Nuclear Medicine and Biology 39 (2012) 1213–1218

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Nuclear Medicine and Biology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n u c m e d b i oTransport mechanism of 11C-labeled L- and D-methionine in human-derived
tumor cells☆,☆☆
Masato Kobayashi a, b, 1, Fumiya Hashimoto a, 1, Kazuyo Ohe a, Takahiro Nadamura a, Kodai Nishi a,
Naoto Shikano c, Ryuichi Nishii d, Tatsuya Higashi e, Hidehiko Okazawa b, Keiichi Kawai a, b,⁎aSchool of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-0942, Japan
Biomedical Imaging Research Center, University of Fukui, Fukui 910-1193, Japanc
Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, Ibaraki 300-0331, Japand
Department of Radiology, School of Medicine, Miyazaki University, Miyazaki 889-2192, Japane
Division of PET Imaging, Shiga Medical Center Research Institute, Shiga 524-8524, Japanba r t i c l e

i n f oArticle history:
Received 25 April 2012
Received in revised form 19 May 2012
Accepted 7 June 2012
Keywords:
Methionine
PET
Tumor
Amino acid transport system
Gene expressiona b s t r a c t
Introduction: S-methyl- 11C-labeled L- and D-methionine (11C-L- and D-MET) are useful as radiotracers fortumor imaging. However, it is not known whether the transport mechanism of 11C-D-MET is the same as thatfor 11C-L-MET, which is transported by the amino acid transport system L. In this study, we investigated thetransport mechanism of 11C-L- and D-MET by analyzing the expression of transport system genes in humanderived tumor cells.
Methods: The expression of transport system genes in human-derived tumor cells was quantitatively
analyzed. The mechanism of MET transport in these cells was investigated by incubating the cells with [Smethyl- 3H]-L-MET (3H-L-MET) or [S-methyl- 3H]-D-MET (3H-D-MET) and the effect of 2-amino-2- norbornane-carboxylic acid, a system L transport inhibitor, or α-(methylamino)isobutyric acid, a system A transport
inhibitor, on their transport was measured. The transport and metabolic stability of [S-methyl- 14C]-L-MET(14C-L-MET) and 3H-D-MET was also analyzed using bearing mice with H441 or PC14 tumor cells.
Results: 3H-D-MET was mainly transported by both systems L and alanine–serine–cysteine (ASC), while
system L was involved in 3H-L-MET transport. There was a high correlation between both 3H-L-MET and 3H-DMET uptake and the expression of amino acid transport system genes. In the in vivo study, H441-cellaccumulation of 3H-D-MET was higher than that of 14C-L-MET. Hepatic and renal accumulation of 3H-D-MET
was lower than that of 14C-L-MET.
Conclusion: The transport mechanism of 3H-D-MET was different from that of 3H-L-MET. Since 3H-D-MET hashigh metabolic stability, its accumulation reﬂects the transporter function of system L and ASC.
© 2012 Elsevier Inc. All rights reserved.

1. Introduction
Amino acid transporter genes are highly expressed in tumor cells
and enable these cells to take up a large amount of amino acids [1].
These transporters are categorized into at least 17 distinct classes [2].
Neutral amino acids are considered to be mainly transported by threesystems: systems L, A, and alanine–serine–cysteine (ASC) [3]. [Smethyl- 11C]-L-methionine ( 11C-L-MET), a natural amino acid, hasbeen used as a positron emission tomography (PET) tracer to detect☆ Conﬂicts of interest: There are no conﬂicts of interest.
☆☆ Financial support: This study was partly funded by Grants-in-Aid for ScientiﬁcResearch from the Japan Society for the Promotion of Science (Nos. 21659268,
22591372, 22791180, and 23300361).
⁎ Corresponding author. Tel.: + 81 76 265 2527; fax: + 81 76 234 4366.
E-mail address: kei@mhs.mp.kanazawa-u.ac.jp (K. Kawai).
1
Masato Kobayashi and Fumiya Hashimoto are co-ﬁrst authors and contributed
equally to this work.
0969-8051/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nucmedbio.2012.06.006

tumors. 11C-L-MET is transported into cells via the amino acidtransport system L. 11C-L-MET accumulates in tumors due to theenhanced protein synthesis and transmethylation metabolic functionsof tumors that allow 11C-L-MET to become incorporated into thetumor molecules [4]. In a clinical study, 11C-L-MET has been used in
the diagnosis and follow-up of glioma patients and is superior to 218
F-ﬂuoro-2-deoxy-D-glucose ( 18F-FDG) for delineating tumor margins [5,6]. In addition, there are reports that 11C-L-MET is as usefulas 18F-FDG in the differential diagnosis of lung cancer [7]. Currently,
there are limitations to the use of 18F-FDG for the diagnosis of cerebral
tumors because 18F-FDG also accumulates in normal brain tissue.
Furthermore, 11C-L-MET cannot be applied to the detection of
abdominal tumors because it accumulates in metabolically normal
tissues and is affected by enzymatic metabolism.
D-methionine (D-MET) is considered to be less affected byenzymatic metabolism than L-MET. Since there is very little metabolism of [S-methyl- 11C]-labeled D-MET ( 11C-D-MET), 11C-D-MET has

