Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes (original) (raw)
In our experimental system, hepatocytes or cholangiocytes expressing albumin (Alb) or cytokeratin 19 (CK19) in the liver, respectively, were heritably labeled in mutant mice with a tamoxifen-inducible (TM-inducible) Cre/loxP system. We used mouse lines expressing an inducible form of Cre recombinase (CreERT2) from the Alb or CK19 genomic locus (Alb-CreERT2 mice, ref. 8, or CK19-CreERT2 mice [Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI63065DS1], respectively). In mice resulting from crosses with R26RlacZ/lacZ or R26RYFP/YFP reporter mouse lines (9), administration of TM allowed permanent marking of _Alb_- or _CK19_-expressing cells and enabled us to follow the fate of the progeny of hepatocytes or cholangiocytes, respectively (Figure 1A and Supplemental Figures 2 and 3).
Primitive ductules formed in ICC are derived from hepatocytes, rather than cholangiocytes. (A) Experimental procedure to follow the lineage of hepatocytes and cholangiocytes in mouse liver. In the presence of TM, CreERT2 expressed from the Alb or CK19 genomic locus translocates into the nucleus and removes the loxP-flanked stop cassette from the R26R allele, leading to permanent heritable expression of the β-gal or YFP gene. (B) Representative images of liver from mice that drank normal water (left) or TAA-containing water (right) for 30 weeks. Tumoral macronodules only formed after TAA administration. (C and D) Immunohistochemical staining of CK19 (C, top row), X-gal staining (C, bottom row), and co-immunofluorescence staining of β-gal and EpCAM (D) were conducted for neoplastic nodules formed in the liver of Alb-CreERT2;R26RlacZ/+ mice and CK19-CreERT2;R26RlacZ/+ mice after 30 weeks’ TAA administration. DNA was stained with DAPI. Scale bars: 10 mm (B), 100 μm (C), and 50 μm (D).
At 1 week after TM injection, we administered thioacetamide (TAA) to Alb-CreERT2;R26RlacZ/+ and CK19-CreERT2;R26RlacZ/+ mice to induce ICC (10, 11). After 30 weeks of TAA administration, all of the treated mice developed macronodular liver cirrhosis (Figure 1B). Immunohistochemical and histochemical analyses revealed that many primitive ductules composed of CK19-positive cells and containing cells with cytoplasmic mucin granules were formed into neoplastic nodules, showing the typical histology of ICC (Figure 1C and Supplemental Figure 4). In contrast, the absence of hepatocyte nuclear factor 4α (Hnf4α) expression in these primitive ductular cells eliminated the possibility of the development of hepatocellular carcinoma with pseudoglandular formation (Supplemental Figure 5). X-gal staining conducted to detect β-gal enzyme activity revealed that the primitive ductules found in Alb-CreERT2;R26RlacZ/+ mice, but not in CK19-CreERT2;R26RlacZ/+ mice, consisted of lacZ-positive cells (Figure 1C). Co-immunofluorescence staining of β-gal and epithelial cell adhesion molecule (EpCAM), an antigen for biliary lineage cells in the normal and neoplastic liver (12), revealed that EpCAM-expressing primitive ductules in Alb-CreERT2;R26RlacZ/+ mice, but not in CK19-CreERT2;R26RlacZ/+ mice, were positive for β-gal expression (Figure 1D). In control experiments, we kept Alb-CreERT2;R26RlacZ/+ and CK19-CreERT2;R26RlacZ/+ mice without TAA administration for 30 weeks after TM injection. In these mice, liver tissue morphology was normal, and β-gal was only detectable in hepatocytes and cholangiocytes, respectively (data not shown). These data indicate that the primitive ductules formed into neoplastic nodules of ICC are derived from hepatocytes, rather than cholangiocytes.
Given these unexpected findings, we next examined the mechanism controlling the generation of ICC from hepatocytes. After 14 weeks of TAA administration, CK19-expressing cells appeared in the hepatic lobules around the central veins in the liver, which were distinguished from cholangiocytes lining the intrahepatic biliary apparatus (Figure 2, A and B). Immunofluorescence analyses revealed that these newly generated CK19-positive cells expressed YFP in Alb-CreERT2;R26RYFP/+ mice, but not in CK19-CreERT2;R26RYFP/+ mice (Figure 2, C and D), indicating that hepatocytes residing around the central veins were initially converted into CK19-positive biliary lineage cells. These CK19-positive cells had an elongated morphology, but were clearly distinct from the surrounding desmin-positive stellate cells (Supplemental Figure 6) and were not derived from stellate cells that exhibited unexpected Cre activity (Supplemental Figure 7).
Hepatocytes on the periphery of central veins in the hepatic lobules are converted into biliary lineage cells at the onset of ICC. (A–D) Immunofluorescence staining of CK19 was conducted in the liver of control wild-type mice (A) and the liver of wild-type mice (B), Alb-CreERT2;R26RYFP/+ mice (C), and CK19-CreERT2;R26RYFP/+ mice (D) after 14 weeks of TAA administration. CV, cetral vein; PV, portal vein; BD, bile duct. DNA was stained with DAPI. Scale bars: 500 μm (A and B) and 100 μm (C and D).
