Volume 132, Issue 4, Pages 1495-1503 (April 2007) Sodium Iodide Symporter Is Expressed at the Preneoplastic Stages of Liver Carcinogenesis and in Human Cholangiocarcinoma  Bingkai Liu, Julie Hervé, Paulette Bioulac–Sage, Yannick Valogne, Jérôme Roux, Funda Yilmaz, Raphaël Boisgard, Catherine Guettier, Paul Calès, Bertrand Tavitian, Didier Samuel, Jérôme Clerc, Christian Bréchot, Jamila Faivre  Gastroenterology  Volume 132, Issue 4, Pages 1495-1503 (April 2007) DOI: 10.1053/j.gastro.2007.01.044 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 The NIS glycoprotein is expressed in tumors of a rat model of primary liver cancer. NIS protein immunostaining using specific polyclonal antibodies. (A) Gastric section. NIS is confined in crypt cells of gastric mucosa. Scale bar: 100 μm. (B) Thyroid section. NIS is located on the basolateral side of follicular thyroid cells. Scale bar: 25 μm. (C) Salivary gland section. NIS is located on the apical side of salivary ducts. Scale bar: 50 μm. These observations validate the antibodies used. (D) Section of normal rat liver. Normal hepatocytes do not express NIS. Scale bar: 200 μm. (E) Section of cancerous rat liver. NIS is strongly expressed at the plasma membrane of tumor cells. Scale bar: 200 μm. (F) NIS immunostaining is inhibited in a competition test with NIS peptide. Scale bar: 200 μm. (G) Enlarged view of NIS-expressing tumor cells. Scale bar: 100 μm. (H) Anti-NIS immunoblot of membrane protein extracts from a stomach (St), and 3 different liver tumors (T1, T2, and T3) treated (+) or nontreated (−) with N-glycosidase PNGase-F. The apparent molecular size of NIS protein shifts from about 100 to 50 kilodaltons upon deglycosilation. Gastroenterology 2007 132, 1495-1503DOI: (10.1053/j.gastro.2007.01.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 The NIS glycoprotein catalyses iodide uptake in cancerous liver of DEN rats. (A) 123I scintigraphy of 2 DEN rats (N1 and N2) 2 hours after an iodide injection before and after (+NaClO4) the administration of sodium perchlorate. A strong heterogeneous liver (L) contrast is visible in the 2 DEN rats, but not in the normal rat (control). Gastric (St) and thyroid (*) contrasts are visible in all cases. The administration of NaClO4 results in the diffusion of 123I from the liver to the whole body demonstrating that 123I uptake is NIS dependent. Bl: bladder. (B) Serial 123I scintigraphy of a DEN rat. The first image (8 weeks) corresponds to the end of carcinogen administration. Hepatic contrast increases over time. (C) Average liver and stomach 123I uptake of 5 DEN rats at the end of carcinogen administration (baseline) and 3 months later (end of study). A significant increase of 123I uptake occurred in the liver (but not in the stomach) during tumor growth. (D) Left: microimager visualization of 131I distribution in a liver section at month 6 of the disease. The iodide uptake is homogeneous in Tumor T1, heterogeneous in Tumor T2. Right: H&S staining of the same liver section. Arrowhead: boundary between tumor (T) and nontumor (NT) areas. Scale bar: 5 mm. (E) SPECT, CT, and SPECT/CT (fusion) images of a DEN rat (same rat as in B) showing the localization of 99mTc uptake in 2 well-delimited liver nodules (arrows) and the stomach. Gastroenterology 2007 132, 1495-1503DOI: (10.1053/j.gastro.2007.01.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 NIS expression occurs at an early stage of carcinogenesis in a DEN-induced rat model of liver cancer. (A) NIS antibody (top) and H&S (bottom) staining of 3-μm serial liver tissue sections at the indicated time points (d: days) after initiating carcinogen administration. At day 15, immunostaining reveals NIS expression in isolated hepatocytes, or small hepatocyte clusters (arrows), which are histologically normal. At subsequent time points, NIS-expressing clusters increase in size and are constituted of tumor hepatocytes. Inset: a heterogeneous NIS expression is visible inside the tumor nodule. Scale bars: 100 μm. (B) NIS (left) and GSTp (right) immunostaining of 3-μm serial liver sections. NIS and GSTp stainings overlap in a single cell at day 15, in clusters at day 30 (arrows). (*): hepatocytes stained by GSTp, not by NIS. The scale bars are of 150 μm in the top and bottom rows, and 50 μm in the middle row. Gastroenterology 2007 132, 1495-1503DOI: (10.1053/j.gastro.2007.01.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 NIS-mediated 131I therapy inhibits liver tumor growth. Livers of untreated and treated DEN rats examined 3 months after 131I treatment. (A) Macroscopic views. The untreated livers show multiple nodules, several centimeters in diameter. Treated livers are macroscopically normal, or contain much smaller, and less numerous nodules than untreated livers. (B) Sliced median lobe of a treated liver. Scale bar: 5 mm. (C) H&S, NIS antibody, and TUNEL staining from 3-μm serial liver tissue sections. A high proportion of 131I-treated tumor cells does not express NIS anymore, and is TUNEL positive (see the enlarged view in the inset). T: tumor area. NT: nontumor area. Arrows: apoptotic cells. Scale bar: 100 μm. Gastroenterology 2007 132, 1495-1503DOI: (10.1053/j.gastro.2007.01.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 The NIS glycoprotein is expressed in human liver cancers, chiefly CCA. RT-PCR for NIS/β-glucuronidase mRNA ratio in normal liver (NL, n = 12), tumor (T), and nontumor (NT) areas of CCA (n = 15) and HCC (n = 26). (A) Mean values ± SEM. (B) Individual data for patients presenting with CCA. Data are means over 3 independent experiments. Error bars give extreme values. Immunoblots for NIS protein (C) from thyroid adenoma (Th), CCA tumor areas (patients 10 and 14), and normal liver (NL); (D) from CCA tumor areas (patients 3, 5, and 10 sorted by increasing NIS mRNA copy numbers) before and after NIS peptide competition; (E) from thyroid adenoma (Th) and CCA tumor areas (patients 4 and 5) with (+) and without (−) peptidyl N-glycosidase F (PNGase F) digestion. Arrow: glycosylated NIS. Bracket: deglycosylated NIS. *P < .05. Gastroenterology 2007 132, 1495-1503DOI: (10.1053/j.gastro.2007.01.044) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 NIS 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). Gastroenterology 2007 132, 1495-1503DOI: (10.1053/j.gastro.2007.01.044) Copyright © 2007 AGA Institute Terms and Conditions