brain research 1535 (2013) 24–37

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www.elsevier.com/locate/brainres

Research ReportComparative evaluation of transport mechanismsof trans-1-amino-3-[18F]ﬂuorocyclobutanecarboxylic
acid and L-[methyl-11C]methionine in human glioma
cell lines
Masahiro Onoa,b, Shuntaro Okaa,n, Hiroyuki Okudairaa, David M. Schusterc,
Mark M. Goodmanc, Keiichi Kawaib, Yoshifumi ShirakamiaaResearch Center, Nihon Medi-Physics Co., Ltd., Chiba 299-0266, Japan
Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, Ishikawa 920-0942, Japanc
Division of Nuclear Medicine and Molecular Imaging, Department of Radiology and Imaging Science, Emory University,
Atlanta, GA 30322, USAbar t ic l e in f o

abs tra ct

Article history:

Positron emission tomography (PET) with amino acid tracers is useful for the visualization

Accepted 21 August 2013

and assessment of therapeutic effects on gliomas. Our purpose is to elucidate the transport

Available online 27 August 2013mechanisms of trans-1-amino-3-[18F]ﬂuorocyclobutanecarboxylic acid (anti-[18F]FACBC)
and L-[methyl-11C]methionine ([11C]Met) in normal human astrocytes (NHA), low-grade

Keywords:(Hs683, SW1088), and high-grade (U87MG, T98G) human glioma cell lines. Because the shortanti-FACBC

half-lives of ﬂuorine-18 and carbon-11 are inconvenient for in vitro experiments, trans-1-

Methionine

amino-3-ﬂuoro[1-14C]cyclobutanecarboxylic

Gliomaacid(anti-[14C]FACBC)

andL-[methyl-

14

C]

14

methionine ([ C]Met) were used instead of the PET tracers. Time-course uptake experi-

Amino acid transporter

ments showed that uptake of anti-[14C]FACBC was 1.4–2.6 times higher than that of [14C]

ASCT2

Met in NHA and low-grade glioma cells, and was almost equal to that of [14C]Met in high-LAT1

grade glioma cells. To identify the amino acid transporters (AATs) involved in the transportof anti-[14C]FACBC and [14C]Met, we carried out competitive inhibition experiments usingsynthetic/naturally-occurring amino acids as inhibitors. We found that anti-[14C]FACBC
uptake in the presence of Naþ was strongly inhibited by L-glutamine and L-serine (thesubstrates for ASC system AATs), whereas L-phenylalanine and 2-amino-bicyclo[2,2,1]
heptane-2-carboxylic acid (BCH, the substrates for L system AATs) robustly inhibited Naþindependent anti-[14C]FACBC uptake. Regardless of Naþ, [14C]Met uptake was inhibitedstrongly by L-phenylalanine and BCH. Moreover, the exchange transport activity of Lglutamine for anti-[14C]FACBC was stronger than that of BCH in the presence of Naþ,whereas that for [14C]Met was almost equal to BCH. These results demonstrate that ASC

Abbreviations: AAT(s),18anti-[ F]FACBC,
Met,

L-[methyl-amino acid transporter(s); anti-[14C]FACBC,trans-1-amino-3-ﬂuoro[1-14C]cyclobutanecarboxylic acid;18trans-1-amino-3-[ F]ﬂuorocyclobutanecarboxylic acid; BCH,
11

14C]methionine; [ C]Met,

L-[methyl-

14C]methionine; MeAIB,

N-ethylmaleimide; PET, positron emission tomographynCorresponding author. Fax: þ81 438 62 6969.
E-mail address: shuntaro_oka@nmp.co.jp (S. Oka).
0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.brainres.2013.08.037

2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid; [11C]
2-(methylamino)-isobutyric acid; NEM,

25

brain research 1535 (2013) 24–37

and L are important transport systems for anti-[18F]FACBC uptake, while system L is
predominantly involved in [11C]Met transport in human astrocytes and glioma cells.
& 2013 Elsevier B.V. All rights reserved.

1.

