Intrahepatic cholangiocarcinoma is a treatment refractory malignancy with a high mortality and an increasing incidence worldwide. Recent studies have observed that activation of Notch and AKT signaling within mature hepatocytes is able to induce the formation of tumors displaying biliary lineage markers, thereby raising the suggestion that it is hepatocytes, rather than cholangiocytes or hepatic progenitor cells that represent the cell of origin of this tumor. Here, we use a cholangiocyte-lineage tracing system to target p53 loss to biliary epithelia and observe the appearance of labeled biliary lineage tumors in response to chronic injury. Consequent to this, upregulation of native functional Notch signaling is observed to occur spontaneously within cholangiocytes and hepatocytes in this model as well as in human intrahepatic cholangiocarcinoma. These data prove that in the context of chronic inflammation and p53 loss, frequent occurrences in human disease, biliary epithelia are a target of transformation and an origin of intrahepatic cholangiocarcinoma. Cancer Res; 74(4); 1005–10. ©2013 AACR.

The unexplained increase in incidence of intrahepatic cholangiocarcinoma (1, 2), coupled with its poor response to chemotherapeutics and high mortality, necessitates a greater understanding of the biology of this aggressive malignancy, in which the cell of origin remains unclear. The historic assumption that these tumors arise from the oncogenic transformation of mature biliary epithelia has been based on a glandular histologic morphology, location within and adjacent to the biliary network, and expression of cholangiocyte-specific proteins, including mucin and biliary cytokeratins 7 and 19 (3). Substantive evidence for this origin, however, has been lacking. Patients with primary sclerosing cholangitis and liver fluke infection, diseases characterized by chronic biliary inflammation and epithelial proliferation, are up to 161 and 27 times more likely to develop biliary tract cancers compared with the general population (4, 5). Bipotential hepatic progenitor cells (HPC) have also been considered as a cellular source of intrahepatic cholangiocarcinoma in light of the existence of combined hepatocellular cholangiocarcinoma (6), tumors with features of both cholangiocarcinoma and hepatocellular carcinoma, as well as cholangiolocellular carcinoma, characterized by ductular reaction and cords resembling the Canals of Hering (7).

Interestingly, the incidence of intrahepatic cholangiocarcinoma is increased in chronic hepatocellular injury such as hepatitis C virus and heptatitis B virus (8) infection, indicating a more complex cellular origin of these cancers. Recent work has demonstrated that mature hepatocytes possess potential for transdifferentiation into intrahepatic cholangiocarcinoma, a phenomenon dependent on intracellular Notch signaling (9, 10). This concurs with the known role of Notch in the specification of hepatoblasts during ontogeny as well as observations that Notch is able to reprogram postnatal and terminally differentiated hepatocytes into biliary epithelia with the capacity to form ductular structures (11–13).

In recent fate-tracing experiments, chemically induced tumors were established in transgenic mice carrying an inducible heritable label for either hepatocyte (Alb-CreERT2) or biliary (CK19-CreERT2) lineages. Unexpectedly, in the absence of transgenic Notch overexpression, labeled neoplastic nodules positive for epithelial cell adhesion molecule were observed in tumors arising in Alb-CreERT2 but not CK19-CreERT2 animals, which suggested that intrahepatic cholangiocarcinoma arose from hepatocytes rather than cholangiocytes in that model (10). Given the unexpected nature of these findings within the clinical context of this disease and their implications for the development of future therapy, we set out to assess whether targeted loss of tumor suppressor function within cholangiocytes can precipitate intrahepatic cholangiocarcinoma formation using an independent transgenic strategy and, hence, whether biliary epithelia should still be considered a cell of origin of intrahepatic cholangiocarcinoma.

Mice

CK19CreERTR26ReYFP mice on a mixed genetic background (a kind gift from Guoqiang Gu, Vanderbilt University Medical Center, Nashville, TN) and Trp53tm1Brn (The Jackson Laboratory) were used in this study.