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M. Kobayashi et al. / Nuclear Medicine and Biology 39 (2012) 1213–1218

been suggested as a potentially useful PET tracer for tumor imaging[8–10]. According to Tsukada et al. [10], who evaluated 11C-D-MET
accumulation using human cervical carcinoma-derived HeLa cells, theaccumulation of 11C-D-MET in tumors is superior to that of 11C-L-MET.
However, the transport mechanism by which 11C-D-MET accumulates
in tumors has not been identiﬁed. In this study, we determined themechanism of accumulation of 11C-D-MET and reinvestigated themechanism of 11C-L-MET accumulation in human-derived tumor cells.
Because 11C-L-MET has a short half-life, S-methyl- 3H or S-methyl- 14C,
both of which act in the same way as 11C-L-MET, was used insteadof 11C-L-MET. Real-time reverse transcription-polymerase chain
reaction (qRT-PCR) was used to measure the gene expression ofneutral amino acid transport systems. Correlations between tumor
accumulation of L-MET and D-MET and the gene expression of neutralamino acid transport systems were evaluated in an in vitro study. In
addition, since the stability of L-MET and D-MET in normal tissue has
not been thoroughly investigated, the metabolic stability of L-MET and
D-MET was assessed in the mouse liver and kidney, which areresponsible for their metabolism.
2. Materials and methods
2.1. Materials[S-methyl- 3H]-L-methionine ( 3H-L-MET: 2.96 GBq/mmol), [Smethyl- 3H]-D-methionine ( 3H-D-MET: 2.96 GBq/mmol), [S-methyl14C]-L-MET ( 14C-L-MET: 2.22 GBq/mmol) and [S-methyl-14C]-D-MET
14( C-D-MET: 2.22 GBq/mmol) were purchased from American Radiolabeled Chemicals Co. (St. Louis, MO, USA).
2.2. Tumor cells
The cultured human tumor cell lines H441, PC14 and MDA-MB435
were got from the University of Texas, MD Anderson Cancer Center,
Houston, TX, USA. The LS180 cell line was purchased from Dainippon
Sumitomo Pharma Co., Ltd (Osaka, Japan). H441 and PC14 are
pulmonary adenocarcinoma cell lines, MDA-MB435 is a breast cancer
cell line and LS180 is a colon cancer cell line. These cancer cells wereselected as representative cells to evaluate transport mechanism of Land D-MET. All tumor cells (2.0×10 5 cells/ml/well) were incubated in
24-well plates with 2 ml of Dulbecco's modiﬁed Eagle's medium (pH7.4; Sigma Chemical Co., St. Louis, MO, USA) in a 10% CO2 atmosphere.
A cell proliferation curve was prepared to determine the duration of
the logarithmic growth phase.
2.3. qRT-PCR analysis of human-derived tumor cells
Total RNA was harvested from each tumor cell using an RNeasy
Mini kit (QIAGEN K. K, Tokyo, Japan). The quality of the total RNA wasevaluated using a bioanalyzer (Agilent Technologies, Santa Clara, CA,
USA). Complementary DNA (cDNA) was ampliﬁed only from highquality total RNA using an AfﬁnityScript qRT-PCR cDNA Synthesis kit(Agilent Technologies). The thermal proﬁle of the reaction was asfollows: 5 min at 25°C for 1 cycle, 15 min at 42°C for 1 cycle and 55
min at 95°C for 1 cycle. The genes analyzed by qRT-PCR were Na +dependent transport systems A (SNAT1, SNAT2, SNAT4) and ASC(ASCT1, ASCT2), and Na +-independent transport systems L (LAT1,
LAT2, LAT3, LAT4) and 4F2hc. Two different housekeeping genes,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta actin(ACTB) were used as internal controls to compensate for thedifferences between the initial RNA and cDNA amounts. An
Mx3005P thermocycler (Agilent Technologies) was used for the
qRT-PCR reactions. Primers were synthesized by Nihon Gene ResearchLaboratories, Inc. (Miyagi, Japan). The artiﬁcial plasmid used for thecalibration curve was synthesized by GenScript (Piscataway, NJ, USA).
The qRT-PCR reagent used in this study was Brilliant II Fast SYBRGreen qRT-PCR Master Mix (Stratagene Products Division, Agilent
Technologies). The thermal proﬁle of the reaction was as follows: 2
