✔
✔
EAAT1
SLC1A3
✔
EAAT2
SLC1A2
✔
EAAT3
SLC1A1
✔
EAAT4
SLC1A6
✔
7, 16, 25
amino acid exchanger; associated with 4F2hc;
handles basic AAs and zwitterionic AAs
16, 17
more active for glutamate at acidic pH
✔
without Na
―
EAAT5
SLC1A7
SLC7A11
PAT1
SLC36A1
✔
✔
✔
PAT2
SLC36A2
✔
✔
✔
✔
―
26, 27
cotransport with Na +/H+/ K+
5, 28
sensitive mRNA expression to pH-change
amino acid exchanger; associated with 4F2hc
7, 16
strong active at acidic pH
cotransporter with H+
29, 30
AAs: amino acids, AAT(s): AA transporter(s), BCH: 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid, MeAIB; 2-(methylamino)-isobutyric acid, NEM: N-ethylmaleimide, SLC: solute carrier, 4F2hc: 4F2 heavy chain
2
16, 18
+
―
xCT/4F2hc
10
amino acid exchanger; associated with rBAT
✔
SLC7A6
―
23
16, 24
more active for glutamate at acidic pH
✔
CAT1
21
22
✔
y LAT2/4F2hc
PAT
amino acid exchanger with H ;
insensitive
SLC43A1
0.+
9, 13
in place of Na +
✔
LAT3
b
dependent
✔
✔
LAT2/4F2hc
B0.+
9, 11
+
L
independent
9, 10
9, 12
✔
✔
5-7
5, 7, 8
―
✔
✔
1, 3
1, 4
―
LAT1/4F2hc
+
more active at alkaline pH
+
y LAT2/4F2hc
Ref
✔
✔
yL
+
notes
MeAIB
✔
B AT2
IMINO
BCH NEM
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 M and 50 U/mL, respectively. To activate T cells, concanavalin A (Con A) was
added at 0.25 g/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 g/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 M 2-ME, 100 g/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
g/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
3
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 g/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-TCR (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 M 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 g/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
4
macrophages. Briefly, 100 L 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.
c
5
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.
6
Table S2. Validation of activation status of T cells, B cells, granulocytes, and macrophages
Cell
T cells
Parameters
Viability
%
TCR
%
FSC
MFI
%
CD25
MFI
Viability
%
CD45RA
%
sIgM
%
FSC
MFI
B cells
%
CD86
MFI
%
MHCⅡ
MFI
Viability
%
RP-1
%
%
Granulocytes
DCF
MFI
%
CD11b/c
MFI
spherical
%
elongated
%
cell morphology
Macrophages
Nitrite production in supernant
M
of culture medium
Stimulation
NS
mean
SD
91.2 ± 2.6
Con A
87.8 ± 4.3
NS
Con A
NS
Con A
NS
Con A
NS
Con A
NS
LPS
NS
LPS
NS
LPS
NS
LPS
NS
LPS
NS
LPS
NS
LPS
NS
LPS
NS
PMA
NS
PMA
NS
PMA
NS
PMA
NS
PMA
NS
PMA
NS
LPS
NS
LPS
NS
LPS
96.5
95.7
272.7
520.2
24.7
85.0
4.1
620.5
89.0
84.6
77.9
72.8
77.7
80.2
380.0
531.9
31.8
85.8
12.0
332.2
99.3
99.4
1847.4
4574.6
87.2
85.0
79.7
79.4
17.6
90.4
12.7
119.1
99.4
99.8
426.0
1439.2
87.4
50.8
12.6
49.2
2.9
6.6
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
1.5
2.5
61.1
109.4
14.5
9.1
3.2
420.2
4.6
4.7
3.1
3.8
5.4
6.6
23.8
35.1
9.5
7.3
7.0
52.0
0.4
0.4
326.0
754.5
7.3
5.7
5.8
9.3
21.2
19.9
7.2
97.2
1.2
0.4
163.8
179.1
2.2 *
4.4 *
2.2 *
4.4 *
1.0 *
0.9 *
ratio
P -values
1.0
<0.05
1.0
>0.05
1.9
<0.01
3.4
<0.01
150.9
<0.01
1.0
<0.05
0.9
<0.01
1.0
>0.05
1.4
<0.01
2.7
<0.01
27.6
<0.01
1.0
>0.05
2.5
<0.01
1.0
<0.05
1.0
>0.05
5.1
<0.01
9.4
<0.01
1.0
<0.01
3.4
<0.01
0.6
<0.01
3.9
<0.01
2.3
<0.05
* mean ± standard error of the mean (SEM)
Con A: concanavalin A, DCF: 2',7'-dichlorofluorescein, FSC: forward scattering, LPS: lipopolysaccharide,
MFI: mean fluorescence intensity, MHCⅡ: major histocompatibility complexⅡ, NS: non-stimulated, PMA:
phorbol myristate acetate, TCR: T cell receptor
7
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.
8
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 M
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.
9
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 M
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.
10
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.
11
Figure S5 Competitive inhibition of anti-[14C]FACBC (FACBC) and [14C]Met (Met) transport in a rat glioma
cell line (C6) 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 2–7 independent experiments (n = 6–21).
* P < 0.05, ** P < 0.01.
12
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