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Sodium Iodide Symporter Is Expressed at the Preneoplastic Stages of Liver Carcinogenesis and in Human Cholangiocarcinoma

2007; Elsevier BV; Volume: 132; Issue: 4 Linguagem: Inglês

10.1053/j.gastro.2007.01.044

1528-0012

Bingkaï Liu, Julie Hervé, Paulette Bioulac‐Sage, Yannick Valogne, J. Roux, Funda Yılmaz, Raphaël Boisgard, Catherine Guettier, Paul Calès, Bertrand Tavitian, Didier Samuel, Jérôme Clerc, Christian Bréchot, J Faivre,

Pancreatic and Hepatic Oncology Research

Background & Aims: The ability of thyroid cells to take up iodide, which enables 131I radiotherapy for thyroid cancer, is due to the expression of the sodium iodide symporter at their plasma membrane. Expression of this symporter has been found in some nonthyroid cancers. However, it is mostly accumulated in the cytoplasm, and its functionality has not been demonstrated. We have investigated sodium iodide symporter expression and functionality in human liver cancer, and in a diethylnitrosamine induced Wistar rat model of primary liver cancer at different stages of carcinogenesis. Methods: Sodium iodide symporter mRNA and protein were studied in tissues from patients with hepatocellular- or cholangio-carcinomas using reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemistry. We studied the dynamics of hepatic iodine uptake in the animal model using nuclear imaging. Results: Sodium iodide symporter expression showed up in all 20 cholangiocarcinomas, but in only 2 of the 26 hepatocellular carcinomas, investigated. It was also found in normal bile duct cells and in the ductular reaction present in cirrhotic tissues. It was located at the plasma membrane in 10 of 20 cholangiocarcinoma. In rat liver cancer, a functional sodium iodide symporter expression was triggered as from the early preneoplastic steps, and was amplified during clonal tumor cell expansion, allowing complete tumor suppression after 131I radiotherapy. Conclusions: A significant proportion of human cholangiocarcinomas expresses membrane sodium iodide symporter, which may permit radioiodine therapy. Our data also suggest that 131I acts on a crucial target for liver cancer development. Background & Aims: The ability of thyroid cells to take up iodide, which enables 131I radiotherapy for thyroid cancer, is due to the expression of the sodium iodide symporter at their plasma membrane. Expression of this symporter has been found in some nonthyroid cancers. However, it is mostly accumulated in the cytoplasm, and its functionality has not been demonstrated. We have investigated sodium iodide symporter expression and functionality in human liver cancer, and in a diethylnitrosamine induced Wistar rat model of primary liver cancer at different stages of carcinogenesis. Methods: Sodium iodide symporter mRNA and protein were studied in tissues from patients with hepatocellular- or cholangio-carcinomas using reverse-transcription polymerase chain reaction, immunoblot, and immunohistochemistry. We studied the dynamics of hepatic iodine uptake in the animal model using nuclear imaging. Results: Sodium iodide symporter expression showed up in all 20 cholangiocarcinomas, but in only 2 of the 26 hepatocellular carcinomas, investigated. It was also found in normal bile duct cells and in the ductular reaction present in cirrhotic tissues. It was located at the plasma membrane in 10 of 20 cholangiocarcinoma. In rat liver cancer, a functional sodium iodide symporter expression was triggered as from the early preneoplastic steps, and was amplified during clonal tumor cell expansion, allowing complete tumor suppression after 131I radiotherapy. Conclusions: A significant proportion of human cholangiocarcinomas expresses membrane sodium iodide symporter, which may permit radioiodine therapy. Our data also suggest that 131I acts on a crucial target for liver cancer development. The thyroid gland is well known for its ability to take up and retain iodide, which forms the basis for the 131I radiotherapy of thyroid cancer and some types of hyperthyroidism. This is due to the expression of the sodium iodide symporter (NIS) at the basolateral plasma membrane of thyroid follicular cells.1Dai G. Levy O. Carrasco N. Cloning and characterization of the thyroid iodide transporter.Nature. 