min at 95°C for 1 cycle, 5 s at 95°C followed by 20 s at 60°C for 40
cycles, 1 min at 95°C followed by 30 s at 55°C and 30 s at 95°C for 1
cycle. In addition, serial dilutions of artiﬁcial plasmids were
performed. The samples used were created using a dilution series
ranging from 1×10 2 to 1×10 8 copies per tube for the calibration curve.
All reactions were performed in triplicate.
2.4. Assay of MET transport in human-derived tumor cells
Transport assays were performed according to the method
developed by Shikano et al. [11]. In brief, tumor cells were seeded
on 24-well cell culture multiwell plates at a density of 2×10 5 cells/ml/
well. The assays were conducted 24 h after seeding.
The sodium-containing incubation medium used was based on
phosphate-buffered saline (Na +-PBS), consisting of 137 mM NaCl, 2.7
mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 5.6 mM D-glucose, 0.9 mM
CaCl2 and 0.5 mM MgCl2. In the sodium-free incubation medium(Na +-free PBS), NaCl and Na2HPO4 were replaced with the equivalentconcentration of choline chloride and K2HPO4, respectively. After theculture medium was removed, each well was incubated with 1 ml ofincubation medium for 10 min at 37°C. The cells were then incubatedwith 0.5 ml of incubation medium containing the 3H-L-MET or 3H-DMET (18.5 kBq) for 0.5 min at 37°C as a control condition. For thecompetitive inhibition assay, the cells were incubated with 3H-L-MET
or 3H-D-MET and 1.0 mM of inhibitors, 2-amino-2-norbornanecarboxylic acid (BCH; Sigma Chemical Co.), a substrate speciﬁc tosystem L transporters, and α-(methylamino)isobutyric acid (MeAIB;
Sigma Chemical Co.), a substrate speciﬁc to system A transporters. At
the end of the incubation, each well was rapidly washed twice with 1
ml of ice-cold incubation medium. The cells were then solubilized in
0.5 ml of 0.1 N NaOH, mixed with an ASC-II Scintillation Cocktail (GEHealthcare UK Ltd, Little Chalfont, England), and radioactivity wasthen measured with a liquid scintillation counter (Aloka, Tokyo,
Japan; LSC-5100). Subsequently, the cells were detached from one
another with trypsin to facilitate further counting. All experimental
conditions were examined in quadruple assays. Reproducibility was
conﬁrmed by repeating the same experiment.
To measure the contribution of amino acid transport systems to
MET transport, we used the method devised by Shotwell [12] andNakajima et al. (Fig. 1) [13]. Brieﬂy, MET uptake in the absence of
inhibitors was calculated as a percentage of the control value, which
was set at 100%. System A transport was calculated by subtracting
MET uptake in the presence of MeAIB from uptake in the control Na +PBS. System ASC transport was calculated by subtracting the value of
MET uptake in control Na +-free PBS from MET uptake in the presenceof MeAIB. Finally, system L participation was calculated by subtracting
MET uptake in the presence of BCH from MET uptake in control Na +free PBS. The rates of tumor uptake of 3H-L-MET and 3H-D-MET were
calculated as uptake rates per 10 5 tumor cells (% injected dose (ID)/
10 5 cells). The correlation between tumor uptake of 3H-L-MET and 3HD-MET and expression of neutral amino acid transport system genes
was also examined. The result for each set of experimental conditions
represents the mean of four wells on the cell culture multiwell plates.
2.5. Biodistribution of 14C-L-MET and 3H-D-MET in tumor-bearing mice
Animal studies were approved by the Animal Care Committee atKanazawa University and were conducted in accordance with theinternational standards for animal welfare and institutional guidelines. Adequate measures were taken to minimize pain anddiscomfort. Male KSN/Slc nude mice from Japan SLC Inc. (Hamamatsu,
Japan) were housed for 1 week under a 12-h light/12-h dark cyclewith free access to food and water. The mice were fasted with no foodovernight with water supplied ad libitum before experiments.

