Expression of OATP1B3 determines uptake of Gd-EOB-DTPA
in hepatocellular carcinoma
Narita, Masato; Hatano, Etsuro; Arizono, Shigeki; Miyagawa-
Hayashino, Aya; Isoda, Hiroyoshi; Kitamura, Koji; Yasuchika,
Kentaro; Nitta, Takashi; Uemoto, Shinji
CitationJournal of Gastroenterology (2009), 44(7): 793-798
Rightc Springer 2009.
KURENAI : Kyoto University Research Information Repository
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Expression of OATP1B3 determines uptake of Gd-EOB-DTPA
in hepatocellular carcinoma
Masato Narita, MD*
Etsuro Hatano, MD, PhD*
Shigeki Arizono, MD†
Hiroyoshi Isoda, MD, PhD†
Koji Kitamura, MD*
Kojiro Taura, MD, PhD*
Kentaro Yasuchika, MD, PhD*
Takashi Nitta, MD, PhD*
Iwao Ikai, MD, PhD*
Shinji Uemoto, MD, PhD*
Department of Surgery* and Department of Diagnostic Imaging and Nuclear
Medicine†, Graduate School of Medicine, Kyoto University
Department of Diagnostic Pathology, Kyoto University Hospital, Kyoto, Japan‡
54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan
Correspondence and reprints request to
Etsuro Hatano, MD, PhD
Department of Surgery, Graduate school of Medicine, Kyoto University
54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
Telephone number: 81-75-751-4323, Fax number: 81-75-751-4348
Uptake of GD-EOB-DTPA in HCC
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Background: Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid
(Gd-EOB-DTPA) is an MRI contrast agent with perfusion and hepatoselective properties.
The purpose of the study was to examine uptake of Gd-EOB-DTPA in the hepatobiliary
phase in hepatocellular carcinoma (HCC).
Methods: A retrospective analysis of 22 patients with HCC who underwent preoperative
Gd-EOB-DTPA-enhanced MRI was performed. Enhancement ratios (ERs) and expression
levels of the organic anion transporter (OATP) 1B3 protein were examined.
Results: Gd-EOB-DTPA accumulated in the hepatobiliary phase in 6 of the 22 cases. All 6
Gd-EOB-DTPA-positive cases were moderately differentiated HCC, but 11 other moderately
differentiated HCCs did not show Gd-EOB-DTPA uptake. Histopathologically, 4
Gd-EOB-DTPA-positive HCCs and 5 Gd-EOB-DTPA-negative HCCs produced bile. HCCs
with Gd-EOB-DTPA uptake overexpressed OATP1B3 compared with HCCs without
Gd-EOB-DTPA uptake, and OATP1B3 levels were significantly correlated with ERs (r=0.91,
Conclusions: Uptake of Gd-EOB-DTPA in HCC is determined by expression of OATP1B3
rather than by tumor differentiation or bile production.
Keywords: Green hepatoma, Gd-EOB-DTPA, MRI, OATP1B3, HCC
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In the past decade, several liver-specific magnetic resonance imaging (MRI) contrast
media have been investigated in clinical studies with the goal of increasing the performance
of liver MRI, especially for lesion detection.1 Gadolinium-ethoxybenzyl-diethylenetriamine
pentaacetic acid (Gd-EOB-DTPA) is a recently developed liver-specific MRI contrast agent
with combined perfusion and hepatocyte-selective properties. A bolus injection of
Gd-EOB-DTPA provides a dual mode of action that allows imaging in the early dynamic
phase (as with standard gadolinium chelates) and in the delayed phase (hepatobiliary phase)
that begins 20 min after injection and lasts until 90 min after injection.2, 3 In the hepatobiliary
phase, hepatic lesions lacking normally functioning hepatocytes are imaged as a defect of
hepatocyte-selective enhancement compared with normal parenchyma,4 and evaluation of
vascularity and hepatocyte-specific uptake enables accurate detection and characterization of
focal liver lesions. Therefore, Gd-EOB-DTPA-enhanced MRI is potentially superior to
standard imaging using gadolinium chelates or spiral CT, especially for detection of small
metastases and differential diagnosis of hypervascular lesions.5
A differential diagnosis of a dysplastic nodule and well-differentiated HCC can be based
on findings with or without uptake of Gd-EOB-DTPA.6 However, some HCCs may show
paradoxical uptake of Gd-EOB-DTPA and are recognized as iso- or hyperintense lesions in
the hepatobiliary phase, compared with normal parenchyma.7,8 The characteristics of such
Gd-EOB-DTPA-positive HCCs in the hepatobiliary phase has not been defined. Therefore,
the aim of this study was to investigate the frequency of HCCs with uptake of
Gd-EOB-DPTA in the hepatobiliary phase and to clarify the mechanism of uptake.