1996; 379: 458-460Crossref PubMed Scopus (949) Google Scholar Several reports have noted that endogenous NIS expression in nonthyroid epithelial cells is generally stronger in proliferating (lactating mammary gland, fibroadenoma, cancers) than in nonproliferating cells.2Knostman K.A. Cho J.Y. Ryu K.Y. Lin X. McCubrey J.A. Hla T. Liu C.H. Di Carlo E. Keri R. Zhang M. Hwang D.Y. Kisseberth W.C. Capen C.C. Jhiang S.M. Signaling through 3',5'-cyclic adenosine monophosphate and phosphoinositide-3 kinase induces sodium/iodide symporter expression in breast cancer.J Clin Endocrinol Metab. 2004; 89: 5196-5203Crossref PubMed Scopus (22) Google Scholar, 3Kogai T. Kanamoto Y. Che L.H. Taki K. Moatamed F. Schultz J.J. Brent G.A. Systemic retinoic acid treatment induces sodium/iodide symporter expression and radioiodide uptake in mouse breast cancer models.Cancer Res. 2004; 64: 415-422Crossref PubMed Scopus (53) Google Scholar, 4Spitzweg C. Joba W. Eisenmenger W. Heufelder A.E. Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa.J Clin Endocrinol Metab. 1998; 83: 1746-1751Crossref PubMed Scopus (273) Google Scholar, 5Spitzweg C. Joba W. Schriever K. Goellner J.R. Morris J.C. Heufelder A.E. Analysis of human sodium iodide symporter immunoreactivity in human exocrine glands.J Clin Endocrinol Metab. 1999; 84: 4178-4184Crossref PubMed Scopus (102) Google Scholar, 6Tazebay U.H. Wapnir I.L. Levy O. Dohan O. Zuckier L.S. Zhao Q.H. Deng H.F. Amenta P.S. Fineberg S. Pestell R.G. Carrasco N. The mammary gland iodide transporter is expressed during lactation and in breast cancer.Nat Med. 2000; 6: 871-878Crossref PubMed Scopus (404) Google Scholar, 7Wapnir I.L. van de Rijn M. Nowels K. Amenta P.S. Walton K. Montgomery K. Greco R.S. Dohan O. Carrasco N. Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections.J Clin Endocrinol Metab. 2003; 88: 1880-1888Crossref PubMed Scopus (230) Google Scholar In breast cancers, mammary NIS glycoprotein is functional,6Tazebay U.H. Wapnir I.L. Levy O. Dohan O. Zuckier L.S. Zhao Q.H. Deng H.F. Amenta P.S. Fineberg S. Pestell R.G. Carrasco N. The mammary gland iodide transporter is expressed during lactation and in breast cancer.Nat Med. 2000; 6: 871-878Crossref PubMed Scopus (404) Google Scholar, 8Upadhyay G. Singh R. Agarwal G. Mishra S.K. Pal L. Pradhan P.K. Das B.K. Godbole M.M. Functional expression of sodium iodide symporter (NIS) in human breast cancer tissue.Breast Cancer Res Treat. 2003; 77: 157-165Crossref PubMed Scopus (40) Google Scholar, 9Wapnir I.L. Goris M. Yudd A. Dohan O. Adelman D. Nowels K. Carrasco N. The Na+/I- symporter mediates iodide uptake in breast cancer metastases and can be selectively down-regulated in the thyroid.Clin Cancer Res. 2004; 10: 4294-4302Crossref PubMed Scopus (92) Google Scholar is stimulated by retinoic acid,3Kogai T. Kanamoto Y. Che L.H. Taki K. Moatamed F. Schultz J.J. Brent G.A. Systemic retinoic acid treatment induces sodium/iodide symporter expression and radioiodide uptake in mouse breast cancer models.Cancer Res. 2004; 64: 415-422Crossref PubMed Scopus (53) Google Scholar, 10Dentice M. Luongo C. Elefante A. Romino R. Ambrosio R. Vitale M. Rossi G. Fenzi G. Salvatore D. Transcription factor Nkx-2.5 induces sodium/iodide symporter gene expression and participates in retinoic acid- and lactation-induced transcription in mammary cells.Mol Cell Biol. 2004; 24: 7863-7877Crossref PubMed Scopus (41) Google Scholar and is induced through the cAMP and phosphoinositide-3 kinase signaling.2Knostman K.A. Cho J.Y. Ryu K.Y. Lin X. McCubrey J.A. Hla T. Liu C.H. Di Carlo E. Keri R. Zhang M. Hwang D.Y. Kisseberth W.C. Capen C.C. Jhiang S.M. Signaling through 3',5'-cyclic adenosine monophosphate and phosphoinositide-3 kinase induces sodium/iodide symporter expression in breast cancer.J Clin Endocrinol Metab. 