M. Kobayashi et al. / Nuclear Medicine and Biology 39 (2012) 1213–1218

100
90
80

% of control

70

System A
Na+-dependent

60
50

System ASC

40
30
20

Na+-independent
System L

1215

renal samples in the acid-soluble fraction. Because 3H-D-MET will not
be appropriated in thin-layer chromatography, 14C-D-MET was usedinstead as it has the same labeling position and similar uptake
properties as 3H-D-MET. Tissue homogenates to which trichloroacetic
acid had been added were centrifuged. The supernatant was spottedon a thin-layer chromatography plate, and the signal was then
expanded using a developing solvent (butanol/acetic acid/water,
4:1:1) followed by exposure on an imaging plate (BAS-SR 2025; Fuji
Film, Tokyo, Japan). BAS5000 (Fuji Film) was then used to evaluate theamount of unmetabolized 14C-L-MET or 14C-D-MET in the acid-soluble
fraction. The amount of 14C-L-MET or 14C-D-MET that was metabolized
was determined by calculating the rate of 14C-L-MET or 14C-D-MET
uptake into protein fractions and the level of the parent 14C-L-MET
and 14C-D-MET component.

10

2.6. Statistical analysis

0

Control MeAIB Control BCH
in Na+in Na+PBS
free-PBS
Fig. 1. Calculation of the contribution of amino acid transport systems to MET uptake.
100%=control uptake of 3H-L-MET or 3H-D-MET in Na+-PBS. System A=(control
uptake of 3H-L-MET or 3H-D-MET in Na+-PBS)−(uptake of 3H-D-MET or 3H-L-MET with
MeAIB inhibitor in Na+-PBS). System ASC=(uptake of 3H-D-MET or 3H-L-MET with
MeAIB inhibitor in Na+-PBS)−(control uptake with Na+ free-PBS). System L=(control
uptake of 3H-L-MET or 3H-D-MET in Na+ free-PBS)−(uptake of 3H-L-MET or 3H-D-MET
with BCH inhibitor in Na+ free-PBS).

H441 cells, in which neutral amino acid transport systems arehighly expressed, and PC14 cells, which have the lowest level ofexpression of amino acid transport systems of the cells assayed in thepresent study, were prepared to a ﬁnal density of 2×10 6 cells per 100
μl. The prepared H441 and PC14 cells (100 μl per mouse) were
subsequently injected subcutaneously into the left and right thighs,
respectively, of ﬁve KSN/Slc nude mice. Approximately 1 week after
transplantation of the H441 and PC14 cells, 14C-L-MET (18.5 kBq)
and 3H-D-MET (92.5 kBq) were simultaneously injected as double
tracers into the tail veins of the tumor-bearing mice. The mice weresacriﬁced at 2, 10, 30 or 60 min postinjection. After blood was sampled
via cardiocentesis, the tumors, pancreas, kidney, muscle and liver
were excised. Approximately 100 mg of each excised organ was
weighed, and SOLVABLE (Perkin-Elmer, Waltham, MA, USA) was
added. The samples were then shaken for 3H at 50°C. After the
samples were dissolved, Ultima Gold (Perkin-Elmer) was added.
Sample radioactivity was measured using a liquid scintillation
counter, and a quenching correction was performed. To compare
the accumulation of 14C-L-MET and 3H-D-MET in organs and tumors,
the unit of each organ and tumor accumulation was calculated as thepercentage of ID per gram (%ID/g). We also evaluated the level of MET
accumulated in the tumor relative to that accumulated in each organ
by dividing the amount of MET accumulated in H441 or PC14 tumor
cells by the amount of MET accumulated in the muscle, liver andplasma of the corresponding tumor-bearing mice. These values areshown as H441 or PC14/muscle, liver or plasma in Table 2.
We also analyzed the levels of 14C-L-MET or 3H-D-MET metabolites
in hepatic and renal samples from the tumor-bearing mice. These
tissue samples were homogenized in a bicarbonate buffer, and 100%
trichloroacetic acid (Nacalai Tesque, Kyoto, Japan) was then added tothe homogenate to a ﬁnal concentration of 5% and mixed. The
precipitated fraction was collected in a glass ﬁlter (ADVANTEC, Tokyo,
Japan) and washed with ice-cold 5% trichloroacetic acid. Protein in the
precipitated fraction was ﬁxed using thermal processing at 150°C for 1
h. Radioactivity was measured and was used to evaluate the uptakeof 14C-L-MET or 3H-D-MET into the protein fraction. In a further
experiment, 18.5 kBq of 14C-L-MET or 14C-D-MET was injected into
ﬁve normal mice to evaluate the metabolic stability of hepatic andData are presented as means and standard deviations (S.D.s). P
values were calculated using a two-tailed paired Student's t test forcomparison between two groups. A P value less than 0.01 and 0.05
was considered signiﬁcant.
3. Results
3.1. Gene expression of amino acid transport systems in human-derived
tumor cells
The expression of amino acid transport system genes in thehuman-derived tumor cell lines H441, MDA-MB435, LS180 and PC14
was analyzed using qRT-PCR. The high quality of the total RNA
harvested from each tumor cell was conﬁrmed using a bioanalyzer(data not shown). The gene expression levels of neutral amino acidtransport systems in each cell line were calculated using a calibration
curve for each gene (Table 1). Transporters of the Na +-dependenttransport system, system A and system ASC were expressed (in orderfrom the highest to the lowest levels of expression) in H441, MDAMB435, LS180 and PC14 cells. Transporters of the Na +-independenttransport system, system L and the coupling factor, 4F2hc, were highly
expressed in H441 cells, followed by MDA-MB435, and then LS180
cells, but little expression was observed in PC14 cells.
3.2. Assay of MET transport in human-derived tumor cells
The contribution of each amino acid transport system to tumor cell
uptake of 3H-L-MET and 3H-D-MET was analyzed using speciﬁc
inhibitors and measured at 0.5 min after 3H-L-MET or 3H-D-MET
injection (Fig. 2). System L was involved in tumor uptake of 3H-L-MET
and mediated over 50% of the total uptake of 3H-L-MET that was