Experimental protocol

For induction of Cre activity, 6-week-old CK19CreERTeYFPp53f/f mice were administered three intraperitoneal injections of 4 mg tamoxifen (Sigma) reconstituted in olive oil (Sigma) at a concentration of 30 mg/mL on alternate days. Mice were harvested 72 hours following tamoxifen administration to assess efficiency of Cre recombination. A separate cohort went on to receive 600 mg/mL thioacetamide (TAA; Sigma) in drinking water for 26 weeks to induce tumor formation. To assess noncarcinogenic injury models, mice received 1 μL/g carbon tetrachloride (Sigma) or olive oil (Sigma) intraperitoneally for 16 weeks or 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC; Sigma) diet (0.1% Purina 5015 Mouse chow) for 14 days.

Immunohistochemistry

Livers were fixed overnight in 4% aqueous buffered formalin, embedded in paraffin, and cut into 5-μm sections. The tissue underwent microwave antigen retrieval using Tris-EDTA with 0.1% Tween20 (Sigma) and blocked with H202 and Protein Block (Invitrogen). Sections were incubated overnight with the following primary antibodies: GFP, CK19, Cyp2D6, Sox9 and Notch1 (Abcam), Nanog (eBiosciences), or Oct4 (Santa Cruz Biotechnology). After washing in PBS, the directly conjugated Alexa 488 and Alexa 555/564–conjugated secondary antibodies (Invitrogen) were used according to species with DAPI (4′,6-diamidino-2-phenylindole)-Fluoromount (SouthernBiotech). Day-8.5 mouse embryos and the murine epistem cell line C2 were used as positive controls for Oct4 and Nanog immunostaining.

Study approval

All animal experiments were approved by the University of Edinburgh animal ethics committee and conducted with the U.K. Home Office approval. Human specimens were collected prospectively from patients undergoing hepatic resection at the Royal Infirmary of Edinburgh with local ethical approval and informed patient consent.

Up to 26% of patients with intrahepatic cholangiocarcinoma carry mutations in the TRP53 gene [up to 44.4% in fluke-associated intrahepatic cholangiocarcinoma (14)], primarily single-base substitutions at CpG sites, resulting in loss of tumor suppressor function (15). In our tamoxifen inducible experimental system, we have, therefore, targeted functional loss of p53 in CK19-expressing cells that are synchronously labeled with a Cre inducible eYFP reporter (CK19-CreERT;R26ReYFP;Trp53loxP), in which Cre recombination induces excision of exons 2 to 10 of the Trp53 gene, and also the stop locus upstream of eYFP (Fig. 1A). In the healthy and injured mouse liver, CK19 expression is found on cholangiocytes lining medium and large-sized bile ducts as well as terminal ductules in the Canals of Hering, but not in hepatocytes (Fig. 1B). In the absence of Cre or tamoxifen, no eYFP expression was seen in either healthy or injured liver, but following induction with tamoxifen at 6 weeks old, eYFP positivity was observed in 14% of all cholangiocytes (Fig. 1C). No cell types other than biliary epithelia were labeled.

Figure 1.

Transgenic system of tamoxifen-inducible, Cre-mediated cell tracking with Trp53 deletion in CK19CreERTeYFPR26p53f/f mice. A, transgenic construct of fluorescent labeling and tumor suppressor deletion in CK19+ cells in response to tamoxifen in 6-week-old mice. B, in the presence of Cre and tamoxifen (TM), eYFP activity is seen within small ductules as well as large bile ducts. The eYFP+ population expands following 14 days of dietary DDC; scale bars, 50 μm. Quantitative analysis of Cre efficiency 72 hours post injection in Cre mice exposed to tamoxifen (n = 5), Cre+ mice without tamoxifen (n = 3), and Cre+ mice exposed to tamoxifen (n = 8). C, following tamoxifen injection, eYFP positivity is seen only in CK19-expressing cells. These are cholangiocytes that also express the biliary markers Sox9. No colocalization is seen with the mature hepatocyte marker Cyp2D6; scale bars, 50 μm.

Figure 1.