2004; 89: 5196-5203Crossref PubMed Scopus (22) Google Scholar Moreover, a transcription factor (nkx 2.5) activates NIS specifically in lactating and cancer cells.10Dentice M. Luongo C. Elefante A. Romino R. Ambrosio R. Vitale M. Rossi G. Fenzi G. Salvatore D. Transcription factor Nkx-2.5 induces sodium/iodide symporter gene expression and participates in retinoic acid- and lactation-induced transcription in mammary cells.Mol Cell Biol. 2004; 24: 7863-7877Crossref PubMed Scopus (41) Google Scholar These observations have led to radioiodine therapy being proposed as a therapeutic option for breast cancer. In the other organs where attempts have been made to detect NIS, its expression mostly accumulates in the cytoplasm.7Wapnir I.L. van de Rijn M. Nowels K. Amenta P.S. Walton K. Montgomery K. Greco R.S. Dohan O. Carrasco N. Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections.J Clin Endocrinol Metab. 2003; 88: 1880-1888Crossref PubMed Scopus (230) Google Scholar Whether or not NIS is functional or implicated in the cell transformation in these organs is unknown. We have investigated NIS expression and functionality in human liver cancers, and in a chemically induced rat model of primary liver cancer at different stages of carcinogenesis. We have thus demonstrated strong membrane NIS expression restricted to tumor cholangiocytes in human cholangiocarcinoma (CCA). Using the diethylnitrosamine (DEN) rat model of liver cancer, we also show that functional NIS expression occurs at a very early stage of liver carcinogenesis and is amplified during clonal tumor cell expansion, which enables the effective 131I radiotherapy of the tumors. Overall, our study has identified CCA as an additional human cancer expressing the NIS glycoprotein, which may permit radioiodine therapy. It has also demonstrated a close and intriguing association between NIS and liver cell transformation. Paraffin-embedded and deep-frozen samples from 26 patients presenting with HCC and 20 patients presenting with intrahepatic CCA were investigated. Among the 26 patients presenting with HCC, 10 were chronically infected with hepatitis B virus, 7 with hepatitis C virus, and 9 with non-hepatitis B virus, non-hepatitis C virus. For each patient we analyzed tissues originating from tumor areas and nontumor areas distant from tumors. Ten normal liver samples were also included in the study. All patients gave written informed consent. The study was approved by the local institutional review boards. Wistar male rats (150–180 g) were obtained from Charles River (France), bred, and kept at the animal facility of Necker University. Experiments were performed under the institutional and European Union guidelines for laboratory animal care. Hepatic carcinogenesis was induced in rats by administering 10 mg/kg of DEN daily in their drinking water for 8 weeks. Their drinking water was supplemented with 50 μg/L thyroxin 2 weeks before nuclear imaging and 131I therapy to reduce thyroid iodine uptake. Six to 12 MBq (0.16 to 0.32 mCi) of Na123I (Schering AG, Berlin, Germany) were injected in rats via the intraperitoneal route. Scintigraphic 256 × 256 images were obtained using a parallel-collimator DSX gamma camera (GE Corp., Syracuse, NY) with a spatial resolution of 5 mm. An aliquot of the tracer administered was also imaged as an internal standard. The same procedure was applied serially for kinetic studies, and biologic uptakes and half-lives were computed as described previously.12Faivre J. Clerc J. Gerolami R. Herve J. Longuet M. Liu B. Roux J. Moal F. Perricaudet M. Brechot C. Long-term radioiodine retention and regression of liver cancer after sodium iodide symporter gene transfer in Wistar rats.Cancer Res. 2004; 64: 8045-8051Crossref PubMed Scopus (65) Google Scholar After an initial image acquisition, rats were injected via the intraperitoneal route with 100 mg sodium perchlorate, and image acquisition was resumed at 1-minute intervals for 30 minutes. Thirty-seven MBq (1 mCi) of [99mTc] pertechnetate were injected into the tail vein of the rats. SPECT/CT acquisition was performed using a γ-IMAGER-S/CT (Biospace Mesures, France) with a spatial resolution of 2 mm and a 10-cm field of view. The spatial resolution of the CT subsystem was 250 μm. The acquisition time was 40 minutes per animal. 