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doi:10.1111/jgh.12692
H E PAT O L O G Y
Epithelial cell adhesion molecule-positive human hepatic neoplastic cells: development of combined hepatocellular-cholangiocarcinoma in mice Sachiko Ogasawara,* Jun Akiba,* Masamichi Nakayama,* Osamu Nakashima,† Takuji Torimura‡ and Hirohisa Yano* *Department of Pathology, Kurume University School of Medicine, †Department of Clinical Laboratory Medicine, Kurume University Hospital, and ‡ Division of Gastroenterology, Department of Medicine, Kurume University, Kurume, Japan
Key words combined hepatocellular-cholangiocarcinoma, epithelial cell adhesion molecule, hepatic stem/progenitor cell. Accepted for publication 14 July 2014. Correspondence Dr Sachiko Ogasawara, Department of Pathology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Email: [email protected]
Abstract Background and Aim: Human combined hepatocellular-cholangiocarcinoma (CHC) expresses several hepatic stem/progenitor cell (HSPC) markers, suggesting this neoplasm originates from HSPCs. We examined the significance of HSPC marker in CHC using a human CHC cell line. Methods: We used a human CHC cell line (KMCH-1) previously established in our laboratory. The original tumor was classified as CHC, showing areas of typical hepatocellular carcinoma (HCC) and cholangiocarcinoma (ChC). We examined the expression of HSPC markers and hepatocyte markers in KMCH-1 by flow cytometry (FCM) and quantitative real-time polymerase chain reaction. EpCAM(+) and EpCAM(−) KMCH-1 cells were isolated. Subsequently, their morphological features, HSPC marker expression, and biological characteristics were examined in vitro and in vivo. Results: FCM showed expression of EpCAM, K7, K19, and ABCG2 in KMCH-1, with various degrees. EpCAM(+) cells expressed K19 mRNA, but did not express α-fetoprotein (AFP). In contrast, EpCAM(−) cells expressed AFP mRNA, but did not express K19. EpCAM(+) cells produced both EpCAM(+) and EpCAM(−) cells, but EpCAM(−) cells produced only EpCAM(−) cells in vitro. EpCAM(+) cells showed higher tumorigenicity and formed larger tumors than EpCAM(−) cells. Inoculation of EpCAM(+) and EpCAM(−) cells produced both ChC and HCC-like component and HCC-like component only, respectively. Conclusion: It is speculated that some CHCs may originate from EpCAM(+) neoplastic cells, and that these cells may affect malignant behavior and progression in such CHCs.
Introduction Combined hepatocellular-cholangiocarcinoma (CHC) is defined as a tumor containing unequivocal, intimately mixed elements of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (ChC). ChC also known as mixed tumor of the liver. According to the latest World Health Organization (WHO) classification (2010)1, CHC is classified into two major categories, that is classical type and subtypes with stem cell features. The latter often express hepatic stem/progenitor cell (HSPC) markers; therefore, HSPC origin of this tumor is suspected. It is known that HSPC (also referred to as oval cells in animal models) can differentiate into both hepatocytes and bile duct epithelial cells. No pathological or immunological methods have yet been established to identify these HSPC, but they are found near the canals of Hering where interlobular bile ducts and hepatocytes intersect. They are thought to be small cells similar to canals of Hering or cholangiole
cells.2 When liver damage occurs in the mature liver, it is hypothesized that hepatocytes located near the canals of Hering are activated and become oval cells, which then help to regenerate the damaged liver tissue. In animal models, if severe liver injury is introduced when hepatocyte proliferation is suppressed, oval cells positive for albumin (Alb), α-fetoprotein (AFP), as well as biliary type keratin (K) appear in the portal region and are considered to possess the features of both hepatocytes and bile duct cells.3 Previous reports have shown that c-kit, epithelial cell adhesion molecule (EpCAM), neural cell adhesion molecule (NCAM, CD56), CD133, and CXCR4 are HSPC markers.4,5 With the recent advance of molecular biology, the cancer stem cell (CSC) theory is now widely accepted not only in hematopoietic but also solid neoplasms. According to CSC theory, tumor cells develop from a morphologically and functionally heterogeneous subset of tumor cells that include CSCs with features and characteristics similar to those of ordinary stem cells. These
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tumor-initiating cells initiate a hierarchical organization of cancer cells and are thought to be deeply involved in carcinogenesis, malignancy, metastases, and recurrence. They also play a role in resistance to chemotherapy and radiotherapy and evasion of immunosurveillance, and are therefore suggested to be an important target for cancer therapy.6–8 Marquardt et al.9 have identified high efficiency self-renewal, differentiation along at least two independent lineages, resistance to traditional genotoxic therapy, and capacity to establish and recapitulate the original tumor as criteria for recognition of CSCs. In HCC, HSPC and cancer stem markers have been observed to overlap. Human CHC is thought to be the best example of an HSPCderived tumor.10 Moreover, while CHC is reported to be biologically more malignant than HCC,11 no studies have been done to examine expression of HSPC markers or CSC characteristics in CHC. In the present study we examined studied the expression of HSPC markers in a KMCH-1 cell line developed at our department, and focused particularly on EpCAM as a means of evaluating the biological significance of these markers.