Introduction

With their invasive and proliferative potential, gliomas arefatal lesions that are the most frequent primary brain tumorsin adult humans (Swanson et al., 2002). The current standardof clinical treatment comprises surgical resection followed byradiation therapy and chemotherapy. Although improvements in surgery, radiotherapy, and chemotherapy are associated with an improved prognosis (Clarke et al., 2010; Gorlia
et al., 2008), a recent study reported that the 2-year survival
rate of grade III patients is 65%, and that of grade IV is 31%(Cho et al., 2010). In particular, inﬁltrative and diffuse tumor
that cannot be completely surgically excised are believed tobe responsible for glioma treatment failure (Nazzaro and
Neuwelt, 1990; Swanson et al., 2002). Therefore, precise
identiﬁcation of tumor size and extent is required for effective cancer therapy.
Positron emission tomography (PET) is useful for thedetection and delineation of gliomas, and L-[methyl-11C]
methionine ([11C]Met) is well studied as a PET tracer (Kracht
et al., 2004). However, because of the short half-life of 11C(20 min), the use of 11C-labeled PET tracers is restricted tothose facilities with an on-site cyclotron. To overcome these
problems, 18F (half-life: 110 min)-labeled amino acid analogs
such as trans-1-amino-3-[18F]ﬂuorocyclobutanecarboxylic
acid (anti-[18F]FACBC) were developed (Shoup et al., 1999),
and the feasibility of anti-[18F]FACBC and [11C]Met for imaging
human gliomas has been reported (Akhurst et al., 2006).
Anti-[18F]FACBC is a synthetic amino acid and is therefore
believed to be transported into cells by amino acid transporters(AATs) (Oka et al., 2012; Okudaira et al., 2011). To date, morethan 40 types of AATs have been identiﬁed. They are divided
into two groups by whether the driving force for transport
needs Naþ, and some Naþ-dependent AATs (e.g. the N and yþL
systems) can maintain transport activity when Liþ is substituted for Naþ (Fei et al., 2000; Kanai et al., 2000; Nakanishi et al.,2001a, 2001b). Moreover, it is known that AAT activities areaffected by extracellular pH. For example, system L activities
increase in low pH conditions, but system N activities decrease(Fei et al., 2000; Nakanishi et al., 2001a, 2001b). In terms oftransport types, ASC, yþL, asc, b0.þ, X
,C and L AAT systems areexchangers that exchange their extracellular and intracellular
substrates with a 1:1 stoichiometry (SLC tables, 2013, see“References”). Among these AATs, we have established thatsystems A, ASC, N, and L are integral in trans-1-amino-3ﬂuoro[1-14C]cyclobutanecarboxylic acid (anti-[14C]FACBC) uptake
in human prostate cancer cell lines (Oka et al., 2012; Okudaira
et al., 2011). However, the exact mechanisms of anti-[18F]FACBCtransport into glioma cells remain to be determined. Furthermore, although anti-[18F]FACBC and [11C]Met can image gliomas
equally well in humans (Akhurst et al., 2006), differences in themechanisms transporting these two tracers in gliomas have notbeen elucidated. In the present study, we set out to clarify thedifferences between the transport mechanisms for anti-[18F]
FACBC and [11C]Met by using normal human astrocytes (NHA),
low-grade (Hs683, SW1088), and high-grade (U87MG, T98G)
human glioma cell lines (Kadowaki et al., 2012; Le Mercier
et al., 2009) through a series of experiments, including timecourse experiment, pH dependency, competitive inhibition,exchange transport, and ﬂow cytometry. Here, anti-[14C]FACBC
and L-[methyl-14C]methionine ([14C]Met), which differ from anti[18F]FACBC and [11C]Met, respectively, only in the isotopes
incorporated, were used as tracers, because the short halflives of ﬂuorine-18 and carbon-11 are impractical forin vitro study.

2.

Results

2.1.
Comparison between anti-[14C]FACBC and [14C]Metuptake in NHA and glioma cells
The time course of anti-[14C]FACBC and [14C]Met uptake wasmeasured in NHA and glioma cell lines. As shown in Fig. 1,
the uptake of anti-[14C]FACBC was substantially higher thanthat of [14C]Met in NHA and low-grade glioma cell lines(Hs683 and SW1088), and was almost equal to that of [14C]
Met in high-grade glioma cell lines (U87MG and T98G).
Although signiﬁcant differences between anti-[14C]FACBC
and [14C]Met uptake were shown by 10 min in NHA, Hs683,
and SW1088 cells, they were not seen until 30 or 60 min in
U87MG and T98G cells. The intracellular amount of anti-[14C]
FACBC decreased moderately between 15 and 60 min in all
cell lines, but [14C]Met decreased only in SW1088 cells.

2.2.
Ion dependency and pH dependency of anti-[14C]
FACBC and [14C]Met uptake
Sodium, choline, and lithium buffers were used in thisexperiment because (1) all AATs work in sodium buffer, (2)only Naþ-independent AATs work in choline buffer, and (3)all Naþ-independent AATs and the Naþ-dependent N and yþL
AAT systems work in lithium buffer (Fei et al., 2000; Kanai
et al., 2000; Nakanishi et al., 2001a, 2001b). Knowing this, thecontributions of Naþ-dependent and Naþ-independent AATs
to the uptake of anti-[14C]FACBC and [14C]Met into cells were
estimated. If tracer uptake in choline or lithium buffer was
higher than that in sodium buffer at each pH value, thecontribution was recorded as 100%.
Figs. 2 and 3 show the changes in the contributions ofNaþ-dependent and Naþ-independent AATs to the anti-[14C]
FACBC uptake in NHA and human glioma cell lines at acidic,
neutral, and alkaline pH.
As shown in Fig. 2, the uptake of each tracer in sodium
buffer at pH 7.3 was normalized to 100%. The intracellularamount of anti-[14C]FACBC in sodium buffer at pH 7.3 was26

brain research 1535 (2013) 24–37

Fig. 1 – Time course of anti-[14C]FACBC and [14C]Met uptake in NHA and human glioma cell lines (Hs683, SW1088, U87MG, andT98G). Each point represents the mean7SD (n ¼6). * Po0.05 vs. [14C]Met.

signiﬁcantly higher than that in choline and lithium buffers
in all cell lines (3.6–36.8% and 6.4–40.8% of the uptake insodium buffer at pH 7.3, respectively). At pH 6.0, the uptake ofanti-[14C]FACBC in sodium buffer dropped to 32.1–69.1%,
whereas that in choline and lithium buffers increased to
10.0–58.4% and 9.2–56.8% compared with each buffer at pH
7.3, respectively. At pH 8.5, the uptake of anti-[14C]FACBC in
sodium buffer decreased to 83.4% and 80.6% in Hs683 andT98G, respectively, but not in the other cell lines.