3D images were displayed using the Amira 3.1.1 analysis tool (Mercury Computer Systems Inc, Chelmsford, MA). 131I labeled frozen liver sections were analyzed with a microimager with a 24 × 32 mm2 field of view and 25-μM/L resolution (Biospace Mesures, France). Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) and then reverse transcribed into cDNA using a RevertAid™ First-Strand cDNA Synthesis Kit (Fermentas, Burlington, Ontario), amplified and quantified by detection of SYBR Green (Roche Diagnostics, Indianapolis, IN). They were quantified by spectrometry, and their quality was assessed by electrophoresis. RT-PCR and melting curve analysis were done in a LightCycler 3.0 (Roche). We used a pcDNA5 vector carrying human NIS cDNA as a calibration standard for quantification, and β-glucuronidase as a housekeeping gene for normalization. Primer sequences were 5 ′-CTCTCTCAGTCAACGCCTCTGG-3 ′ (forward) and 5′-CCTGTGTTGGACATGATGGTGGT-3′ (reverse) for human NIS, and 5 ′-CTCATTTGGAATTTTGCCGATT-3′ (forward) and 5′-CCGAGTGAAGATCCCCTTTTTA-3 ′ (reverse) for human β-glucuronidase. Formalin-fixed paraffin-embedded human and rat sections were stained with hematoxylin-eosin and examined under an optical microscope. Rat tissues were incubated with polyclonal rabbit antibodies directed against rat 600–618 NIS peptide, generated in our laboratory. Human tissues were incubated with monoclonal NIS antibodies (FP5A and 14F, Labvision, Fremont, CA). Polyclonal rabbit antibody against glutathione S-transferase P was purchased from MBL, Japan. Incubation with primary antibody was followed by incubation with peroxidase-conjugated donkey anti-rabbit or mouse antibody. Plasma membrane and total protein extracts from liver, thyroid, and stomach tissues were prepared in a buffer containing 250 mmol/L sucrose, 1 mmol/L EDTA, 20 mmol/L HEPES pH 7.5 and mixture of protease inhibitors (Complete Protease Inhibitor, Roche), and then ultracentrifuged at 105×g and 4°C for 1 hour. Proteins were separated on 8% SDS-PAGE and transferred onto nitrocellulose. Incubations with the above antibodies were performed in 20 mmol/L Tris–HCl pH 7.5, 137 mmol/L NaCl, 0.05% Tween 20, 5% nonfat dry milk. NIS immunoreactivity was competitively inhibited in the presence of 5 μg of the NIS peptide used to generate antibodies. Twenty micrograms of membrane protein extracts were incubated for 1 hour at 37°C with 25 UI peptidyl N-glycosidase F (Biolabs, Ipswich, MA) in 50 mmol/L Sodium Phosphate pH 7.5, 1% Nonidet P-40. The chemoluminescence ECL Plus reagent was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Apoptosis was measured using an Apoptag detection kit according to the manufacturer's instructions (Chemicon Inc., Temecula, CA). We performed statistical analysis using the Student unpaired t test and Stat View software. Wistar rats treated with diethylnitrosamine for 2 months (referred to as DEN rats) developed dysplastic nodules and then multifocal hepatocellular carcinoma (HCC), and generally died within 6 months of initiating carcinogen administration. Using 2 polyclonal antibodies (R10 and R14) against the rat NIS protein, we verified that these antibodies recognized gastric, thyroid, and salivary-gland NIS by immunohistochemistry (IHC) and immunoblot (Figure 1). NIS was confined to the crypt and surface epithelial cells at the apex of the gastric mucosa (Figure 1A). NIS was located on the basolateral side of rat follicular thyroid cells (Figure 1B), and on the apical side of rat salivary glands (Figure 1C). We never observed any NIS expression in the hepatocytes of normal rats (Figure 1D). DEN rats sacrificed 4 months after the start of carcinogen administration exhibited clear NIS staining at the plasma membrane of tumor hepatocytes (Figure 