Methods Cell line and reagents. We used a CHC cell line, KMCH-1, which had been established in our department.12 Detailed information on the cell line, patient records, and pathological features of the original tumor were reported previously. Expression of HSPC and hepatocyte markers in the original tumor. We performed immunohistochemistry (IHC) to examine the expression of HSPC markers, such as CD34, CD56, CD133, K 7, K19, c-kit, and EpCAM, and hepatocyte markers, such as hepatocyte paraffin (HepPar)-1. IHC staining was performed using the streptavidin-biotin-peroxidase method. Detailed information regarding the primary antibodies used in this study was listed in Table 1. Alcian blue staining was also performed to assess mucin production.
Table 1
Primary antibodies
Primary
Clone
Source
Antibody ABCG2 Albumin α-fetoprotein CD34 CD56 CD133 CD133 K7 K19 c-kit
BXP-21 polyclonal polyclonal QB-END/10 1B6 AC133 293C3 OV-TL 12/30 BA17 K963
EpCAM (EpEX) EpCAM (EpICD) EpCAM HepPar-1 MUC1
HEA125 E144 EBA-1 OCH1E5 Ma695
EDM Millipore Co. DAKO DAKO Leica Microsystem Leica Microsystem Miltenyi Biotec K.K. Miltenyi Biotec K.K. DAKO DAKO Immuno-Biological Laboratories Co., Ltd. Abcam Abcam Becton Dickinson DAKO Leica Microsystem
Dilution IHC
FCM
1:25 1:500 1:100 1:25 1:50 1:50 — 1:100 1:50 1:200
1:50 — — — 1:50 — 1:10 1:50 1:5 1:50
1:20 1:350 — 1:200 1:100
— 1:1 — —
—, indicate not performed; EpEX, EpCAM extracellular domain; EpICD, EpCAM intracellular domain; FCM, flow cytometry; IHC, immunohistochemistry.
Soft agar colony formation assay. Fifty thousand EpCAM(−) or EpCAM(+) cells were plated in 2 ml of medium containing 0.36% (w/v) top agar layered over a basal layer of 0.72% (w/v) agar in 35 mm dishes (n = 5) and allowed to grow for 2 weeks. Number of colonies was counted using light microscopy.
Sphere formation assay. Sphere formation assay of EpCAM(−) or EpCAM(+) cells was performed according to our previous reports14,15 with minor modification.
Flow cytometric (FCM) analysis and cell isolation. FCM analyses for HSPC markers in KMCH-1 were performed as described in our previous report.13 KMCH-1 cells were stained with antibody for ATP-binding cassette transporter G2 (ABCG2), CD56, CD133, K7, K19, c-kit, and PE conjugated mouse anti-EpCAM (Table 1). In addition, EpCAM-negative (EpCAM[−]) and EpCAM-positive (EpCAM[+]) KMCH-1 cells were isolated with FACS Aria II (Becton Dickinson, San Jose, CA, USA).
Proliferation and drug resistance. Growth speed of EpCAM(−) or EpCAM(+) cells was examined with colorimetry using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kits (Chemicon, Temecula, CA, USA) as described elsewhere.16 In order to examine drug resistance, the cells were seeded and cultured for 24 h, and the medium was changed to medium containing pegylated interferon α-2b (PEG-IFN) (2000 or 4000 IU/ mL; MSD Co., Ltd, Tokyo, Japan), 5-fluorouracil (5-FU) (0.5 or 1 μM; Kyowa Hakko Kirin Co., Ltd, Tokyo, Japan), cisplatin (1.25, 2.5, or 5 μg/mL; Nippon Kayaku Co., Ltd, Tokyo, Japan), or sorafenib (1.25 or 2.5 μM; Cell Signaling Technology., Inc., Danvers, MA, USA). After 72 h of culture, the number of viable cells was examined.