Table 1
Expression of genes of neutral amino acid transport systems in human-derived
tumor cells.

Na+-dependent

SystemFamilyH441

MDAMB435

LS180

PC14

A

SNAT1
SNAT2
SNAT4
ASCT1
ASCT2
LAT1
LAT2
LAT3
LAT4
4F2hc

91.66
30.55
0.02
23.6
65.95
568.83
17.87
0.62
0.80
1008.52

321.56127.840.63
30.6
323.37167.93ND
3.66
1.45
608.77

91.49
20.34
0.38
8.27
89.33
76.83
11.1
5.08
3.69
217.08

76.71
81.41
0.01
3.35
29.98
6.35
ND
0.73
0.24
50.80

ASC
+

Na -independent

L

Coupling
factorData are expressed as copy number per 1000 copies of housekeeping genes. ND:
not determined.

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M. Kobayashi et al. / Nuclear Medicine and Biology 39 (2012) 1213–1218

A

B

Uptake (% of control)

100

80

60

40

20

0

Uptake (% of control)

0

*

20

40

60

H441
*

LS-180*100*

MDAMD435

*

80

*

PC14
System L*

System A

System ASC

Fig. 2. Contribution of each amino acid transport system to 3H-L-MET and 3H-D-MET uptake in human-derived tumor cells. Tumor uptake at 0.5 min after 3H-L-MET (A) and 3H-D-MET(B) addition is shown. System L played a signiﬁcant role in tumor transport of 3H-L-MET, whereas both systems L and ASC played a large role in tumor transport of 3H-D-MET,
signiﬁcantly PC14 cells. The asterisks indicate Pb0.01 between the uptake of 3H-L-MET and 3H-D-MET in each tumor cell.

observed in each of the control tumor cells. System L was also
involved in tumor uptake of 3H-D-MET and mediated over 46% of thetotal 3H-D-MET uptake that was observed in control H441, MDAMB435 and LS180 cells. However, in PC14 cells, system ASC mediatedabout 70% of the total 3H-D-MET uptake. System A was not involved intumor uptake of 3H-L-MET or 3H-D-MET.
3.3. Correlation between MET uptake and transporter gene expression inhuman-derived tumor cells
The correlation between tumor uptake of MET and the expressionof amino acid transport system genes is shown in Fig. 3. The above
data (Fig. 2) showed that transport system L was the main mediator of
3
H-L-MET transport and that both system L and ASC mediated 3H-DMET transport. We therefore evaluated correlations between tumor
uptake of 3H-L-MET and expression of system L genes, and between
tumor uptake of 3H-D-MET and expression of both system L and ASC
genes. There was a high correlation between both 3H-D-MET and 3H-LMET uptake and the expression of amino acid transport system genes.
3.4. Biodistribution of 14C-L-MET and 3H-D-MET in tumor-bearing mice
The biodistribution of 14C-L-MET and 3H-D-MET in H441 and PC14
tumor-bearing mice is shown in Table 2. Tumor accumulation of 3H-DMET was signiﬁcantly greater than that of 14C-L-MET in H441-bearing
mice. Tumor accumulation of 14C-L-MET and 3H-D-MET in PC14bearing mice was signiﬁcantly lower than that of H441-bearing mice.
Furthermore, 3H-D-MET accumulation in the PC14 tumors was
signiﬁcantly lower than that of 14C-L-MET. There was high accumulation of both 14C-L-MET and 3H-D-MET in the pancreas and kidney ofIn this study, we propose a transport mechanism of MET,
particularly of D-MET, into human-derived tumor cells that is basedon accurate qRT-PCR evaluation of the gene expression levels ofneutral amino acid transport systems in tumor cells. We reconﬁrmed previous reports [12,14–16] demonstrating that system L
was the main system for transport of 3H-L-MET (Figs. 2A and 3A).
Shotwell et al. [12] divided the uptake of L-MET by Chinese hamster
ovary (CHO)-K1 cells into four components: approximately 52% bysystem L, 21% by system ASC, 20% by nonsaturable processes and 7%
by system A. Oxender and Chistensen [14] reported the involvement of systems L and A in the uptake of 14C-L-MET by Ehrlich cells.
Guerino and Baumrucker [15] estimated that 14C-L-MET wastransported into cattle small intestine as follows: 54% throughsystem L, 28% through system ASC, 25% through nonsaturable