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The study was performed as a retrospective review of patients with HCC who received
Gd-EOB-DTPA-enhanced MRI as a preoperative examination and underwent liver resection
at Kyoto University Hospital from May 2008 to October 2008. Patients who received
pretreatment with transarterial chemoembolization (TACE) or percutaneous ethanol injection
therapy (PEIT) in the preoperative period were excluded.
MR imaging was performed at 3 or 1.5 T using commercially available MR systems in
the Magnetom series (Siemens, Erlangen, Germany): Magnetom Trio Tim (3 T), Magnetom
Avanto (1.5 T) or Magnetom Symphony (1.5 T). All images were obtained in the axial plane.
Dynamic and delayed hepatobiliary phase T1-weighted three-dimensional spoiled gradient
echo images were obtained using a volumetric interpolated breath-hold examination (VIBE)
sequence with chemically selective fat saturation (TR, 3 to 5.21 msec; TE, 1.06 to 2.13
msec; flip angle, 9° to 15°; matrix, 205 × 256, 198 × 320 or 259 × 320 mm; field of view,
240 × 320 to 270 × 360 mm; number of signals acquired, 1; slice thickness, 3 to 5 mm; slice
number, 40 to 80; acquisition time, 14 to 22 sec). In pre-contrast imaging at 1.5 T, parallel
acquisition techniques were not used. In pre-contrast imaging at 3 T and in post-contrast
imaging, iPAT reconstruction was performed using the GRAPPA algorithm with a parallel
acquisition technique factor of two. Parameters varied according to the machines used and
the patient physique. Images with the initial VIBE sequence were acquired before
administration of the contrast agent. A bolus of 25 μmol/kg body weight Gd-EOB-DTPA
(Bayer, Germany) was then injected and flushed with 40 ml of sterile saline solution from
the antecubital vein. A power injector (Sonic Shot, Nemoto-Kyorindo, Tokyo, Japan) was
used for injection of the contrast agent and saline. After the dynamic study, images were
obtained with a T1-weighted VIBE sequence in the delayed hepatobiliary phase at 20-25 min
after injection of the contrast agent.
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The enhancement ratio (ER) of each lesion diagnosed as HCC was calculated according
to Saito et al.8 Briefly, the signal intensity (SI) of the lesion was measured on T1-weighted
images collected before injection of the contrast agent and 20-25 min after the injection. The
SI of the tumor was measured by defining an oval region of interest (ROI) that covered as
much of the lesion as possible. ERs were calculated with the formula: ER = [(SI after
enhancement-SI before enhancement)/SI before enhancement].
Evaluation of bile production
Bile production was evaluated blindly by a liver-specialized pathologist (A. M-H.) using
hematoxylin and eosin (HE) staining. Positive bile production was indicated by the presence
of bile pigment inside the pseudo-cholangiole, bile canaliculi, or cytoplasm of tumor cells. 9
To perform semi-quantitative evaluation of bile production, bile production was stratified
into 3-scale according to the degree of it: Mild (< 5%); Moderate (5-25%); Severe (> 25%).
Western blot analysis
Tissue samples were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 6.8),
10% glycerol and 2% sodium dodecylsulfate. After the concentration of the sample was
determined, 0.1% bromophenol blue and 5% 2-mercaptoethanol was added. For
immunoblotting, protein (24 μg) was subjected to SDS-PAGE and then transferred to a
polyvinylidene difluoride membrane. The membranes were blocked with Blocking-One
(Nacalai Tesque, Kyoto, Japan) and incubated with a primary antibody against OATP1B3
(#651140; Progen Biotechnik, Heidelberg, Germany) at a dilution of 6 μg/mL in
Blocking-One or an anti-actin antibody (#sc-1615; Santa Cruz Biotechnology, Santa Cruz,
CA, USA) at 1:1000 dilution overnight at 4°C. After washing, membranes were reacted with
horseradish peroxidase-conjugated antibodies (Santa Cruz). Chemiluminescence was
detected with Immobilon Western HRP Substrate (Millipore, Billerica, MA, USA) and the
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intensity of the bands was quantified with Quantity One imaging analysis software (Bio-Rad
Laboratories, Hercules, CA, USA).