Furthermore, in NHA and T98G, the anti-[14C]FACBC uptake
dropped to 18.0% and 27.9% in choline buffer at pH 8.5, and to
20.7% and 31.8% in lithium buffer at pH 8.5, respectively, but
not in the other cell lines. On the other hand, there was no
signiﬁcant difference for [14C]Met uptake among each buffer
at pH 7.3, except in NHA and Hs683 (90.4% and 56.3% incholine buffer, and 118% and 86.5% in lithium buffer, respectively). The [14C]Met uptake at pH 6.0 was higher than that at
pH 7.3, especially in choline buffer (111–169% that of thebrain research 1535 (2013) 24–37

27

Fig. 2 – Ion and pH dependencies of anti-[14C]FACBC uptake and [14C]Met in NHA and human glioma cell lines (Hs683, SW1088,
U87MG, and T98G). The percentage of uptake is expressed as the mean7SD (n ¼ 5–15). *Po0.05 vs. sodium buffer at pH 7.3;†
Po0.05 vs. choline buffer at pH 7.3; §Po0.05 vs. lithium buffer at pH 7.3; #Po0.05 vs. indicated bar; N.D.: not done, because
U87MG detached from the culture plates at pH 6.0.

uptake in sodium buffer at pH 7.3). However, the intracellularamount of [14C]Met at pH 8.5 was not changed in all the celllines compared with that using each buffer at pH 7.3.
Furthermore, Fig. 3 shows the AAT contribution that wascalculated from Fig. 2, using the formulas described in
Section 4.4. In NHA at pH 7.3, Naþ-dependent and Naþindependent AATs transported 71.5% and 28.5% of the total
anti-[14C]FACBC uptake, respectively. Similarly, the contributions of Naþ-dependent and Naþ-independent AATs to anti[14C]FACBC uptake were 96.4% and 3.6% in Hs683, 85.1% and14.9% in SW1088, 80.8% and 19.2% in U87MG, and 63.2% and36.8% in T98G, respectively. Thus, the contribution of theNaþ-dependent transport of anti-[14C]FACBC tends to behigher in the low-grade oligodendroglioma cell line lines(Hs683, SW1088) than in the high-grade glioblastoma celllines (U87MG, T98G). Among Naþ-dependent AATs, the contributions of the N and yþL systems to anti-[14C]FACBCtransport were less than 5% in all cell lines. At pH 7.3,
therefore, it would seem that Naþ-dependent AATs could
transport anti-[14C]FACBC except for the N and yþL systems.
On the other hand, the contributions of Naþ-dependent AATsfor [14C]Met uptake were 0.0–11.4% at the neutral pH in all cell
types except for Hs683 (43.7%); the majority of the [14C]Metuptake was mediated by Naþ-independent AATs (Hs683,
56.3%; others, 88.6–100%) (Fig. 3). Therefore, unlike anti-[14C]
FACBC uptake, Naþ-independent AATs were largely responsible for [14C]Met uptake at pH 7.3.
It is reported that transport activity via system L is
facilitated in acidic conditions, while that via system N is
diminished (Fei et al., 2000; Nakanishi et al., 2001a, 2001b). As
shown in Figs. 2 and 3, the transport of anti-[14C]FACBC and[14C]Met via Naþ-independent AATs was facilitated at pH 6.0
in all cell lines except U87MG, while the uptake proﬁles of theboth tracers were almost unchanged in the alkaline condition. These results support the idea that system L mediates
the transport of anti-[14C]FACBC and [14C]Met.

2.3.

Competitive inhibition transport assay

To narrow down the AATs involved in anti-[14C]FACBC and[14C]Met uptake, the inhibitory effects of synthetic/naturallyoccurring amino acids on tracer uptake were investigated.

28

brain research 1535 (2013) 24–37

Fig. 3 – AAT contribution percentages of anti-[14C]FACBC and [14C]Met uptake in NHA and human glioma cell lines (Hs683,
SW1088, U87MG, and T98G). The mean contribution of AATs in each buffer was calculated from Fig. 2, using the formulas in
Section 4.4 (n ¼ 5–15). N.D.: not done, because U87MG detached from the culture plates at pH 6.0.

Intensity of inhibition was categorized into four classes basedon the average inhibitory rates: null (o20%), weak (20–49.9%),
moderate (50–79.9%) and strong ( Z80%).
As shown in Fig. 4A–D, null to weak inhibitory effects by
2 mM 2-(methylamino)-isobutyric acid (MeAIB, a substrate forsystems A, IMINO, and PAT), L-arginine (Arg, a substrate forsystems B0,þ, yþL, b0,þ, and yþ), L-glutamate (Glu, a substrate forsystems X-C and X-A,G), and L-proline (Pro, a substrate for systems
A, ASC [ASCT1], G-like, IMINO, B0 [B0AT2], B0,þ, and PAT) wereobserved on the uptake of anti-[14C]FACBC and [14C]Met insodium and choline buffers. There were no inhibitory effects
on the transport of either tracer if MeAIB and Arg were
employed as inhibitors, even at high concentration (5 or
10 mM) (data not shown). These results suggest that thecontributions of AATs, including systems A, IMINO, PAT, B0,þ,yþL, b0,þ, yþ, X-C, and/or X-A,G, to the transport of anti-[14C]FACBC
and [14C]Met are low. In the absence of Naþ, L-phenylalanine(Phe, a substrate for systems L and T) and 2-amino-bicyclo[2,2,1]
heptane-2-carboxylic acid (BCH, a substrate for system L)
showed strong inhibitory effects on the uptake of anti-[14C]
FACBC and [14C]Met in all cell lines (Fig. 4C and D). Moreover, Nethylmaleimide (NEM), a substrate for systems yþ and L (exceptLAT1), induced weak to moderate inhibition of anti-[14C]FACBC
and [14C]Met uptake only in Hs683, with or without Naþ(Fig. 4A–D). Therefore, it is possible that within system L,