Morphological features and IHC. EpCAM(−) or EpCAM(+) cells were seeded in Lab-Tek chamber slides, cultured for a few days until achieving subconfluency, fixed, and stained with HE, or fixed and IHC for ABCG2 and K19 (Table 1) was performed using CSA II kit (DAKO, Carpinteria, CA, USA).
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The expression of HSPC markers at the mRNA level of EpCAM(−) or EpCAM(+) cells was examined as described previously.14 qRT-PCR was carried out with TaqMan technology using the ABI 7500
Morphologic features of KMCH-1 cell line. The cells were cultured on Lab-Tek chamber slides (Nunc, Inc., Roskilde, Denmark), then fixed in Carnoy’s solution for 20 min, and stained with hematoxylin and eosin (HE) or Alcian blue.
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Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression assays primer and probe mixes were used for ABCG2 (Hs00184979_m1), AFP (Hs00173490_ m1), Alb (Hs00609411_m1), c-kit (Hs00174029_m1), CD34 (Hs00156373_m1), CD56 (Hs00169851_m1), CD133 (Hs00195682_m1), K19 (Hs00761767_m1), and β-actin (Hs99999903_m1). β-actin was used for normalization. Relative quantitation was carried out using the ΔΔCt method. Expression of EpCAM after isolation. EpCAM(−) or EpCAM(+) cells were plated at 5 × 104 cells/10 cm in dishes and EpCAM expression was examined weekly from week 1 to week 7 by FCM. To facilitate continued observation of EpCAM expression, every week, the plating process was repeated with the remaining cells at 5 × 104 cells/dish, and chronological changes in EpCAM expression were observed through the end of week 7. Tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. EpCAM(−) or EpCAM(+) cells (106 cells/mouse) were subcutaneously injected into the left and right backs, respectively, of 4-week-old female NOD/SCID mice. The NOD/SCID mice were sacrificed after 16 weeks. Resected tumors were weighed and
Hepatic progenitor cell in mixed tumor
subjected to morphological studies (e.g. HE staining and IHC). The expressions of AFP, Alb, K19, EpCAM (extracellular domain (EpEX) and intracellular domain [EpICD]), HepPar-1, and MUC1 (Table 1) were examined. All procedures were approved by the Ethics Review Committee for Animal Experimentation of Kurume University School of Medicine. EpCAM expressions of human CHC tissues. The expression of EpCAM was examined using two antibodies for its EpEX and EpICD (Table 1). Nine cases previously diagnosed as CHC, classical type were used in this study. Statistical analysis. Data are expressed as the mean ± SD. Comparisons between groups were performed using Student’s t-test. Differences were considered significant at P < 0.05. This study was approved by the ethical committee of Kurume University (approved #10229 and #10294).
Results Morphology and IHC. The original KMCH-1 tumor was composed of both HCC (Fig. 1a), with cancer nests of tumor cells
Figure 1 Histological findings of original tumor. (a) The hepatocellular carcinoma (HCC) portion showing a trabecular proliferative pattern (hematoxylin and eosin staining). (b) The hepatocellular-cholangiocarcinoma (ChC) portion showing gland formation. (c) Transition between HCC and ChC. (d) Mucin production is observed inside the rim of the glands in the ChC portion (Alcian blue staining). (e) K19 expression is found in the glands of the ChC component. (f) The expression of EpCAM is observed in both the HCC and ChC components although the intensity is stronger in ChC components than HCC components. Histological findings of KMCH-1 cells. (g) Hematoxylin and eosin staining. (h) Mucin production is observed. (i) Flow cytometric analyses revealed 65.3%, 28.7%, 29.4%, and 25.1% of KMCH-1 cells were positive for EpCAM, K7, K19, and ABCG2, respectively. CD56, CD133, and c-kit were not expressed. Staining with control antibody or specific antibodies is shown by black line or red line, respectively.