For immunohistochemistry of OATP1B3, paraffin sections were pretreated with 0.3%
H2O2 in methanol and then subjected to antigen retrieval in citrate buffer (10 mM, pH 6.0) in
a pressure cooker. After blocking with 3% BSA-10% normal serum for 1 h, the sections were
incubated with a primary antibody recognizing OATP1B3 (Progen Biotechnik) overnight at
4°C. Subsequently, the sections were incubated with Labeled Polymer in an Envision +
System HRP Kit (Dako, Tokyo, Japan) at room temperature for 1 h. The sections were
examined after incubation with a Liquid DAB Substrate Chromogen System (Dako) and
counterstained with hematoxylin.
Statistical analysis was performed using SPSS v. 11.0.1 (SPSS Inc., Chicago, IL, USA).
Data are expressed as means ± SD. Differences in measured variables between each group
were assessed using a Mann-Whitney U test with P < 0.05 considered to indicate statistical
significance. Pearson correlation analysis was used to evaluate the relationship between ERs
and expression levels of OATP1B3 protein, with a correlation coefficient (r) of ≥ 0.8
indicating a strong correlation.
From May 2008 to October 2008, 30 consecutive patients with HCCs underwent liver
resection at Kyoto University Hospital. Five of these patients did not receive preoperative
Gd-EOB-DTPA-enhanced MRI and 3 were treated with TACE (n = 3) and PEIT (n = 1)
before surgery, leaving 22 patients for evaluation. No hepatocyte-specific enhancement in
Gd-EOB-DTPA-enhanced MRI in the hepatobiliary phase was observed in 16 cases (Figs.
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1A, B), whereas hyperintensive lesions were found in 6 cases (Figs. 1C, D). The patients
were divided into two groups based on the ER (Table 1) and all cases with high ER (≥ 1)
were found to be moderately differentiated HCCs. Macroscopically, HCCs producing bile
were identified by a greenish color in HE staining; so-called green hepatoma (Fig. 2). HCCs
with bile production were observed in 5 of 16 cases (31.3%) with a low ER (< 1) and 4 of 6
cases (66.7%) with a high ER (P = 0.231). In the low ER group, the details of the bile
production in 5 cases were described as follows: Mild; 2, Moderate; 1, and Severe; 2. On the
other hand, all cases diagnosed as bile production HCCs were stratified into the Mild in the
high ER group.
Oatp1 is involved in gadoxetate uptake into rat liver10 and therefore expression of
OATP1B3 was examined in tumor and corresponding non-tumor tissues by Western blotting.
Non-tumor liver parenchyma were diagnosed as normal parenchyma (n = 4), chronic
hepatitis (n = 6), liver cirrhosis (n = 8), liver fibrosis (n = 3), and steatosis (n = 1). There was
no significant difference in non-tumor liver tissues. On the other hand, OATP1B3 was hardly
detectable in HCCs with a low ER, but strongly expressed in HCCs with a high ER (P <
0.0001; Fig. 3A). Immunohistochemical analysis indicated that OATP1B3 was highly
expressed in tumor cell membrane and weakly expressed in tumor cytoplasm in HCCs with a
high ER (Fig. 3B). In contrast, expression of OATP1B3 was hardly observed in HCCs with a
low ER (Fig. 3C). Furthermore, ERs were significantly correlated with OATP1B3 levels (r =
0.91, P < 0.0001; Fig. 3D).
The mechanism of uptake of Gd-EOB-DTPA in HCCs and the characteristics of
Gd-EOB-DTPA-positive HCCs have not been clarified to date. In the present study, 6 of 22
HCCs (27.3%) accumulated Gd-EOB-DTPA in the hepatobiliary phase. This raises questions
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regarding the kind of HCC that accumulates Gd-EOB-DTPA and the clinical importance of
uptake of Gd-EOB-DTPA. Well-differentiated HCCs have been shown to be more enhanced
than the surrounding liver parenchyma in the hepatobiliary phase, suggesting that MRI with
Gd-EOB-DTPA can be used to predict the extent of differentiation of tumors11, but it has also
been suggested that positive enhancement does not depend on tumor differentiation10. These
studies were both performed in animals and the situation in human HCC remains unclear. In
the current study, uptake of Gd-EOB-DTPA in the hepatobiliary phase was only observed in
moderately differentiated HCCs. However, 11 other moderately differentiated HCCs did not
accumulate Gd-EOB-DTPA. The frequency of bile production in HCCs with a high ER was
similar to that of HCCs with a low ER. These results suggest that neither tumor
differentiation nor bile production are correlated with Gd-EOB-DTPA enhancement in MRI
in HCCs in the hepatobiliary phase.