Transgenic system of tamoxifen-inducible, Cre-mediated cell tracking with Trp53 deletion in CK19CreERTeYFPR26p53f/f mice. A, transgenic construct of fluorescent labeling and tumor suppressor deletion in CK19+ cells in response to tamoxifen in 6-week-old mice. B, in the presence of Cre and tamoxifen (TM), eYFP activity is seen within small ductules as well as large bile ducts. The eYFP+ population expands following 14 days of dietary DDC; scale bars, 50 μm. Quantitative analysis of Cre efficiency 72 hours post injection in Cre mice exposed to tamoxifen (n = 5), Cre+ mice without tamoxifen (n = 3), and Cre+ mice exposed to tamoxifen (n = 8). C, following tamoxifen injection, eYFP positivity is seen only in CK19-expressing cells. These are cholangiocytes that also express the biliary markers Sox9. No colocalization is seen with the mature hepatocyte marker Cyp2D6; scale bars, 50 μm.

Close modal

One week following Cre induction, mice were initiated on TAA to induce tumor formation (Fig. 2A; ref. 16). After 26 weeks, multifocal tumors were observed in the livers of CK19CreERTeYFPp53−/− (80%) but not CK19CreERTeYFPp53+/− (0%) or CK19CreERTeYFPp53+/+ (0%) animals (Fig. 2B). eYFP positivity was observed in all histologically identified neoplastic nodules, and this colocalized with expression of the ductular markers CK19 and Sox9. No cells were dually positive for eYFP and the mature hepatocyte marker Cyp2D6 (Fig. 2C).

Figure 2.

Intrahepatic cholangiocarcinoma is derived from CK19+ cholangiocytes. A, experimental strategy of tamoxifen induction in CK19CreERTeYFPR26p53f/f mice followed by oral administration of 600 mg/mL TAA for 26 weeks. B, multifocal tumors developed only in CK19CreERTeYFPp53−/− (homozygous for p53 deletion; n = 5) and not CK19CreERTeYFPR26p53+/− (n = 14) or CK19CreERTeYFPR26p53+/+ (n = 5) animals, and only following TAA administration. C, coimmunofluorescent staining of eYFP with the biliary lineage markers CK19 and Sox9. All eYFP+ cells were seen to be CK19+. eYFP positivity did not overlap with the mature hepatocyte marker Cyp2D6. Nuclei are stained with DAPI; scale bars, 50 μm.

Figure 2.

Intrahepatic cholangiocarcinoma is derived from CK19+ cholangiocytes. A, experimental strategy of tamoxifen induction in CK19CreERTeYFPR26p53f/f mice followed by oral administration of 600 mg/mL TAA for 26 weeks. B, multifocal tumors developed only in CK19CreERTeYFPp53−/− (homozygous for p53 deletion; n = 5) and not CK19CreERTeYFPR26p53+/− (n = 14) or CK19CreERTeYFPR26p53+/+ (n = 5) animals, and only following TAA administration. C, coimmunofluorescent staining of eYFP with the biliary lineage markers CK19 and Sox9. All eYFP+ cells were seen to be CK19+. eYFP positivity did not overlap with the mature hepatocyte marker Cyp2D6. Nuclei are stained with DAPI; scale bars, 50 μm.