1E and G). Peptide competition tests confirmed the specificity of this staining (Figure 1F). Western blots of gastric extracts yielded the expected broad signal ranging from about 75 to 110 kDa (Figure 1H). The apparent molecular size of NIS (about 90 to 65 kDa) was within the range of the mature glycosylated form of NIS, in line with its observed membrane localization (see below); comparison with gastric extracts suggested a tissue-dependent post-translational maturation of NIS.11Levy O. De la Vieja A. Ginter C.S. Riedel C. Dai G. Carrasco N. N-linked glycosylation of the thyroid Na+/I-symporter (NIS) Implications for its secondary structure model.J Biol Chem. 1998; 273: 22657-22663Crossref PubMed Scopus (162) Google Scholar The peptidyl N-glycosydase F digestion of tumor membrane extracts yielded 45 to 55 kilodaltons species, indicating complete digestion of the NIS glycoprotein. We injected 123I via the intraperitoneal route in liver cancer-bearing rats, and performed serial planar scintigraphy analyses at various time points during the disease. The image was taken 2 hours after 123I injection, when the injected activity was completely resorbed, and urinary excretion negligible. Dynamic studies showed that, at this time point, tracer activity has long ceased to circulate in the blood, and is trapped in stomach, thyroid (which are both known NIS-expressing organs), and liver; in contrast, organs with high blood flow, but without a strong NIS expression such as the kidney, brain, or normal liver show no tracer activity.12Faivre J. Clerc J. Gerolami R. Herve J. Longuet M. Liu B. Roux J. Moal F. Perricaudet M. Brechot C. Long-term radioiodine retention and regression of liver cancer after sodium iodide symporter gene transfer in Wistar rats.Cancer Res. 2004; 64: 8045-8051Crossref PubMed Scopus (65) Google Scholar Thus, the contrast, at 2 hours essentially reflects NIS activity. A clear, heterogeneous hepatic contrast appeared after about 3 months (the initiation of DEN administration being taken as time 0), and increased over time (Figure 2A and B). Whole-liver 123I uptake reached 1.5% ± 0.7% of the injected dose (%ID) in normal control rats. It had the same value in DEN rats at month 3 (n = 35), rose to 9.1 ± 2.5 %ID at month 4 (n = 22), and to 14.2 ± 1.7 %ID at month 6 (n = 3). The calculated biologic half-life of iodide was 39 ± 29 hours, consistent with our previous observations using 131I.12Faivre J. Clerc J. Gerolami R. Herve J. Longuet M. Liu B. Roux J. Moal F. Perricaudet M. Brechot C. Long-term radioiodine retention and regression of liver cancer after sodium iodide symporter gene transfer in Wistar rats.Cancer Res. 2004; 64: 8045-8051Crossref PubMed Scopus (65) Google Scholar These values were similar to those found in the stomach (123I uptake and biologic half-life of 23.5 ± 6 %ID and 42 ± 38 hours, respectively). The injection of sodium perchlorate, a specific inhibitor of NIS-dependent iodide transport, led to extinction of the hepatic contrast and whole-body diffuse 123I distribution 5 minutes after injection, confirming that the hepatic iodide uptake was essentially NIS mediated (Figure 2A). We then selected 5 rats presenting with clearly delimited nodules at 5 months, defined 9 regions of interest (ROI), each including a single nodule, and measured the 123I uptake in these ROI at time intervals of 0.5 month from month 2 to month 5. As a comparison, we measured simultaneously 123I uptake in gastric ROI in the same rats. 123I uptake increased significantly (from 4 ± 0.7 to 7 ± 0.9 %ID; P = .015) in liver ROI, while remaining unchanged in gastric ROI (Figure 2C). This confirmed that liver iodide uptake was closely related to tumor volume. Microimager-based analysis performed at a late cancer stage (month 6) confirmed iodide uptake confined inside the nodules. Some nodules exhibited a heterogeneous intranodular iodide distribution, in line with their heterogeneous NIS expression (Figure 2D). We performed 2-mm resolution-SPECT/CT (single photon emission computed tomography