LAT1 is involved mainly in the transport of anti-[14C]FACBC
and [14C]Met in all cell lines, while the other subtypes of system
L (LAT2, 3, and/or 4), except for LAT1, also contribute to anti[14C]FACBC and [14C]Met uptake in Hs683. Although the contribution of system T to anti-[14C]FACBC and [14C]Met uptake
should be considered in light of the inhibitory effects of Phe,
its contribution is unexpected because system T selectivelytransports aromatic amino acids (e.g. Phe, L-tryptophan, and14
L-tyrosine) (SLC tables, 2013, see “References”), and anti-[ C]
FACBC and [14C]Met do not possess an aromatic ring.
In the presence of Naþ, both L-glutamine (Gln) and L-serine(Ser), which are preferentially transported by systems A, ASC(especially ASCT2 for Gln transport), B0 (B0AT1), N, yþL, L, asc,
and/or B0,þ, showed the strongest inhibitory effect on anti-[14C]
FACBC uptake in all cell lines (Fig. 4A). In contrast, although[14C]Met uptake was inhibited strongly by Gln in sodium buffer
in all cell lines except for Hs683, the inhibitory effect of Ser was
weak to moderate in all cell lines, except T98G (Fig. 4B). The
results so far narrow down the AATs involved in transport ofanti-[14C]FACBC and [14C]Met to systems ASC (especially ASCT2),B0 (especially B0AT1), N, L, and asc. However, because previous
studies reported that expression of systems B0 and asc cannot
be detected in astrocytes (Bröer, 2006; Shao et al., 2009), thecontributions of systems B0 and asc to anti-[14C]FACBC and [14C]
Met transport could be negligible.

brain research 1535 (2013) 24–37

29

Fig. 4 – Competitive inhibition proﬁles of NHA and human glioma cell lines (Hs683, SW1088, U87MG, and T98G) on anti-[14C]
FACBC and [14C]Met. Incubation of anti-[14C]FACBC (A, C, E) or [14C]Met (B, D, F) in sodium (A, B), choline (C, D), or lithium (E, F)
buffer with the indicated amino acids (2 mM). The average inhibitory rates (n¼ 5–9) were categorized into 4 classes.

In lithium buffer, the inhibitory effects of Arg and Gln on
the uptake of [14C]Met were strong compared with those on
anti-[14C]FACBC, that is, systems N and yþL mediate theuptake of [14C]Met rather than that of anti-[14C]FACBC. The
contributions of these AATs on the uptake of both tracers
may be insigniﬁcant, however, because Liþ-dependent transport of anti-[14C]FACBC and [14C]Met was a small proportion of
the total uptake of either tracer (Figs. 2 and 3).
In NHA, the overall tendencies in the inhibitory effects ofsynthetic and naturally occurring amino acids on anti-[14C]
FACBC and [14C]Met uptake were similar to those in SW1088.
Because both cells are from an astroglial lineage, similarities
in the transport of anti-[14C]FACBC and [14C]Met imply theproperties are tissue speciﬁc in nature.
Taken together with the results listed in Section 2.2(Figs. 2 and 3), the ASC and L systems, especially ASCT2 and
LAT1, could be central players in the transport of anti-[14C]
FACBC, while system L is the critical AAT for [14C]Met uptake.

2.4.

Tracer exchange transport

The ASC and L (LAT1 and LAT2) systems are amino acidexchangers that exchange their intracellular and extracellular

substrates (SLC tables, 2013, see “References”). To conﬁrm thatthese AATs are responsible for anti-[14C]FACBC and [14C]Mettransport, we conducted exchange transport studies. The
results at 37 1C and 4 1C in the absence of synthetic or
naturally occurring amino acids indicated active and passive
diffusion, respectively.
As shown in Fig. 5A, without amino acids, 31.9% of theintracellular anti-[14C]FACBC in NHA was efﬂuxed in 13 min at
37 1C, and this dropped to 12.9% at 4 1C. The corresponding
values for the other cell lines were 19.3–62.0% and 10.0–16.2%
at 37 1C and 4 1C, respectively (Fig. 5B–E). Similar results wereobserved for [14C]Met efﬂux in the absence of amino acids(efﬂux at 13 min: 27.0–67.3% at 37 1C and 7.2–16.2% at 4 1C)(Fig. 6), indicating that the active diffusion rates varied
between the cells used in this study, while the passive
diffusion rates did not.
When Gln or BCH was added to the sodium buffer, transstimulation of anti-[14C]FACBC efﬂux was observed in all celllines, with Gln having the greater effect (after 13 min: Gln
93.0–98.2% vs. BCH 56.5–74.3%) (Fig. 5). Along with anti-[14C]
FACBC efﬂux, the addition of Gln or BCH to the sodium buffer
caused trans-stimulation of [14C]Met efﬂux, with the degree ofstimulation at the 13-min point being almost same in all cell30

brain research 1535 (2013) 24–37

Fig. 5 – Exchange transport of anti-[14C]FACBC in NHA and human glioma cell lines (Hs683, SW1088, U87MG, and T98G) in sodium
buffer. At the 4-min point, the indicated amino acids (2 mM) were added. Each point represents the mean7SD (n¼ 3–6).

lines (Gln, 76.9–89.0%; BCH, 74.3–88.7%) (Fig. 6). Arg had notrans-stimulating effect on anti-[14C]FACBC or [14C]Met efﬂux
in any cell line (Figs. 5 and 6).
As shown in Fig. 7, in the absence of Naþ in Hs683 andT98G, the efﬂux without inhibitor at 13 min for anti-[14C]
FACBC was 50.8% and 40.3%, respectively, at 37 1C, and thisdecreased to 8.5% and 6.0%, respectively, at 4 1C (Fig. 7A and C).