Close modal

In light of the emerging role for Notch in driving cholangiocarcinogenesis, we then looked to identify the cellular expression of the Notch 1 receptor within this model. Membranous and nuclear positivity of activated Notch1 was observed widely in the epithelium of the malignant ducts and frequently colocalized with eYFP staining (Fig. 3A). Interestingly, positivity was also seen to occur within nuclei of hepatocytes, particularly those located adjacent to the cancerous stroma (Fig. 3B). We went on to assess whether Notch1 was also expressed in nonmalignant models of liver injury and observed strong ductular positivity in the context of the DDC biliary injury dietary model, but none during chronic hepatocyte regeneration with carbon tetrachloride or in the uninjured mouse liver (Fig. 3C). Furthermore, this pattern of Notch activity is recapitulated in human resected intrahepatic cholangiocarcinoma specimens, in which the strongest positivity is observed within malignant ducts, as well as in hepatocytes adjacent to the invasive front of the tumors (Fig. 3D). Hepatic lineage-tracing experiments have proved problematic; indeed the CK19CreERTR26RYFP mouse has hitherto not been widely adopted for cell-specific gene deletion experiments due to poor efficiency. p53 deletion at the point of tamoxifen administration does not result in increased labeling efficiency, but is likely to cause a preferential expansion of the eYFP+ compartment in response to TAA-induced injury, making it more probable that a transforming event will occur in this population of cells compared with labeled cells in a similar fate-tracing system without p53 deletion. We believe this to be a robust and representative model of biliary carcinogenesis, given the frequent combination of p53 loss and chronic biliary inflammation observed in human disease. eYFP positivity was observed in all animals in which tumors arose as well as in each and every focus of malignancy. We observed colocalization between eYFP and the M3 acetylcholine receptor, a marker of mature cholangiocytes, occasional colocalization with CD44 and no colocalization with the stem cell markers Nanog and Oct 4 (17; Supplementary Fig. S1). A likely cell of origin is, therefore, the mature cholangiocyte, although we cannot eliminate the possibility of stem cells, progenitors or intermediates as targets of transformation. Interestingly, given the lineage-tracing system used here, these would be CK19+ cells. Given the CK19CreERTR26RYFP mouse has not hitherto exhibited lineage labeling of hepatocytes, we can conclude that the eYFP+ tumor cells here, arise from cholangiocytes rather than hepatocytes. It is unclear why labeled tumors were not observed after 30 weeks of TAA administration in the CK19CreERT2R26RYFP system published by Sekiya and colleagues; however, our data clearly and definitively attest that biliary epithelia can be a cell of origin of intrahepatic cholangiocarcinoma in an independent CK19-based transgenic system.

Figure 3.

Native Notch signaling is activated in intrahepatic cholangiocarcinoma (ICC). A, immunofluorescent staining of activated Notch1 in the membranes of malignant ductules of TAA-induced intrahepatic cholangiocarcinoma in CK19CreERTeYFPp53−/− mice frequently colocalizes with eYFP positivity (filled arrowheads; scale bars, 50 μm). B, immunostaining of activated Notch1 within nuclei of peritumoral hepatocytes [open arrowheads; scale bars, 50 μm and 125 μm (second photomicrograph taken under oil)]. C, activated Notch1 immunostaining in uninjured mouse liver; CCl4 induced fibrosis (16 weeks) and DDC diet; scale bars, 50 μm). D, activated Notch1 immunostaining in human intrahepatic cholangiocarcinoma specimens. Staining in malignant biliary epithelia (filled arrowheads) and peritumoral hepatocytes (open arrowheads); scale bars, 50 μm.

Figure 3.

Native Notch signaling is activated in intrahepatic cholangiocarcinoma (ICC). A, immunofluorescent staining of activated Notch1 in the membranes of malignant ductules of TAA-induced intrahepatic cholangiocarcinoma in CK19CreERTeYFPp53−/− mice frequently colocalizes with eYFP positivity (filled arrowheads; scale bars, 50 μm). B, immunostaining of activated Notch1 within nuclei of peritumoral hepatocytes [open arrowheads; scale bars, 50 μm and 125 μm (second photomicrograph taken under oil)]. C, activated Notch1 immunostaining in uninjured mouse liver; CCl4 induced fibrosis (16 weeks) and DDC diet; scale bars, 50 μm). D, activated Notch1 immunostaining in human intrahepatic cholangiocarcinoma specimens. Staining in malignant biliary epithelia (filled arrowheads) and peritumoral hepatocytes (open arrowheads); scale bars, 50 μm.

Close modal

Primary liver cancers are a phenotypically and molecularly heterogeneous group of malignancies without a stereotypical mutational signature. It has been suggested that such heterogeneity reflects in part the diversity of the underlying cells of origin (17), although this remains unproved. What is evident, however, is the plasticity of hepatic lineages. Following oncogenic transduction, mature hepatocytes, HPCs, and hepatoblasts all have potential for reprogramming into tumor-initiating cells with acquisition of CD133+ expression, side population fractions as well as tumor-forming and metastatic capacity (18). Cellular differentiation seems to trigger distinct transcriptional programs in response to the same oncogenic stimulus; however, all transduced cells independent of origin, are able to form tumors of multiple lineages.