coupled with high-powered computerized tomography) after an intraperitoneal injection of 99mTc. Three-dimensional analysis revealed multiple, well-delimited volumes located in the liver area, accounting for the heterogeneous contrast observed in scintigraphy images (Figure 2E). We checked by IHC that these volumes coincided with the NIS expressing nodules (not shown). We investigated the kinetics of NIS expression in DEN-induced rat liver carcinogenesis during and after carcinogen administration (Figure 3A and B). As early as 15 days after the initiation of DEN administration, liver sections exhibited a single, or a very small cluster (less than 5 cells) of NIS-stained cells. Although these NIS-stained cells were morphologically normal, they were glutathione S-transferase P stained, demonstrating that they had already been driven into the carcinogenetic process (Figure 3B). The size and density of NIS-stained clusters increased dramatically as the duration of carcinogen administration lengthened. As from 30 days of administration, NIS-stained clusters successively exhibited a clear dysplastic and then neoplastic morphology. This strongly suggests that each tumor nodule originated from a single NIS-expressing cell, from which it clonally expanded. We administered an 18 mCi dose of 131I to 8 DEN rats at month 2, and then followed their liver morphology by Doppler ultrasonography (not shown). Three months later, we sacrificed these rats together with 5 nonirradiated DEN rats at the same stage of the disease (month 5). Five of the 8 irradiated rats exhibited no tumor growth, while all nonirradiated controls exhibited multinodular tumor progression. More precisely, 5 of the 8 irradiated rats had macroscopically normal livers, while the other 3 only exhibited a few small nodules less than 1 cm in diameter (Figure 4A and B). The livers showed no evidence of any tumor cell proliferation after irradiation on histologic evaluation; the degree of apoptosis varied from liver to liver, ranging from a few apoptotic cells to a ballooning and massive apoptosis of formerly tumor cells, which had lost NIS expression. Non-irradiated livers showed very little apoptosis at the same stage of the disease (Figure 4C). Such a delayed apoptosis following internal 131I irradiation was previously conjectured to account for the incidence of hypothyroidism after radioiodine therapy of thyrotoxicosis,13Malone J.F. Cullen M.J. Two mechanisms for hypothyroidism after 131I therapy.Lancet. 1976; 2: 73-75Abstract PubMed Scopus (19) Google Scholar but not directly observed. Thus, 131I administered at an early stage of carcinogenesis exerted significant antitumor efficacy. We analyzed tissues from 15 patients with cholangiocarcinoma using real-time polymerase chain reaction (PCR). The average NIS mRNA level normalized for the β-glucuronidase housekeeping gene was significantly higher in CCA tumors than in HCC tumors (average NIS/β-glucuronidase ratios of 147 and 7, respectively). In cholangiocarcinoma, tumor areas expressed NIS mRNA more strongly than the adjacent nontumor areas (Figure 5A and B). NIS mRNA was virtually undetectable in normal liver (NIS/β-glucuronidase ratio <3.5). The NIS protein appeared at ∼100 kilodaltons in Western blots of all the CCA tumors studied (Figure 5C). Signal intensity varied from sample to sample, more or less proportionally with NIS mRNA levels measured by real-time PCR (RT-PCR). Using NIS peptide competition, we verified the specificity of NIS staining (Figure 5D). Also, peptidyl N-glycosydase F digestion of membrane CCA tumor extracts yielded 55 to 65 kilodaltons species, indicating complete digestion of the NIS glycoprotein (Figure 5E). We then analyzed 20 tumor–nontumor pairs of CCA samples by IHC. We found that NIS was expressed specifically in the cholangiocytes of bile duct epithelia (biliary canals and ductules) in all patients presenting with CCA (Figure 6A–G). Very weak NIS expression was found in the biliary canals of normal livers (Figure 6H), contrasting with the strong