Previous studies have shown that activation of Notch signaling is important for induction of the biliary program not only in hepatic progenitor cells during development but also in postnatal hepatocytes (13, 14). Thus, our findings suggested that Notch signaling is also important for the conversion of hepatocytes into biliary lineage cells in the early stages of ICC formation. To address this issue, we investigated the process of ICC formation in the liver of TM-administered Alb-CreERT2;R26RNotch/+ mice, in which the hepatocytes express an intracellular fragment of mouse Notch1 (NotchIC) (15) that exhibits constitutive Notch signaling activity, and Alb-CreERT2;Hes1fl/fl mice, in which the hepatocytes lack the gene encoding the Notch effector Hes1 (16). After 14 weeks of TAA administration, immunofluorescence analyses revealed that the number of CK19-positive cells in the hepatic lobules was significantly increased or decreased in Alb-CreERT2;R26RNotch/+ or Alb-CreERT2;Hes1fl/fl mice, respectively (Figure 3, A–D). Lineage-tracing analyses using Alb-CreERT2;R26RYFP/Notch mice confirmed that the CK19-positive cells that were increased in response to Notch signal activation were derived from hepatocytes (Figure 3E). Moreover, neoplastic nodules rapidly formed in the liver of Alb-CreERT2;R26RNotch/+ mice even after 14 weeks of TAA administration, but were not observed in the liver of R26RNotch/+ and Alb-CreERT2;Hes1fl/fl mice (Figure 3, F and G, and Supplemental Table 1). Indeed, the number of developing primitive ductules formed by CK19-positive cells was considerably increased in the liver of Alb-CreERT2;R26RNotch/+ mice after 14 weeks of TAA administration (Supplemental Figure 8). Thus, the results of our gain-of-function and loss-of-function experiments demonstrate that ICC arises through Notch-mediated conversion of hepatocytes into biliary lineage cells and that its malignancy and progression depend on the intensity of Notch signaling in hepatocytes.
Notch signal activation is significant not only for conversion of hepatocytes into biliary lineage cells at the onset of ICC, but also the malignancy and progression of ICC. (A–C) Immunofluorescence staining of CK19 was conducted in the liver of R26RNotch/+ mice (A), Alb-CreERT2;R26RNotch/+ mice (B), and Alb-CreERT2;Hes1fl/fl mice (C) after 14 weeks of TAA administration. (D) The percentages of cells immunoreactive for CK19 in the liver of R26RNotch/+, Alb-CreERT2;R26RNotch/+, and Alb-CreERT2;Hes1fl/fl mice were calculated after counting approximately 3,000 cells per field of vision in 3 discontinuous liver tissue slides for 3 individual mice after 14 weeks of TAA administration. The data represent mean ± SD. (E) Immunofluorescence staining of CK19 was conducted in the liver of Alb-CreERT2;R26RYFP/Notch mice after 8 weeks of TAA administration. (F and G) Representative images of the liver from R26RNotch/+ mice (F) and Alb-CreERT2;R26RNotch/+ mice (G) after 14 weeks of TAA administration. Developing neoplastic nodules are only found in the liver of Alb-CreERT2;R26RNotch/+ mice. CV, central vein; PV, portal vein. DNA was stained with DAPI. Scale bars: 500 μm (A–C), 100 μm (E), and 5 mm (F and G).
Although it has been generally accepted that ICC is a tumor derived from cholangiocytes, our present data indicate that, at least in our mouse TAA model, ICC actually arises from hepatocytes, rather than cholangiocytes, through Notch-mediated conversion of hepatocytes into biliary lineage cells. However, it remains unclear why hepatocytes residing around the central veins are actively converted into biliary lineage cells in this model of ICC. In the portal areas of the hepatic lobule, it has been reported that hepatocytes can change into biliary lineage cells when intrahepatic bile duct regeneration is induced, but cholangiocytes cannot proliferate owing to toxic influences (17). Thus, hepatocytes may be capable of changing their fate to biliary lineage cells regardless of their position in the hepatic lobule, while the particular location of lineage-converting hepatocytes is likely decided by the nature of the toxins used. In the case of TAA, its toxicity depends on the activity of cytochrome P450 2B and 2E1, which are expressed in hepatocytes around the central veins in the liver (18–22). This may likely explain why CK19-positive biliary lineage cells appeared in the pericentral zone of the hepatic lobule after TAA administration.
Although it remains uncertain whether human ICC also arises from hepatocytes, our data may provide an answer to the question of why patients with viral hepatitis often develop ICC. In these patients, hepatocytes infected with hepatitis viruses may be converted into biliary lineage cells via Notch signal activation and become the source of ICC. Therefore, suppression of Notch signaling, which could inhibit the conversion of hepatocytes into biliary lineage cells at the onset of ICC, would be a novel strategy in the treatment of ICC. The identification of the cellular origin of ICC will be instrumental not only for elucidating the pathogenic mechanism of ICC, but also developing therapeutic approaches to this refractory disease.