Our data support the published evidence for Notch as driver of biliary oncogenesis (19) by demonstrating active signaling within the ductular epithelium in intrahepatic cholangiocarcinoma in both human and mouse. The observation of strong Notch1 intracellular domain expression within hepatocyte nuclei adjacent to the desmoplastic stroma substantiates previous experiments that have shown Notch1 activation within these cells acts as a transdifferentiating factor (9, 10). Moreover, this model of reprogramming is further strengthened by the capacity of constitutively activated Notch2 in albumin-expressing cells to induce intrahepatic cholangiocarcinoma formation and accelerate DEN-induced hepatocellular carcinoma, which is less differentiated than wild-type controls (20). This Notch high state, able to prime the peritumoral parenchyma for transdifferentiation, has significant therapeutic implications for the many patients who develop intrahepatic cholangiocarcinoma on a background of chronic hepatocellular injury (8). We believe that these findings unify and clarify previous reports and explain how chronic biliary damage can lead to cholangiocarcinoma arising from biliary epithelium. In conclusion, we have definitively shown that even in the absence of transgenic Notch activation, intrahepatic cholangiocarcinoma can arise from the biliary epithelia. Future therapeutic strategies should target the Notch pathway as a driver of tumorigenesis in this aggressive malignancy.

No potential conflicts of interest were disclosed.

Conception and design: R.V. Guest, L. Boulter, S.E. Minnis-Lyons, S.J. Wigmore, O.J. Sansom, S.J. Forbes

Development of methodology: R.V. Guest

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R.V. Guest, T.J. Kendall, R. Walker, S.J. Wigmore

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.V. Guest, L. Boulter, T.J. Kendall, S.J. Wigmore, S.J. Forbes

Writing, review, and/or revision of the manuscript: R.V. Guest, L. Boulter, S.E. Minnis-Lyons, S.J. Wigmore, S.J. Forbes

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. Walker, S.J. Wigmore

Study supervision: S.J. Wigmore, S.J. Forbes

The authors thank Guoqiang Gu, Vanderbilt University Medical Center, Nashville, TN for the provision of mice.

This work was supported by the MRC Centre for Regenerative Medicine, University of Edinburgh, United Kingdom. Funding was provided by the Wellcome Trust, the Medical Research Council, and Cancer Research UK. R.V. Guest is supported by a Wellcome Trust Clinical Research Training Fellowship; L. Boulter is supported by a Cancer Research UK project grant and an MRC research grant; T.J. Kendall is supported by a Wellcome Trust Intermediate Clinical Fellowship; S.E. Minnis-Lyons is supported by an MRC Scottish Clinical Pathology Fellowship; R. Walker is supported by a Cancer Research UK project grant; S.J. Wigmore is supported by the Scottish Higher Education Funding Council; O.J. Sansom is supported by Cancer Research UK and the European Research Council; and S.J. Forbes is supported by the MRC and a Cancer Research UK project grant.