B

1.0

R2 = 0.88

0.8
0.6
0.4
0.2
0.0

4. Discussion

Uptake (%ID/105 cells)

Uptake (%ID/105 cells)A

the tumor-bearing mice, but 3H-D-MET accumulated to a higher level
than 14C-L-MET at early time points after injection. The accumulationof 3H-D-MET was lower than that of 14C-L-MET in the liver, but higher
in plasma of the tumor-bearing mice.
Fig. 4 shows the percentage of the injected 14C-L-MET and 14C-DMET incorporated into proteins and nonproteins and the percentage ofnonmetabolized 14C-L-MET and 14C-D-MET in the liver (A) and kidney(B) of normal mice at 10 min after injection. Approximately 25% to 50%of the injected 14C-L-MET was incorporated into protein, whereas thelevel of 14C-D-MET incorporation was much lower at approximately
3%. The incorporation of 3H-D-MET into nonprotein molecules was
lower than that of 14C-L-MET. Approximately 20% to 40% of 14C-L-MET,
but more than 80% of 14C-D-MET, remained unincorporated.

0

100

200

300

400

500

600

Gene expression (system L)(arb. units)

700

1.0

R2 = 0.99

0.8
0.6
0.4
0.2
0.0

0

100

200

300

400

500

600

700

Gene expression (system L+ASC)(arb. units)

Fig. 3. Correlation between tumor uptake of 3H-L-MET and 3H-D-MET at 0.5 min after the addition and the expression of amino acid transport system genes in human-derived tumor
cells. The ﬁgures show the relationship between 3H-L-MET uptake and expression of system L genes (A) and that between 3H-D-MET uptakes and expression of system L and ASC
genes (B). Cell lines analyzed were as follows: H441 (●), MDA-MB435 (▲), LS180 (■) and PC14 (○). The tumor uptake of 3H-D-MET showed a higher correlation with expression oftransport genes than that of 3H-L-MET.

M. Kobayashi et al. / Nuclear Medicine and Biology 39 (2012) 1213–1218

1217

Table 2
Biodistribution of 14C-L-MET and 3H-D-MET in H441 and PC14 tumor-bearing mice.
14

3

C-L-MET

H-D-MET

min

2

10

30

60

2

10

30

60

H441
PC14
Pancreas
KidneyMuscleLiver
PlasmaH441/muscle
H441/liver
H441/plasma
PC14/muscle
PC14/liver
PC14/plasma

3.94±0.84
1.24±0.38
29.53±2.32
11.99±0.99
2.24±0.13
6.21±0.78
4.31±0.34
1.76±0.41
0.63±0.11
0.91±0.22
0.55±0.11
0.20±0.11
0.29±0.08

7.02±0.75
3.24±0.45
45.60±3.61
12.62±1.73
2.50±0.38
10.23±2.11
1.94±0.07
2.81±0.48
0.69±0.13
3.62±0.10
1.30±0.25
0.32±0.08
1.67±0.31

7.42±1.27
3.90±0.71
43.13±5.96
8.50±0.87
1.68±0.20
10.42±1.48
1.31±0.09
4.42±0.56
0.71±0.18
5.66±0.19
2.32±0.42
0.37±0.13
2.98±0.15

5.46±0.69
3.15±0.58
32.79±7.01
6.52±0.73
1.30±0.21
8.13±0.84
1.02±0.14
4.20±0.35
0.67±0.10
5.35±0.15
2.42±0.24
0.39±0.47
3.09±0.11

4.56±0.65**
1.15±0.25
30.61±2.53
32.83±2.90*
2.03±0.24
3.40±0.32*
8.26±1.16*
2.25±0.39**
1.34±0.18*
0.55±0.15*
0.57±0.22
0.34±0.15**
0.14±0.07**

