The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
Cholangiocarcinomas can originate
from hepatocytes in mice
Biao Fan,1,2 Yann Malato,3,4 Diego F. Calvisi,5 Syed Naqvi,3 Nataliya Razumilava,6 Silvia Ribback,5
Gregory J. Gores,6 Frank Dombrowski,5 Matthias Evert,5 Xin Chen,1,7 and Holger Willenbring3,4,7
1Department of Bioengineering and Therapeutic Sciences, UCSF, San Francisco, California, USA. 2Department of Surgery,
Beijing Cancer Hospital and Institute, Peking University School of Oncology and Key Laboratory of Carcinogenesis and Translational Research
(Ministry of Education), Beijing, China. 3Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research and 4Department of Surgery,
Division of Transplantation, UCSF, San Francisco, California, USA. 5Institut für Pathologie, Ernst-Moritz-Arndt-Universität, Greifswald, Germany.
6Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA. 7Liver Center, UCSF, San Francisco, California, USA.
Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of
effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit
heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably
from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack
of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been
considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model
of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal
hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a
previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy.
Cholangiocarcinomas (CCs) are adenocarcinomas that can be
distinguished by their anatomic location. Extrahepatic CCs may
cause symptoms such as jaundice and are therefore occasionally
detected at resectable stages (1). In contrast, intrahepatic CCs
(ICCs) long remain asymptomatic and are therefore typically
diagnosed at therapy-resistant advanced stages. The resulting
poor prognosis and increasing incidence of ICCs necessitate a bet-
ter understanding of their pathogenesis.
ICCs frequently grow alongside or into the lumen of bile ducts
and are thought to originate from biliary epithelial cells (BECs).
The heterogeneous histology of ICCs suggests that they can derive
from both cylindrical BECs lining large bile ducts (ductal ICCs)
or cuboidal BECs lining small bile ducts or ductules (peripheral
ICCs) (2). Ductules also contain liver progenitor cells (LPCs),
which can give rise to both BECs and hepatocytes and are also
being considered as an origin of ICCs, particularly of rare tumors
such as cholangiolocellular carcinomas that exhibit both ICC and
hepatocellular carcinoma (HCC) characteristics.
An alternative mechanism explaining the formation of ICC
could be lineage conversion occurring during malignant trans-
formation of hepatocytes. To our knowledge, this possibility has
so far not been considered. However, the common developmental
origin of BECs and hepatocytes, and the previous finding that
forced activation of NOTCH signaling can convert hepatocytes
into cells resembling BECs in mice, provide indirect support for
this concept (3). The clinical observation that peripheral ICCs can
form masses in the liver parenchyma, where mainly hepatocytes
reside, is also compatible with a hepatocyte origin (4). Further-
more, ICCs can be caused not only by diseases affecting BECs,
such as parasitic infections, primary sclerosing cholangitis, bile
duct cysts, and hepatolithiasis, but also by diseases that cause
hepatocyte injury, such as hepatitis C or B infection, alcohol
abuse, and nonalcoholic steatohepatitis (5).
The first direct evidence in support of a hepatocyte origin of
ICCs was provided by our previous finding that hydrodynamic
tail vein injection of a human AKT overexpression plasmid into
mice produced mainly HCCs, but also a small number of chol-
angiocellular lesions (6). However, although this technique deliv-
ers plasmids mainly to hepatocytes in the liver (7), we could not
rule out plasmid entry and AKT overexpression in BECs or LPCs,
especially because AKT was under the control of a ubiquitous pro-
moter. Thus, a hepatocyte origin of the cholangiocellular lesions
could not be definitely established. In addition, our finding of
only benign tumors, but not ICCs, suggested that overexpression
of AKT alone is not sufficient for ICC formation.
Here, we hypothesized that activated NOTCH signaling can con-
vert hepatocytes into ICC precursors because of its ability to induce
biliary differentiation of hepatocytes in mice (3) and its emerg-
ing role as a driver and prognostic marker in human CCs (8, 9).