Similar tendencies for [14C]Met were observed at 13 min in
Hs683 and T98G (42.8% and 29.5% at 37 1C, and 7.1% and 5.2%
at 4 1C, respectively; Fig. 7B and D). In the presence of Gln or
BCH, trans-stimulation of the anti-[14C]FACBC and [14C]Met
efﬂuxes was observed in Hs683 and T98G. Unlike the efﬂux insodium buffer, the efﬂux degrees were almost equal ineach tracer (anti-[14C]FACBC efﬂux at 13 min in Hs683 andbrain research 1535 (2013) 24–37

31

Fig. 6 – Exchange transport of [14C]Met in NHA and human glioma cell lines (Hs683, SW1088, U87MG, and T98G) in sodium
buffer. At the 4-min point, the indicated amino acids (2 mM) were added. Each value represents the mean7SD (n ¼ 3–6).

T98G: Gln 62.9% and 99.2% vs. BCH 71.3% and 94.4%, respectively; [14C]Met efﬂux at 13 min in Hs683 and T98G: Gln 68.0%
and 86.7% vs. BCH 71.9% and 85.9%, respectively) (Fig. 7).
Considering that (1) Gln has afﬁnity for the ASC (especially
ASCT2), N, L, yþL, asc and b0.þ systems; (2) Arg is a representative substrate for the yþL and b0.þ systems, and; (3) BCH
is exclusively recognized by system L, a review of the transstimulation effects in all cell lines suggests that anti-[14C]

FACBC is mainly transported via the ASC and L systems,
while the L system predominates in [14C]Met transport, as
described in Section 2.3.

2.5.

AAT expression in NHA and glioma cell lines

The expression proﬁles of ASCT1, ASCT2, LAT1, and 4F2
heavy chain (4F2hc, a regulatory subunit of LAT1) in NHA

32

brain research 1535 (2013) 24–37

Fig. 7 – Exchange transport of anti-[14C]FACBC and [14C]Met in human glioma cell lines (Hs683 and T98G) in choline buffer. At
the 4-min point, the indicated amino acids (2 mM) were added. Each value represents the mean7SD (n ¼ 3–9).

Fig. 8 – AAT expression proﬁle of NHA and human glioma cell lines (Hs683, SW1088, U87MG, and T98G). Fluorescenceintensity (horizontal axis) and cell number (vertical axis) were analyzed using a ﬂow cytometer. Each histogram represents
the ﬂuorescence intensity of AAT expression (closed histogram) and isotype control (open histogram), respectively (n ¼3).

brain research 1535 (2013) 24–37

and glioma cell lines were analyzed using ﬂow cytometry. As
shown in Fig. 8, the three AAT subtypes and 4F2hc wereexpressed in all cell lines.

3.

Discussion

We set out to determine the differences between the transport mechanisms for anti-[14C]FACBC and [14C]Met in NHA
and human glioma cell lines by several in vitro experiments,
including time-course experiments, Naþ dependency, pH
dependency, competitive inhibition, and exchange transport.
First, we found that the uptake of anti-[14C]FACBC was
substantially higher than that of [14C]Met in NHA and lowgrade glioma cell lines (Hs683, SW1088), but was almost equal
to that of [14C]Met in high-grade glioma cell lines (U87MG and
T98G: glioblastoma). Akhurst et al. (2006) reported that theaccumulation rate of anti-[18F]FACBC in glioblastoma andhigh-grade astrocytoma is faster than that of [11C]Met, andthat the ratio of tumor-to-normal brain accumulation ishigher with anti-[18F]FACBC than with [11C]Met. Furthermore,
PET imaging with [11C]Met has the potential to visualize not
only high-grade but also low-grade gliomas (Herholz et al.,1998). Therefore, our results suggest that anti-[18F]FACBC as
well as [11C]Met would be expected to be effective in visualization, evaluation, and follow up for low-grade gliomas.
Second, we showed that the Naþ-dependent ASC system(especially ASCT2) and the Naþ-independent L system (especially LAT1) play a central role in anti-[14C]FACBC transport,
and that the L system (especially LAT1) is mainly involved in[14C]Met transport in NHA and glioma cell lines. We also used
ﬂow cytometry to conﬁrm that ASCT2, LAT1, and 4F2hc are
expressed in all the cell lines. These results are in accord withthe results from our previous studies, which included an
ASCT2-silenced transport study using human prostate cancer
cell lines (Oka et al., 2012; Okudaira et al., 2011), as well aswith the results from the kinetic assay for ASCT2 and LAT1
(Okudaira et al., 2013) and the results of others (Kanai et al.,
1998; Yu et al., 2009). The fact that anti-[14C]FACBC is a
substrate for both the ASC and L systems is not surprisingbecause naturally occurring amino acids are transported into
cells by more than one transport system, and ASCT2 andLAT1 share several substrates, such as L-threonine, L-leucine,and L-phenylalanine. Furthermore, the recognition of anti[14C]FACBC by both systems might lead to its higher uptake ascompared with [11C]Met, because ASCT2 and LAT1 are upregulated in brain tumor cells (Fuchs and Bode, 2005), andthere are thus two routes for anti-[14C]FACBC transportcompared with one for [14C]Met. Indeed, the uptake of anti[14C]FACBC in some glioma cell lines was higher than that of[14C]Met, as described in Section 2.1.
While the ASC and L systems are involved in the transportof anti-[14C]FACBC in glioma and prostate cancer cells, a Naþdependent system A, which participates in anti-[14C]FACBC
uptake in prostate cancer cell lines (Okudaira et al., 2011), is
not involved in the transport of anti-[18F]FACBC in glioma
cells, because MeAIB, a speciﬁc inhibitor of system A, showed
no inhibitory effect on anti-[14C]FACBC uptake in this study(Fig. 4). The difference between prostate cancer and glioma
cells may be attributed to the fact that the expression of