11.03±1.52*
1.84±0.21*
57.56±5.17*
30.70±3.55*
2.33±0.63
3.52±0.44*
4.76±0.26*
4.73±0.58*
3.13±0.41*
2.32±0.27**
0.79±0.21**
0.52±0.13*
0.39±0.10*

13.50±1.98*
2.11±0.35*
26.51±6.08*
13.86±0.38*
1.94±0.21**
2.62±0.19*
2.44±0.18*
6.96±0.50*
5.15±0.43*
5.53±0.49
1.09±0.18*
0.81±0.10*
0.86±0.09*

10.20±1.09*
1.73±0.39*
10.20±1.09*
7.20±0.12**
1.82±0.33*
1.83±0.12*
1.45±0.08**
5.60±0.25*
5.57±0.18*
7.03±0.32*
0.95±0.06*
0.95±0.05*
1.19±0.04*

Data are expressed as means±.S.D. Values of 3H-D-MET are signiﬁcantly different than those of 14C-L-MET at each time point after injection (*Pb0.01, **Pb0.05, paired t test). The
units are the percentage of injected dose per gram (%ID/g) of MET accumulated in the tumor cells and in each organ. The values of H441(PC14)/muscle, H441(PC14)/liver andH441(PC14)/plasma indicate the values for MET accumulation in the tumor tissue, divided by MET accumulation in the indicated organ of the corresponding tumor-bearing mice.

processes and 15% through system A. Shikano et al. [16] reported
that system L mediated approximately 85% of [1- 14C]-labeled L-MET
transport in CHO-K1 cells, system A mediated approximately 10% of
its intracellular transport and nonsaturable processes mediatedapproximately 5% of its transport. In contrast to all of these studies
that evaluated L-MET transport mechanisms of normal cells
expressing speciﬁc levels of amino acid transport system genes,
our study estimated the L-MET transport mechanism using four
different tumor cell lines, which express different levels of transportsystem genes. The high correlation between tumor uptake of 3H-LMET and the expression levels of system L genes (R 2=0.88; Fig. 3A)
indicated that 3H-L-MET is mainly transported through system L in
the tumor cells.
Regarding 3H-D-MET transport, system L was very much involved
in its transport in H441, MDA-MB435 and LS180 cells, but system ASC
was dominant for its transport in PC14 cells (Fig. 2B). There was astronger correlation between 3H-D-MET uptake and the expression ofsystem L and ASC genes in the tumor cells than that between 3H-LMET uptake and the expression of system L genes (R 2=0.99) (Fig. 3).
Shikano et al. [16] reported that system L mediated approximately
95% of the transport of [1- 14C] labeled D-MET in CHO-K1 cells and thatsystem A mediated approximately 5% of its intracellular transport.
Since the labeling position of 14C on D-MET used in their study, [1- 14C],
differed from that of 3H and 11C used in our study and in clinicalstudies, [S-methyl-3H and 11C], it is important to investigate thetransport mechanism of 3H-D-MET based on the gene expression ofreporters in various kinds of tumor cells. In our study, the tumortransport of 3H-D-MET was associated with both systems L and ASC(Figs. 2B and 3B). Although we used three different human tumor celllines — lung, breast and colon cancers — to investigate the transportmechanism of L- and D-MET, the transport mechanism and uptake of
MET may be different in the brain tumor cells, which is useful forclinical PET study.
Recent nuclear medicine studies have evaluated the correlation
between the uptake of radiotracers and the expression of transportsystem genes in tumor cells [17–19]. These studies were performed inorder to identify the uptake mechanisms of radiotracers in tumor
cells. Forstner et al. [17] reported that the imaging results obtainedwith the three radiotracers, 18F-FDG, 3-deoxy-3- 18F-ﬂuorothymidine
and 18F-ﬂuoroethylcholine, could be explained by the gene expression
patterns of membrane transporters and enzymes that are involved inradiotracer uptake and retention, which were measured in the tumor
cells using qRT-PCR. By comparing 18F-FDG PET imaging with theresults of qRT-PCR analysis of gene expression, Christen et al. [18]showed that human visceral adipose tissue has increased glucose
uptake compared with subcutaneous adipose tissue. By measuring thegene expression levels of amino acid transporters in prostate cancer,
our research group found that the transport mechanism of trans-1amino-3- 18F-ﬂuorocyclobutanecarboxylic acid involves not only
system L but also system ASC [19]. Hence, gene analysis enables
accurate determination of the detailed transport and uptake mechanism of radiotracers.
For in vivo studies, we selected H441 cells, which have highexpression levels of all of the genes of system L and ASC, and PC14 cells,
which display the lowest level of expression of system L and ASC genesof the four tumor lines assayed (Table 1). The transport mechanismof 14C-L-MET and 3H-D-MET in these cells that was identiﬁed in an invitro study was conﬁrmed in H441 and PC14-bearing mice (Table 2).
As shown by the expression levels of the genes of the different
transport systems, tumor accumulation of 14C-L-MET and 3H-D-MET
was signiﬁcantly higher in H441 than in PC14 cells, and in particular,
accumulation of 3H-D-MET was signiﬁcantly increased (Pb0.01). In
PC14 cells, the accumulation of 3H-D-MET was signiﬁcantly lower thanFig. 4. Proportion of 14C-L-MET and 14C-D-MET incorporated into protein andnonprotein metabolites in liver and kidney of normal mice. The percentage of 14C-LMET and 14C-D-MET incorporated into protein and nonprotein metabolites and thepercentage of parent 14C-L-MET and 14C-D-MET in the liver (A) and kidney (B) were
measured at 10 min after radiotracer injection. The combination of all three values
was set at 100% for each organ. The incorporation of 14C-D-MET into protein andnonprotein metabolites was lower than that of 14C-L-MET, and over 80% of the 14C-DMET remained unmetabolized.