Indeed, using our previously reported hepatocyte fate–tracing
model (10), we found that activated NOTCH signaling, not alone,
but in combination with AKT overexpression, causes rapid forma-
tion of ICCs from hepatocytes.
Results and Discussion
To activate NOTCH signaling in the liver, we stably
overexpressed the intracellular domain of the NOTCH1 receptor
(NICD; Myc-tagged). For this purpose, we delivered plasmids for
sleeping beauty transposase–mediated genomic transgene inte-
gration to livers of wild-type FVB/N mice by hydrodynamic tail
vein injection (6). We did not detect histological changes within
10 weeks after NICD plasmid injection (data not shown). By 20
weeks, we found cystic cholangiocellular tumors resembling
human biliary cystadenomas (Supplemental Figure 1, A and B).
Some of these tumors contained cytologically malignant cells
that not only expanded intracystically, but also invaded the sur-
Authorship note: Biao Fan, Yann Malato, and Diego F. Calvisi contributed equally
to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(8):2911–2915. doi:10.1172/JCI63212.
2912 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
rounding liver tissue (Supplemental Figure 1, C–E), consistent
with progression to invasive cystadenocarcinomas. All tumors
were derived from cells that stably overexpressed NICD (Supple-
mental Figure 1F).
Considering the rapid onset of hepatocyte proliferation and for-
mation of premalignant lesions in mice overexpressing AKT in the
liver (6), we decided to inject the NICD plasmid together with an
AKT overexpression plasmid (HA-tagged; combination referred
to as NICD/AKT) into mice. Macroscopically, livers of these mice
appeared normal 1.5 and 2.5 weeks after injection (Figure 1A).
However, small, white, cyst-like lesions were present on the liver
surface after 3.5 weeks. These lesions rapidly expanded and by
4.5 weeks occupied most of the liver surface. At 5 weeks after plas-
mid injection, the lesions had replaced most of the normal liver
tissue, and the mice rapidly deteriorated and either died or needed
to be euthanized (Supplemental Figure 2).
At the microscopic level, single or clusters of cytologically malig-
nant cells could be identified 1.5 weeks after plasmid injection
(Figure 1B and Supplemental Figure 3, A and B). These clusters
progressed into small tumors of ductular phenotype by 2.5 weeks
(Supplemental Figure 3, C and D). The tumors grew markedly
by 3.5 weeks and exhibited either a ductular or cystic phenotype
(Supplemental Figure 3, E and F). Although cytologically malig-
nant, many tumors still had well-defined borders at this stage.
By 4.5 weeks, however, most tumors showed additional signs of
malignancy, including necrosis, high mitotic activity, and inva-
sion of the surrounding liver parenchyma (Supplemental Fig-
ure 3, G and H), characteristics that correspond to human ICCs.
Immunostaining and immunoblotting for the Myc and HA tags
showed that tumors invariably derived from cells stably overex-
pressing NICD/AKT (Supplemental Figure 4, A and B). Additional
immunostainings for the biliary marker cytokeratin 19 (Ck19) and
major urinary protein (Mup), a marker specific for hepatocytes
(10), confirmed that the tumors exhibited exclusively biliary dif-
ferentiation (Supplemental Figure 5, A and B), providing further
support for their classification as ICCs. Along these lines, expres-
sion of Afp, a gene overexpressed in HCCs, remained at low levels,
whereas expression of Epcam, a gene specific for biliary cells in the
liver, markedly increased with time after injection (Supplemental
Figure 6, A and B). Thus, overexpression of NICD/AKT in the liver
induces specifically ICCs.