33

system A in glioma cells is at a lower level than in prostatecancer cells. In fact, it is reported that system A is abundantin prostate cancer cells (Okudaira et al., 2011), but poorly
expressed in astrocytes (Mackenzie and Erickson, 2004), andthat all cell lines used in the current study are derived fromglial cells. We therefore suggest that the main AATs for thetransport of anti-[18F]FACBC are the ASC and L systems, while
system L is mainly responsible for [11C]Met transport. System
L transports both tracers into cells regardless of their tissuesource and/or malignancy, whereas the contributions ofother AATs such as systems A and N vary by cell type.
The transport activities of systems N and yþL are unaffected by the substitution of Liþ for Naþ (Fei et al., 2000; Kanai
et al., 2000; Nakanishi et al., 2001a, 2001b). [14C]Met transportin Hs683 is Liþ tolerant at pH 7.3 and 8.5, indicating thecontribution of the N and yþL systems is higher (approximately 30%) than in other cell lines (Figs. 2 and 3). Moreover,the inhibition study on [14C]Met uptake in the presence of Liþ(Fig. 4F) showed moderate inhibition by Gln, a substrate forsystem N, but not by Arg, a substrate for system yþL. These
results suggest that system N is partly involved in [14C]Mettransport in Hs683, a low-grade glioma cell line. Sidoryk et al.
(2004) reported that the expression of SNAT3, a subtype ofsystem N, is increased in gliomas. Thus, it is possible thatsystem N mediates a portion of the [11C]Met transport in lowgrade gliomas, and this is a reason why they can be detectedby [11C]Met-PET (Herholz et al., 1998). However, system N
cannot be an important AAT in [11C]Met transport inadvanced high-grade gliomas because the pH microenvironment in progressive tumor tissue is relatively acidic (Zhang
et al., 2010), and the activity of system N is inhibited by low
pH (Fei et al., 2000; Nakanishi et al., 2001a, 2001b). The resultsof the current study support these conclusions.
The exchange transport study showed that, apart from
U87MG, it might be possible to correlate differences in the
efﬂux rates of anti-[14C]FACBC and [14C]Met between 37 1C and4 1C with the total tracer uptake. For example, the differencesin SW1088 were smaller than in the other cell lines (Figs. 5
and 6), and the uptake of both tracers was higher (Fig. 1).
These ﬁndings suggest that the total amounts of anti-[14C]
FACBC and [14C]Met in cells were regulated by efﬂux activity
rather than inﬂux activity.
Contrary to our expectations, the total uptake of anti-[14C]
FACBC, but not [14C]Met, in NHA was relatively high compared to that in glioma cell lines (Fig. 1A). It was reported in aclinical study, however, that anti-[18F]FACBC accumulation innormal brain tissue is low, but is constantly high in a glioma(Shoup et al., 1999). A possible explanation for this discrepancy between our in vitro experiments and the clinical study
is as follows: as described in Section 2.3, Gln was one of themost effective inhibitors of anti-[14C]FACBC uptake in thepresence of Naþ, and ASCT2 is thought to be the main AAT
for Gln and anti-[14C]FACBC. It is known that Gln and ASCT2
are indispensable for cell growth, and that Gln itself has thepotential to enhance ASCT2 expression on the surface ofastrocytes by inducing the trafﬁcking of ASCT2 from thecytoplasm (Bode et al., 2002; Bröer and Brookes, 2001;
Gegelashvili et al., 2006). Furthermore, Bröer et al. (1999)
reported that ASCT2 expression in cultured rat astrocytes ismuch higher than that in the adult brain. Considering the34brain research 1535 (2013) 24–37

transport mechanism of anti-[14C]FACBC into cells, it isreasonable to increase the transport of anti-[14C]FACBC
in NHA.
As mentioned above, it is reported that the accumulationof anti-[18F]FACBC in normal brain tissue is lower than that of[11C]Met; the standard uptake values of anti-[18F]FACBC and[11C]Met in the cortex of human brain were 0.570.1 and
1.370.4, respectively (Akhurst et al., 2006). One reason forthe low accumulation of anti-[18F]FACBC in the normal brain
is that anti-[18F]FACBC is not metabolized and incorporated
into protein (Okudaira et al., 2011). According to a report by
O’Kane et al., (2004), Naþ-dependent AATs, including the A, N,
and ASC systems, are expressed on the abluminal blood–
brain-barrier (BBB) membrane, while LAT1 is present on theabluminal membrane, but is predominantly on the luminal
membrane. The presence of Naþ-dependent AATs on theabluminal BBB membrane provides a mechanism by which
neutral amino acid concentrations in the extracellular brain
ﬂuid are maintained at o10% of those of plasma (Laterra
et al., 1999; O’Kane et al., 2004). Thus, it might be speculated
that a small accumulation of anti-[18F]FACBC in the normal
brain is related to the removal of neutral amino acids via
Naþ-dependent AATs such as system ASC on the abluminal
membrane.
In conclusion, we elucidated the differences in transportmechanisms between anti-[14C]FACBC and [14C]Met in NHA
and human glioma cell lines. Our results demonstrate that
uptake of anti-[14C]FACBC was higher than that of [14C]Met in
low-grade glioma cell lines, and was almost equal to that of[14C]Met in high-grade glioma cell lines. Furthermore, ASC
systems (especially ASCT2) as well as L systems (especially
LAT1) play a central role in anti-[14C]FACBC transport, while L
systems (especially LAT1) are predominantly involved in [14C]
Met transport in NHA and glioma cell lines. Because ourresults were obtained from in vitro experiments, further
investigations are necessary to determine if these mechanisms operate in vivo.