expression exhibited by cholangiocytes in both tumor and adjacent nontumor areas in CCA livers (Figure 6A and B). Within bile duct tumor cells, NIS accumulated in the cytoplasm in 11, the plasma membrane in 2, and in both compartments in 7 of the total of 20 patients investigated. Within nontumor bile duct cells, NIS accumulated at the basolateral side of the plasma membrane. Of note was the fact that NIS staining clearly delineated CCA tumor areas, which consisted in stained proliferating ducts (Figure 6A). We next analyzed 26 tumor–nontumor pairs of HCC samples (Figure 6I–M). In contrast with our findings in CCA, IHC did not reveal any NIS expression in tumor hepatocytes (Figure 6I), except in 2 of the 26 patients (Figure 6J and K). We also noted constant cytoplasmic NIS expression in the proliferating ducts from corbelled structures surrounding HCC nodules (Figure 6L), or nontumor cirrhotic areas (Figure 6M). This accounts for the fact that we found some mRNA NIS expression in frozen HCC tissue using real-time PCR (data not shown).Figure 6NIS is expressed in normal and tumor bile duct cells. NIS immunostaining of tissue sections from human cholangiocarcinoma (A–G), human normal liver (H), human hepatocellular carcinoma (I–M). (A) Tumor (T) and nontumor (NT) areas. Arrow: ductular cells. (B) NIS expression at the cell basolateral side of a large bile duct in a nontumor area. (C–F) Tumor bile duct cells showing NIS both at the plasma membrane and in the cytoplasm (C, D), or exclusively at the plasma membrane (E, F). (G) NIS peptide competition on section (E). (H) Normal hepatocytes do not express NIS. Arrow: portal-tract bile duct. (I) Section typical for most HCC cases, in which tumor hepatocytes do not express NIS. (J, K) Section from 1 of the few HCC cases, in which tumor hepatocytes expressed NIS. (L, M) NIS expressing ductular reaction (arrows) forming corbelled structures surrounding (L) a HCC nodule, and (M) cirrhotic nodules. The scale bars are of 100 μm in (A–G, K) and 250 μm in (H–J, L, M).View Large Image Figure ViewerDownload Hi-res image Download (PPT) During this study, we demonstrated a marked NIS expression in human CCA (but not in human HCC) tumor cells. Tumor biliary cells expressed NIS in all (20) studied CCA cases. NIS was located at the plasma membrane in 9 of these patients, and was thus likely to be functional. CCA is a fatal disease (5-year survival of 2%), for which practically no efficient therapy is available at present.14Sirica A.E. Cholangiocarcinoma: molecular targeting strategies for chemoprevention and therapy.Hepatology. 2005; 41: 5-15Crossref PubMed Scopus (262) Google Scholar, 15Malhi H. Gores G.J. Review article: the modern diagnosis and therapy of cholangiocarcinoma.Aliment Pharmacol Ther. 2006; 23: 1287-1296Crossref PubMed Scopus (84) Google Scholar, 16Lazaridis K.N. Gores G.J. Cholangiocarcinoma.Gastroenterology. 2005; 128: 1655-1667Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 17Khan S.A. Thomas H.C. Davidson B.R. Taylor-Robinson S.D. Cholangiocarcinoma.Lancet. 2005; 366: 1303-1314Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar, 18Gores G.J. Baskin-Bey E.S. Baron T.H. Alberts S.R. Treatment endpoints for advanced cholangiocarcinoma.Nat Clin Pract Gastroenterol Hepatol. 2004; 1: 4-5Crossref PubMed Scopus (5) Google Scholar Our observations suggest considering 131I radiotherapy as a therapeutic option for CCA, as was also suggested recently for breast cancer. Several issues need to be addressed before clinical trials can be envisaged. In particular, checks must be made that iodide uptake levels and residence times in NIS expressing tumor cells are sufficient for radiotherapy to be both safe and efficient.19Daniels G.H. Haber D.A. Will radioiodine be useful in treatment of breast cancer?.Nat Med. 2000; 6: 859-860Crossref PubMed Scopus (25) Google Scholar It has been reported that a rapid efflux of iodide from tumor cells may result in a short iodide residence time in some nonthyroid NIS-expressing cells.20Mandell R.B. Mandell L.Z. Link