Many ICCs formed in the central area of the liver lobule, where
normally hepatocytes, but not BECs or LPCs, reside (Supplemen-
tal Figure 3B). Therefore, we hypothesized that the NICD/AKT-
induced ICCs originated from hepatocytes. To rule out migrant
BECs or LPCs as the origin, we used our previously reported
hepatocyte fate–tracing model, in which all hepatocytes and their
progeny, but no other liver cells, express enhanced yellow fluores-
cent protein (EYFP) (10). To generate the model, we intravenously
injected 4 × 1011 viral genomes of a double-stranded adenoasso-
ciated viral vector serotype 8 expressing Cre recombinase from
the hepatocyte-specific transthyretin promoter (AAV8-Ttr-Cre)
into mice that carry EYFP disrupted by a floxed stop codon in the
ubiquitously expressed Rosa26 locus (R26R-EYFP mice) (Figure 2A).
To replicate the experiments described above, we used FVB/N
R26R-EYFP mice. We analyzed a subset of the mice 1 week after
injection to ascertain that AAV8-Ttr-Cre looped out the stop
codon and activated EYFP expression in hepatocytes with the
NICD/AKT-induced ICC formation. (A) Photographic images of mouse livers taken at different time points after NICD/AKT plasmid injection.
Small tumors (arrows) were visible as early as 3.5 weeks after injection. (B) H&E stainings of corresponding liver sections show rapidly
expanding tumors replacing the normal liver parenchyma. Original magnification, ×40. At least 15 liver sections from 3 mice were analyzed
for each time point.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
same efficiency and specificity as previously reported (Figure 2B
and ref. 10). We hydrodynamically injected the remaining mice
with the NICD/AKT plasmids.
As expected, all mice receiving the NICD/AKT plasmids harbored
numerous large ICCs 4.5 weeks later. HA immunostaining showed
that all ICCs originated from cells stably overexpressing NICD/AKT
(Supplemental Figure 7A). Positive EYFP immunostaining
revealed that the cells of origin of ICCs were hepatocytes (Figure 2,
C–E). Confirming our results described above (Supplemental Fig-
ure 5, A and B), the ICCs expressed the biliary markers Sry-box con-
taining gene 9 (Sox9), Ck8, and mucin 1 (Muc1), but were negative
for the hepatocyte marker Mup (Figure 2, C–E, and Supplemen-
tal Figure 7B). Ck8 is expressed in mouse BECs from early devel-
opmental stages onward (Supplemental Figure 7C and ref. 11).
The Muc1 protein is normally localized in the apical membrane of
BECs (Supplemental Figure 7D and ref. 12). High or cytoplasmic
expression of Muc1 is associated with progression and invasive-
ness of human ICCs (13, 14). Indeed, we observed intense, non-
polarized Muc1 labeling in many ICC cells (Supplemental Figure
7B). Furthermore, many cells in NICD/AKT-induced ICCs were
positive for the proliferation marker Ki67 (Supplemental Figure
7E), which is in accordance with what has been shown in human
high-grade ICCs (15). To exclude that ICCs were EYFP positive
because AAV8-Ttr-Cre became expressed in BECs or LPCs during
malignant transformation, we injected AAV8-Ttr-Cre into R26R-
EYFP mice as before, but eliminated this nonintegrating vector
from the liver by 2/3 partial hepatectomy before injection of the
NICD/AKT plasmids (Supplemental Figure 8, A–C, and ref. 16).
We found that ICCs remained EYFP positive in the absence of
potential unspecific AAV8-Ttr-Cre expression (Supplemental Fig-
ure 8, D and E). These results show that hepatocytes give rise to
bona fide ICCs in response to NICD/AKT overexpression.