4.

Experimental procedure

4.1.

Radioisotope-labeled tracers

Medium (L-15), Minimum Essential Medium (MEM), andpenicillin–streptomycin were purchased from Life Technologies Japan (Tokyo, Japan). Cells were maintained in DMEM
(Hs683), L-15 (SW1088), or MEM (U87MG and T98G) at 37 1C in
air (SW1088) or 5% CO2 (Hs683, U87MG and T98G). All media
were supplemented with 10% fetal bovine serum (FBS),
100 units/mL penicillin, and 0.1 mg/mL streptomycin. Normal
human astrocytes (NHA) were purchased from Takara Bio Inc.
(Shiga, Japan) and cultured under 5% CO2 in astrocyte basal
medium supplement with FBS, epidermal growth factor,
ascorbic acid, Gln, gentamicin, and amphotericin B, according
to the manufacturer's protocol.

4.3.

All reagents used in the current study were purchased fromcommercial suppliers (Wako Pure Chemical Industries,
Osaka, Japan; Sigma-Aldrich Japan, Tokyo, Japan; and Nacalai
Tesque, Kyoto, Japan) unless otherwise noted. Cells wereseeded at densities of 3  104 (SW1088), 5  104 (Hs683 andT98G), or 7.5  104 (NHA and U87MG) cells per well in 24-well
ﬂat-bottom tissue culture plates (Becton Dickinson, New
Jersey, USA). After 3 days of cultivation, the cells were washedtwice with sodium buffer (140 mM NaCl, 5 mM KCl, 5.6 mM Dglucose, 0.9 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.3
at 37 1C), and then incubated with 0.3 mL of sodium buffer
containing anti-[14C]FACBC or [14C]Met at a ﬁnal concentrationof 10 μM for 1, 3, 5, 7.5, 10, 15, 30, and 60 min at 37 1C in air.
The uptake was terminated by removing the tracer solution
and rapidly washing the cells twice with ice-cold sodium
buffer. The cells were lysed in 0.3 mL of 0.1 N NaOH, and theradioactivity of each aliquot was measured with a Tri-Carb
2910 TR liquid scintillation counter (PerkinElmer, Massachusetts, USA) by using Ultima Gold (PerkinElmer). The protein
concentration of the cell lysate was determined with a
VersaMax microplate reader (Nihon Molecular Device K.K.,
Osaka, Japan) using the BCA Protein Assay Kit (Thermo Fisher
Scientiﬁc, Kanagawa, Japan). Tracer uptake was expressed as
pmol/mg protein.

4.4.

In all experiments, 14C-labeled amino acids were used because
their long half-lives (5700 years) made them more convenient
for in vitro experiments than 18F (110 min) or 11C (20 min). Trans1-amino-3-ﬂuoro[1-14C]cyclobutanecarboxylic acid (anti-[14C]
FACBC, 2.09 GBq/mmol) was synthesized (Oka et al., 2012) bySekisui Medical Co., Ltd. (Tokyo, Japan), and L-[methyl-14C]
methionine ([14C]Met, 2.04 GBq/mmol) was purchased from
American Radiolabeled Chemicals (Missouri, USA).

4.2.

Cell culture

Human glioma-derived cell lines Hs683 (low-grade oligodendroglioma), SW1088 (low-grade astrocytoma), U87MG (highgrade glioblastoma), and T98G (high-grade glioblastoma)(Kadowaki et al., 2012; Le Mercier et al., 2009) were obtained
from the American Type Culture Collection (Maryland, USA).
Dulbecco's modiﬁed Eagle Medium (DMEM), Leibovitz's L-15

Time-dependent uptake study

Study of ion-dependent and pH-dependent uptake

Experiments were carried out as described previously(Oka et al., 2012) with minor modiﬁcations. Sodium, choline,
and lithium buffers were used in this study. In choline andlithium buffers, sodium chloride was replaced by the equivalent concentrations of choline chloride and lithium chloride,
respectively. The pH of the buffers was adjusted to 6.0 or 8.5,
as follows: HEPES was replaced with the equivalent concentration of either 4-morpholineethanesulfonic acid (for pH 6.0)
or Tris (for pH 8.5).
Cells were cultured as described above. After washing
twice with sodium, choline, or lithium buffer at each pH,
cells were incubated with each buffer containing anti-[14C]
FACBC or [14C]Met at a ﬁnal concentration of 10 μM for 5 min
at 37 1C in air. The radioactivity and protein concentration ofeach sample were measured as described above. The datafrom U87MG at pH 6.0 were not determined because this cellline was easily detached from the tissue culture plate during
the cell washing procedure.

brain research 1535 (2013) 24–37

In this experiment, all AATs functioned in sodium buffer,
while only Naþ-independent AATs worked in choline buffer. In
lithium buffer, both Naþ-independent AATs and the Naþdependent N and yþL systems worked (Fei et al., 2000; Kanai
et al., 2000; Nakanishi et al., 2001a, 2001b). From this knowledge,
the amounts of tracer uptake (pmol/mg protein) mediated byeach amino acid transport system were calculated as follows:Naþ-independent AATs¼VC
Naþ-dependent AATs¼ VSVC (1)
System N and yþL AATs¼VLVC (2)Naþ-dependent AATs except for system N and yþL AATs¼(1)(2)where VS, VC, and VL are the amounts of tracer uptake insodium, choline, and lithium buffers, respectively. In Fig. 2, theamount of each tracer uptake in sodium buffer at pH 7.3 wasnormalized to 100%. In Fig. 3, the transport of tracers in sodium
buffer at each pH was normalized to 100%. If the tracer uptake
in choline buffer was higher than that in sodium buffer at eachpH, then the contribution of Naþ-dependent AATs was taken to
be zero. If the tracer uptake in lithium buffer was higher thanthat in sodium buffer at each pH, then the uptake in lithium
buffer was considered same as that in sodium buffer.