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M. Kobayashi et al. / Nuclear Medicine and Biology 39 (2012) 1213–1218

that of 14C-L-MET (Pb0.01). However, Bergström et al. [20] reported
that the accumulation of 11C-D-MET was lower than that of 11C-L-MET
in a brain tumor. These data suggest that if the transport systems
involved in the transport of 3H-D-MET are highly expressed in tumor
cells, then tumor accumulation of 3H-D-MET would be greater thanthat of 14C-L-MET.
One of the reasons why 11C-D-MET is useful in tumor diagnosis isthat it is metabolically stable in plasma samples [10]. Therefore, in
our study, we examined the incorporation of 14C-L-MET and 14C-DMET, which have similar characteristics to 11C-D-MET, into hepaticand liver proteins and nonprotein molecules, as well as the level ofunincorporated 14C-L-MET and 14C-D-MET (Fig. 4). Approximately
25% to 40% of 14C-L-MET was incorporated into protein, but theincorporation of 14C-D-MET was signiﬁcantly lower at approximately
3%. We also conﬁrmed that more than 80% of 14C-D-MET was
unincorporated; in other words, 14C-D-MET is not highly incorporated into proteins. There have been a few reports of large amountsof D-MET being converted into L-MET by D-amino acid oxidase via αketo-γ-methiobutyrate, and of L-MET being utilized for protein
synthesis [21,22]. However, in our study, only approximately 3%of 14C-D-MET was utilized for protein synthesis (Fig. 4). We
therefore predict that it is unlikely that metabolites of 14C-D-MET
would affect the accumulation of 14C-D-MET in tumor cells. As
shown in Table 2, the accumulation of 3H-D-MET in the liver was
lower than that of 14C-L-MET. Both 14C-L-MET and 3H-D-MET
accumulated in the pancreas, where amino acids are known toaccumulate and protein synthesis is active, suggesting that both 14C3
14
L-MET and H-D-MET are recognized as amino acids.
C-L-METand 3H-D-MET accumulated to the same degree in muscle. The
accumulation of 3H-D-MET was higher than that of 14C-L-MET in
plasma at early time points after injection, but it was subsequently
rapidly cleared. As a consequence, the tumor-to-muscle ratio and thetumor-to-liver ratio of 3H-D-MET were signiﬁcantly increased
compared with the ratios of 14C-L-MET for both H441 and PC14
tumors. On the other hand, the tumor-to-plasma ratios of 14C-L-MET
were higher than those of 3H-D-MET for up to 30 min after injection,
whereas the reverse was the case at 60 min after injection, which isconsistent with a report by Tsukada et al. [10]. Our results show that
11
C-D-MET, unlike 11C-L-MET, is not incorporated into protein, has
few metabolites and is accumulated in tumor cells via the speciﬁc
neutral amino acid transport systems L and ASC. We speculate that
other D-amino acid tumor imaging radiotracers may also betransported by a different transport mechanism from that of theirL-amino acid.

5. Conclusion
The transport mechanism of 3H-L-MET and 3H-D-MET in humanderived tumor cells differed. 3H-D-MET was transported by systems L
and ASC, whereas 3H-L-MET was transported by system L. In an in vivo
study, tumor uptake and accumulation of 3H-D-MET were signiﬁcantly
greater than that of 14C-L-MET. Incorporation of 14C-D-MET into
protein and nonprotein molecules was signiﬁcantly lower than thatof 14C-L-MET. Based on the above in vitro and in vivo studies, weconclude that 11C-D-MET is a potentially useful radiotracer for tumor
imaging in PET studies of metabolic organs.

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