To investigate the initial stages of NICD/AKT-induced ICC
formation, we screened for single cells expressing NICD/AKT at
1.5 weeks after plasmid injection (Figure 3A). Although most cells
expressing the NICD/AKT tags were located in the center of the
liver lobule, they exhibited biliary differentiation, as was evident
from expression of Ck8 (Supplemental Figure 9, A and B). Coim-
munostaining for EYFP, Ck8, and Sox9 revealed that these cells
were hepatocytes in which biliary gene expression had been acti-
vated (Figure 3B). Coimmunostaining for EYFP, Ck8, and the HA
tag showed that all hybrid cells stably overexpressed NICD/AKT
(Figure 3C). Interestingly, we failed to detect cells coexpressing
EYFP, HA, and Ck19 at this time point. However, Ck19 expression
was detectable at 2.5 weeks after plasmid injection, when single
cells had formed small tumors (data not shown). Because Sox9 is
also expressed in bipotential LPCs (17) and Ck8 is expressed in
bile duct development at earlier stages than Ck19 (18), this find-
Hepatocyte origin of NICD/
AKT-induced ICCs. (A) R26R-
EYFP mice were intravenous-
ly injected with 4 × 1011 viral
genomes of AAV8-Ttr-Cre,
followed by hydrodynamic tail
vein injection of the NICD/AKT
plasmids 1 week later. Tumors
were analyzed 4.5 weeks after
plasmid injection. (B) Coim-
munostaining for EYFP (red)
and Ck19 (green) 1 week after
AAV8-Ttr-Cre injection shows
that all hepatocytes, but no
BECs, express EYFP. (C–E)
Immunostainings of tumors
for EYFP (red) show that they
originated from hepatocytes.
(all green) for Sox9 (C), Ck8
(D), and Mup (E) show that
tumors express biliary markers
but lack hepatocyte differentia-
tion. Nuclei were stained with
DAPI (blue). Original magni-
fication, ×100; inset, ×200. At
least 15 liver sections from 3
mice were analyzed for each
2914 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
ing suggests that NICD/AKT-expressing hepatocytes gradually
acquired biliary differentiation. In accordance with this assess-
ment, we found that EYFP and Sox9 double-positive cells gradually
lost hepatocyte differentiation, as indicated by declining levels of
Mup, which is only expressed in mature hepatocytes (Figure 3D).
Furthermore, electron microscopy revealed loss of glycogen stores
in hepatocytes undergoing lineage conversion, and, illustrating
their hepatocyte origin and difference from normal BECs, these
cells showed formation of cell junctions and bile canaliculi with
adjacent hepatocytes, lack of a basement membrane, and cytologi-
cal atypia (Supplemental Figure 10, A–C). These findings show
that NICD/AKT-induced conversion of hepatocytes into biliary
cells occurs already at the single-cell stage and suggest concurrent
initiation of malignant transformation.
In conclusion, our results show that overexpression of two
factors, NICD and AKT, is sufficient for rapid conversion of
fully differentiated, normal hepatocytes into lethal ICCs. Our
finding that ICC formation involves conversion of hepatocytes
into atypical biliary cells at an early, single-cell stage suggests
that lineage reprogramming gives way to or promotes initiation
of malignant transformation, which is then manifested by cell
proliferation. Although the mechanism by which NICD and
AKT cooperate to induce hepatocyte-derived ICCs remains to
be determined, our finding that overexpression of NICD alone
induces invasive cystadenocarcinomas suggests that it is the
driving oncogene. AKT is likely acting to accelerate ICC forma-
tion by providing metabolic and thus proliferation-promoting
support (6). In support of this assessment, overexpression of
AKT alone produces mainly HCCs and a few benign cholangio-
cellular lesions (6), whereas overexpression of both NICD and
AKT induced only ICCs in our model. NOTCH and AKT signal-
ing are frequently coactivated in human ICCs (Supplemental
Figure 11, A–C), which suggests that their cooperation is also
driving human ICC formation.
Conversion of hepatocytes into ICC precursors. (A) R26R-EYFP mice were intravenously injected with 4 × 1011 viral genomes of AAV8-Ttr-Cre,
followed by hydrodynamic tail vein injection of the NICD/AKT plasmids 1 week later. Livers were analyzed 1.5 weeks after plasmid injection. (B)
Coimmunostaining for EYFP (red), Ck8 (green), and Sox9 (white) shows a hepatocyte expressing the early BEC and LPC markers Ck8 and Sox9.