Jr, C.J. Radioisotope concentrator gene therapy using the sodium/iodide symporter gene.Cancer Res. 1999; 59: 661-668PubMed Google Scholar, 21Haberkorn U. Kinscherf R. Kissel M. Kubler W. Mahmut M. Sieger S. Eisenhut M. Peschke P. Altmann A. Enhanced iodide transport after transfer of the human sodium iodide symporter gene is associated with lack of retention and low absorbed dose.Gene Ther. 2003; 10: 774-780Crossref PubMed Scopus (50) Google Scholar On this subject, it is interesting to note that we had previously observed a long iodide residence time in rat liver cancer, probably due to the high hepatic blood flow favoring the reuptake of effluent iodide.12Faivre J. Clerc J. Gerolami R. Herve J. Longuet M. Liu B. Roux J. Moal F. Perricaudet M. Brechot C. Long-term radioiodine retention and regression of liver cancer after sodium iodide symporter gene transfer in Wistar rats.Cancer Res. 2004; 64: 8045-8051Crossref PubMed Scopus (65) Google Scholar In normal liver, where previous reports mentioned NIS in intrahepatic bile canaliculi only,7Wapnir I.L. van de Rijn M. Nowels K. Amenta P.S. Walton K. Montgomery K. Greco R.S. Dohan O. Carrasco N. Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections.J Clin Endocrinol Metab. 2003; 88: 1880-1888Crossref PubMed Scopus (230) Google Scholar we found weak, but constant NIS expression in bile duct cells, consistent with biliary NIS acting as a transporter. Chiefly, we found that NIS expression was significantly stronger in bile ducts and proliferating biliary ductules belonging to the nontumor areas of primary liver cancers than in normal bile ducts. NIS was located at the bile duct plasma membrane, and in ductule cytoplasm. Such NIS up-regulation in proliferating duct cells was also reported in breast tissue.7Wapnir I.L. van de Rijn M. Nowels K. Amenta P.S. Walton K. Montgomery K. Greco R.S. Dohan O. Carrasco N. Immunohistochemical profile of the sodium/iodide symporter in thyroid, breast, and other carcinomas using high density tissue microarrays and conventional sections.J Clin Endocrinol Metab. 2003; 88: 1880-1888Crossref PubMed Scopus (230) Google Scholar We also studied NIS expression in a DEN-induced rat model of hepatocellular carcinoma, in which HCC develops from mature hepatocytes.22Sell S. Cellular origin of hepatocellular carcinomas.Semin Cell Dev Biol. 2002; 13: 419-424Crossref PubMed Scopus (78) Google Scholar We found that, in this model, a functional NIS was expressed specifically in tumors. The reason for which rat HCC expresses NIS, unlike most human HCC, is unknown. We took advantage of this model to study the timing of NIS activation during the multistep process of DEN carcinogenesis. We demonstrated that NIS staining appeared initially in isolated, histologically normal hepatocytes, then in small transformed cell clusters, and eventually in tumor nodules. Thus, NIS expression was triggered at the preneoplastic stage, and was maintained throughout the subsequent clonal expansion of cancer. This accounts for the efficacy of 131I therapy applied to DEN rats at an early stage of the disease. Indeed, a dose of 18 mCi injected at the end of carcinogen administration resulted in a complete suppression of tumors in 5 of the 8 animals studied, and a marked slowing of tumor growth in the others. In particular, this radical antitumor effect occurred when large multifocal HCC tumors tended to develop. Clearly, 131I acted on a crucial target for cancer development, which perhaps means that functional NIS was expressed in progenitor tumor cells. The general validity of these conclusions clearly needs to be investigated in other cancer models. In conclusion, our study suggests that human CCA, which is an increasingly common tumor with a very poor prognosis, could be amenable to 131I therapy. We thank J.M. Correas for performing Doppler ultrasonography imaging, T. Pourcher for providing us with pcDNA5-hNIS vector, and A.E. Sayag for helpful discussions. We are grateful to Biospace Mesures for kindly providing access to a microimager.