4.5.

Competitive inhibition uptake study

Experiments were carried out as described previously (Okaet al., 2012) with minor modiﬁcations. Cells in 24-well ﬂatbottom tissue culture plates were incubated in sodium, choline, or lithium buffer containing 10 μM anti-[14C]FACBC or[14C]Met for 5 min at 37 1C in air in the presence or absence of
2 mM inhibitors. As inhibitors, synthetic and naturally occurring amino acids were used as follows: 2-amino-bicyclo[2,2,1]
heptane-2-carboxylic acid (BCH, for system L, B0, and B0,þ); 2(methylamino)-isobutyric acid (MeAIB, for system A, IMINO
and PAT); N-ethylmaleimide (NEM, for system yþ and LAT2/3/
4); L-phenylalanine (Phe, for system L, T); L-proline (Pro, forsystem A, ASC [ASCT1], G-like, IMINO, B0, B0,þ, and PAT); Lglutamine (Gln, for system A [SNAT1, SNAT2], ASC [ASCT2],
B0 [B0AT1], N, yþL, L, and B0,þ); L-serine (Ser, for system A,
ASC, B0 [(B0AT1]), N [(SNAT5]), yþL, L, and B0,þ); L-arginine(Arg, for system B0,þ, yþL, b0,þ, and yþ); and L-glutamate (Glu,for system X-C, X-A,G). Following that, the procedures werecarried out as described above.
In data analyses, the control transport of anti-[14C]FACBC
and [14C]Met in sodium, choline, and lithium buffers was
normalized to 100%, and the inhibitory effects of the synthetic
and naturally occurring amino acids on the uptake of bothtracers were calculated as a percentage of the control.
To simplify the results, the intensity of the inhibitory effects
was categorized into 4 classes based on the average inhibitory
rates: null (o20%), weak (20–49.9%), moderate (50–79.9%), andstrong (Z80%). A summary of these proﬁles is shown in Fig. 4.

4.6.

Tracer efﬂux study

This study was performed as described previously (Oka et al.,2012). Cells were cultured in 35-mm cell culture dishes

35(Becton Dickinson) and loaded with anti-[14C]FACBC or [14C]
Met in sodium or choline buffer (ﬁnal concentration: 10 μM)
for 15 min at 37 1C in air. After 2 quick washes with ice-cold
sodium or choline buffer, cells were added to 0.5 mL of
sodium or choline buffer and incubated at 37 1C in air. The
efﬂux of each tracer from the cells was measured by thesequential addition and removal of 0.5 mL of buffer at every
minute until 3 min. At the 4-min point, each warmed sodium
buffer (37 1C) with or without 2 mM Arg, Gln, or BCH, or
warmed choline buffer (37 1C) with or without 2 mM Gln or
BCH was added to the dishes, and the buffer exchange wasrepeated at the 5-, 6-, 7-, 9-, 11-, and 13-min time points. In
some experiments, ice-cold sodium or choline buffer without
inhibitors was used in the buffer-exchange procedures. The
radioactivity of the collected buffer and the cell lysate wasmeasured as described above.

4.7.

Flow cytometry

Anti-LAT1 monoclonal antibody (clone 181C, mouse IgG1
mAb) was kindly provided by Dr. Tsurudome (Mie University)(Okamoto et al., 2002). Anti-ASCT1 polyclonal antibody (rabbit
IgG pAb) was purchased from Medical & Biological Laboratories (Nagoya, Japan). Anti-ASCT2 rabbit pAb was obtained
from Santa Cruz Biotechnology (California, USA). Anti-human
4F2hc phycoerythrin (PE)-labeled monoclonal antibody(mouse IgG1 mAb) was purchased from Becton Dickinson.
Puriﬁed mouse IgG1 (Becton Dickinson) and puriﬁed rabbit
IgG (Beckman) were used as isotype controls. PE-labeled antimouse IgG1 (rat IgG1; Becton Dickinson) and PE-labeled antirabbit IgG (goat IgG; Santa Cruz Biotechnology) were used as
secondary antibodies. The staining of cells with antibodies
was performed as described previously (Oka et al., 2012) anddata were acquired using a FACSCalibur ﬂow cytometer(Becton Dickinson).

4.8.

Statistical analysis

All data are presented as the mean7standard deviation (SD).
Statistical analyses were carried out using the two-tailed
unpaired t-test or Tukey–Kramer multiple comparison tests,
if appropriate. Po0.05 was considered signiﬁcant.

Acknowledgments
The authors express their gratitude to Ms. Sachiko Naito for
performing the cell culture and to Mr. Kodai Nishi and Dr.
Masato Kobayashi for their helpful discussions.
Masahiro Ono, Shuntaro Oka, Hiroyuki Okudaira, andYoshifumi Shirakami are employees of Nihon Medi-Physics
Co., Ltd. Mark M. Goodman and Emory University have patentrights for anti-[18F]FACBC and are eligible to receive royalties
on anti-[18F]FACBC from Nihon Medi-Physics Co., Ltd. David
M. Schuster, Mark M. Goodman, and Keiichi Kawai haveongoing research collaboration with Nihon Medi-Physics
Co., Ltd.

36

brain research 1535 (2013) 24–37

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