1.
Taylor-Robinson
SD
,
Toledano
MB
,
Arora
S
,
Keegan
TJ
,
Hargreaves
S
,
Beck
A
,
Khan
SA
, et al
Increase in mortality rates from intrahepatic cholangiocarcinoma in England and Wales 1968–1998
.
Gut
2001
;
48
:
816
20
.
2.
Witjes
CDM
,
Karim-Kos
HE
,
Visser
O
,
de Vries
E
,
Ijzermans
JNM
,
de Man
RA
, et al
Intrahepatic cholangiocarcinoma in a low endemic area: rising incidence and improved survival
.
HPB
2012
;
14
:
777
81
.
3.
Ishak
KG
,
Anthony
PP
,
Sobin
LH
. 
Histological typing of tumours of the liver. WHO International Classification of Tumours
.
Berlin: Springer Verlag
; 
1994
.
4.
Bergquist
A
,
Ekbom
A
,
Olsson
R
,
Kornfeldt
D
,
Loof
L
,
Danielsson
A
, et al
Hepatic and extrahepatic malignancies in primary sclerosing cholangitis
.
J Hepatol
2002
;
36
:
321
7
.
5.
Sripa
B
,
Kaewkes
S
,
Sithithaworn
P
,
Mairiang
E
,
Laha
T
,
Smout
M
, et al
Liver fluke induces cholangiocarcinoma
.
PloS Med
2007
;
4
:
e201
.
6.
Goodman
ZD
,
Ishak
KG
,
Langloss
JM
,
Sesterhenn
IA
,
Rabin
L
. 
Combined hepatocellular-cholangiocarcinoma. A histologic and immunohistochemical study
.
Cancer
1985
;
55
:
124
35
.
7.
Komuta
M
,
Spee
B
,
Vander Borght
S
,
De Vos
R
,
Verslype
C
,
Aerts
R
, et al
Clinicopathological study on cholangiolocellular carcinoma suggesting hepatic progenitor cell origin
.
Hepatology
2008
;
47
:
1544
56
.
8.
Palmer
WC
,
Patel
T
. 
Are common factors involved in the pathogenesis of primary liver cancers? A meta-analysis of risk factors for intrahepatic cholangiocarcinoma
.
J Hepatol
2012
;
57
:
69
76
.
9.
Fan
B
,
Malato
Y
,
Calvisi
DF
,
Naqvi
S
,
Razumilava
N
,
Ribback
S
, et al
Cholangiocarcinomas can originate from hepatocytes in mice
.
J Clin Investig
2012
;
122
:
2911
5
.
10.
Sekiya
S
,
Suzuki
A
. 
Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes
.
J Clin Investig
2012
;
122
:
3914
8
.
11.
Zong
Y
,
Pamikkar
A
,
Xu
J
,
Antoniou
A
,
Raynaud
P
,
Lemaigre
F
, et al
Notch signaling controls liver development by regulating biliary differentiation
.
Development
2009
;
136
:
1727
39
.
12.
Yanger
K
,
Zong
Y
,
Maggs
LR
,
Shapira
SN
,
Maddipati
R
,
Aiello
NM
, et al
Robust cellular reprogramming occurs spontaneously during liver regeneration
.
Genes Dev
2013
;
27
:
719
24
.
13.
Boulter
L
,
Govaere
O
,
Bird
TG
,
Radulescu
S
,
Aucott
RL
,
Van Rooijen
N
, et al
Macrophage derived Wnt signalling opposes Notch signalling in a NUMB mediated manner to specify HPC fate in chronic liver disease in liver and mouse
.
Nat Med
2012
;
18
:
572
9
.
14.
Ong
CK
,
Subimerb
C
,
Pairojkul
C
,
Wongkham
S
,
Cutcutache
I
,
Yu
W
, et al
Exome sequencing of liver fluke-associated cholangiocarcinoma
.
Nat Genet
2012
;
44
:
690
3
.
15.
Khan
SA
,
Thomas
HC
,
Toledano
MB
,
Cox
IJ
,
Taylor-Robinson
SD
. 
p53 Mutations in human cholangiocarcinoma: a review
.
Liver Int
2005
;
25
:
704
16
.
16.
Yeh
C-N
,
Maitra
A
,
Lee
K-F
,
Jan
Y-Y
,
Chen
M-F
. 
Thioacetamide-induced intestinal-type cholangiocarcinoma in rat: an animal model recapitulating the multi-stage progression of human cholangiocarcinoma
.
Carcinogenesis
2004
;
25
:
631
6
.
17.
Komuta
M
,
Govaere
O
,
Vandecaveye
V
,
Akiba
J
,
Van Steenbergen
W
,
Verslype
C
, et al
Histological diversity in cholangiocellular carcinoma reflects the different cholangiocyte phenotypes
.
Hepatology
2012
;
55
:
1876
88
.
18.
Holczbauer
A
,
Factor
VM
,
Andersen
JB
,
Marquardt
JU
,
Kleiner
D
,
Raggi
C
, et al
Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types
.
Gastroenterology
2013
;
145
:
221
31
.
19.
Zender
S
,
Nickeleit
I
,
Wuestefeld
T
,
Sorensen
I
,
Dauch
D
,
Bozko
P
, et al
A critical role for notch signaling in the formation of cholangiocellular carcinomas
.
Cancer Cell
2013
;
23
:
784
95
.
20.
Dill
MT
,
Tornillo
L
,
Fritzius
T
,
Terracciano
L
,
Semela
D
,
Bettler
B
, et al
Constitutive notch2 signaling induces hepatic tumors in mice
.
Hepatology
2013
;
57
:
1607
19
.