(C) Coimmunostaining for EYFP (red), Ck8 (green), and HA (white) shows that hybrid cells express the NICD/AKT plasmids. (D) Coimmunostain-
ing for EYFP (red), Mup (green), and Sox9 (white) shows decreased Mup expression in hybrid cells. Original magnification, ×400. At least 15 liver
sections from 3 mice were analyzed for each immunostaining.
brief report Download full-text
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
placed on Superfrost Plus slides (Fisher Scientif ic). Sections were depa-
raffinized and boiled in Antigen Retrieval Citra Solution (BioGenex) for
10 minutes. After blocking in 10% serum for 1 hour, sections were incu-
bated with primary antibodies overnight at 4°C and secondary antibodies
for 1 hour at room temperature (Supplemental Tables 1 and 2). Nuclear
DNA was stained with 300 nM DAPI (Millipore).
Statistics. Data are expressed as mean ± SEM. Statistical significance was
determined by 2-way ANOVA followed by 2-tailed Student’s t test. A P value
less than 0.05 was considered significant.
Study approval. All mouse procedures were approved by the Institutional
Animal Care and Use Committee at UCSF.
This work was supported by California Institute for Regenerative
Medicine (CIRM) New Faculty Award RN2-00950 to H. Willenbring,
NIH R01 CA136606 to X. Chen, Deutsche Forschungsgemeinschaft
(DFG) Ev168/2-1 to M. Evert, DFG Do622/2-1 to F. Dombrowski,
and P30 DK026743 (UCSF Liver Center). B. Fan was supported by
the China Scholarship Council (contract 2010601079). The authors
thank Lijie Jiang and Jason Au for technical assistance, Aras Mattis for
discussion, and Pamela Derish for manuscript editing.
Received for publication February 3, 2012, and accepted in revised
form June 1, 2012.
Address correspondence to: Xin Chen, UCSF, 513 Parnassus Ave-
nue, Campus Box 0912, San Francisco, California 94143, USA.
Phone: 415.502.6526; Fax: 415.502.4322; E-mail: chenx@pharma-
cy.ucsf.edu. Or to: Holger Willenbring, UCSF, 35 Medical Center
Way, Campus Box 0665, San Francisco, California 94143, USA.
Phone: 415.476.2417; Fax: 415.514.2346; E-mail: willenbringh@
Whether ICCs originate from hepatocytes also in humans
remains to be determined. However, this possibility is strongly sug-
gested by recent findings of intracytoplasmic p62+ hyaline inclu-
sions, which are thought to be specific for injured or malignantly
transformed hepatocytes, in common peripheral ICCs (that is, not
rare, potentially LPC-derived, cholangiolocellular carcinomas)
(19). Most ICCs with p62+ hyaline inclusions emerged in patients
with liver disease due to hepatitis C or B infection or alcohol abuse,
which suggests that chronic hepatocyte injury caused lineage con-
version and ICC formation. In accordance with these observations,
epidemiologic studies have identified viral hepatitis as a risk factor
for ICC (5). By establishing that hepatocytes can give rise to ICCs,
our study sheds light on the pathogenesis of this cancer and sug-
gests molecular targets for much-needed new therapies.
Supplemental Methods are available online with this article.
Mice. Six- to 10-week-old wild-type (Charles River) or R26R-EYFP (20)
mice on the FVB/N strain background were used.
Plasmids. HA-tagged AKT (human AKT1) and hyperactive sleeping
beauty transposase plasmids were described previously (6). AKT was
myristoylated for constitutively active kinase activity. Mouse NOTCH1
receptor NICD sequence with Myc tag was obtained from Addgene.
NICD and AKT were expressed from the EF1α promoter. Transposase
was expressed from the CMV promoter.
Hydrodynamic tail vein injection. Twenty micrograms of NICD, 4 μg AKT, and
1 μg transposase plasmids were diluted in 2 ml 0.9% NaCl, sterile filtered, and
injected into a lateral tail vein within 5–7 seconds as previously described (6).
AAV8-Ttr-Cre. Production, titering, and injection of AAV8-Ttr-Cre was
performed as previously described (10).
Immunostaining. Liver samples were fixed overnight in zinc formalin
(Anatech Ltd.), embedded in paraffin, cut into 5-μm-thick sections, and
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