OATPs are sodium-independent organic anion transporters that are expressed in many
tissues, including the liver, kidney, intestine and brain. OATPs are involved in transport of a
wide range of amphipathic organic anions, including bile salts, thyroid hormones, steroid
hormones and their conjugates, as well as organic cations such as N-methylquinidine.12 In
addition to endogenous compounds, OATPs are capable of transporting xenobiotics and
drugs, and therefore play an important role in drug absorption, disposition and excretion.
OATP1B3, which is expressed in the human liver in the basolateral membrane of
hepatocytes, is especially important for transport of agents such as Gd-EOB-DTPA, as well
as uptake of endogenous substances and xenobiotics into hepatocytes.2, 12-14 Gd-EOB-DTPA
is transported into hepatocytes via OATPs and excreted into bile canaliculi via the multidrug
resistance-associated protein MRP2.15
OATP1B3 protein was overexpressed in all HCCs that showed accumulation of
Gd-EOB-DTPA in the hepatobiliary phase, whereas almost no expression of OATP1B3 was
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observed in most HCCs that did not show uptake of Gd-EOB-DTPA. Furthermore, a strong
correlation was observed between ERs and expression levels of OATP1B3 protein. We also
examined the expression of MRP2 protein by immunoblot analysis, since this protein is
another transporter of bile salts16, but expression of MRP2 was neither correlated with ERs
nor bile production (data not shown). It is of note that OATP1B3 also transports anticancer
drugs such as the antifolate methothrexate,17 and paclitaxel and docetaxel.18 Therefore, HCC
with upregulation of OATP1B3 may take up these drugs as well as Gd-EOB-DTPA, and this
suggests that preoperative Gd-EOB-DTPA-enhanced MRI can be used to predict the
potential efficacy of anticancer drugs transported by OATP1B3.
In conclusion, our results suggest that expression of OATP1B3 determines the uptake of
Gd-EOB-DTPA in the hepatobiliary phase in HCCs, rather than tumor differentiation or bile
production. However, the study is limited by the small number of cases and a further study is
needed to confirm the correlation between expression of OATP1B3 and Gd-EOB-DTPA
uptake and the clinical significance of Gd-EOB-DTPA enhancement in MRI of HCCs.
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Table 1. Comparison of characteristics between patients with HCCs with ER ≥ 1 and < 1
ER ≥ 1
(n = 6)
ER < 1
(n = 16)
Male 5 1
Female 1 3
Age 58.0 ± 10.4 62.3 ± 11.5 0.37
Well differentiated 0 3
Moderately differentiated 6 11 0.294
Poorly differentiated 0 2
Tumors with bile production 4 5 0.231
Mild (< 5%) 4 2
Moderate (5-25%) 0 1
Severe (> 25%) 0 2
OATP1B3 / Actin expression ratio 1.34 ± 0.59 0.34 ± 0.29 < 0.001
Data are shown as numbers or means ± SD.
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Figure 1. (A and B) MRI images of HCC without hepatocyte-selective enhancement in a
56-year-old-woman with a normal liver. Arrows indicate HCC located in a posterior liver
segment. (C and D) MRI images of HCC with hepatocyte-selective enhancement in a
67-year-old-man with mild steatosis. Arrows indicate HCC located in a posterior liver
Figure 2. (A) Surgically removed specimen. The cut surface of the tumor has a greenish
color (green hepatoma). (B) HE staining of HCC (400x). The arrow heads indicate bile
production on the insides of cytoplasm of tumor cells.
Figure 3. (A) Western blot for OATP1B3 and Actin (control) and densitometry ratio of the
OATP1B3 level to that of the control. Non-tumor liver parenchyma were diagnosed as
normal parenchyma (n = 4), chronic hepatitis (n = 6), liver cirrhosis (n = 8), and there was no
significant difference in non-tumor liver tissues. On the other hand, expression of OATP1B3
was significantly higher in HCCs with ER ≥ 1 compared to HCCs with ER < 1. Liver
sections from HCC with a high ER (B) and a low ER (C) were subjected to
immunohistochemical analysis to determine expression patterns of OATP1B3 protein.
Expression of OATP1B3 was observed predominantly in tumor cell membrane in HCC with
a high ER. (D) A strong correlation was observed between expression levels of OATP1B3