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in a trabecular arrangement, and ChC (Fig. 1b), with glandular structures accompanied by abundant fibrous stroma. A transitional area was observed between them (Fig. 1c). Mucin production was confirmed in the rim of the glandular structure of ChC component by Alcian blue stain (Fig. 1d). K7, K19, and EpCAM were positive for tumor cells (Fig. 1e,f), but the expression of CD34, CD56, CD133, c-kit, and HepPar-1 was negative. The expression of EpCAM was stronger in ChC component than in HCC component. These findings indicated that the original tumor corresponded to CHC, classical type, in the latest WHO classification.1
Morphologic features and HSPC marker expression in KMCH-1. KMCH-1 cells proliferated in a pave stone arrangement. They had large, round nuclei containing several nucleoli (Fig. 1g). Mucin production was found in KMCH-1 by Alcian blue stain (Fig. 1h).
FCM analysis. FCM analyses revealed that EpCAM, K7, K19, and ABCG2 were expressed on KMCH-1 cells, and their positive cell rates were 65.3%, 28.7%, 29.4%, and 25.1%, respectively (Fig. 1i). CD56, CD133, and c-kit were not expressed.
Morphologic features and HSPC marker expressions of EpCAM(−) and EpCAM(+) cells. There was no morphologic difference between EpCAM(−) and EpCAM(+) cells with HE stain (Fig. 2a,b). Both EpCAM(+) and EpCAM(−) cells expressed ABCG2; however, the number of ABCG2 positive cell was higher in EpCAM(+) cells than in EpCAM(−) cells (Fig. 2c,d). K19 (Fig. 2e,f) expression was detected only in EpCAM(+) cells.
Soft agar colony formation and sphere formation assay. The average numbers of soft agar colony formation in EpCAM(−) and EpCAM(+) cells were 1215.0 ± 184.5 and 1278.2 ± 284.2 colonies, respectively. There was no significant difference between them (Table 2). Sphere formation was significantly higher in EpCAM(+) cells (24.5 ± 9.3) than EpCAM(−) cells (7.8 ± 4.9) (P < 0.05, Table 2).
Expression of EpCAM after isolation. EpCAM(−) cells generated only EpCAM(−) cells through weeks 1 to 7. EpCAM(+) cells generated both EpCAM(−) and EpCAM(+) cells through all 7 weeks. EpCAM(+) cells rates at the 1st- and 7thweek were 68.8% and 73.5%, respectively (Fig. 2i). Tumorigenicity in NOD/SCID mice. One of five mice inoculated with EpCAM(−) cells developed a tumor that was 169 mg in weight. This tumor showed HCC-like morphological features and lacked a ChC-like component (Fig. 3a,b). Immunohistochemically, the tumor lacked EpCAM expression (Fig. 3c,d), but had HepPar-1 (Fig. 3e,f), Alb (Fig. 3g,h), and AFP (data not shown) expression. The expression of K19 was equivocal (data not shown). In contrast, four of five mice inoculated with EpCAM(+) cells generated tumors, and the average tumor weight was 1758 ± 223 mg. These tumors morphologically mimicked the original tumor of KMCH-1. Tumors were composed of HCC-like and ChC-like components. HCC-like component (Fig. 4arectangle-1, e) lacked EpCAM (Fig. 4b,f) and K19 (Fig. 4d,h) expression, and had HepPar-1 (Fig. 4c,g), Alb, and AFP (data not shown) expression. ChC-like component (Fig. 4a-rectangle-2, i) showed positivity for EpCAM (Fig. 4b,j), K19 (Fig. 4d,l), and MUC-1 (data not shown). Mucin production was found by mucicarmine stain. ChC component was negative for HepPar-1 (Fig. 4c,k). EpEX and EpICD expression tumor obtained from NOD/SCID mice and human CHC tissues. The expression of EpICD was found only in ChC component of tumors obtained from EpCAM(+) cells. The expression of EpICD in ChC component of tumors obtained from EpCAM(+) cells was in accordance with cell membrane, and no expression was observed in cytoplasm or nucleus. EpICD expression was not found in HCC component of tumors obtained from EpCAM(+) cells and a tumor from EpCAM(−) cells (data not shown). The expression pattern of EpEX and EpICD in human CHCs was described in Table 3. There was no apparent correlation between expression patterns and clinicopathologic findings, including microvascular permeation and patient prognosis.
Discussion Proliferation and drug resistance in vitro. No difference was observed in proliferation rates between EpCAM(−) and EpCAM(+) cells at 24, 48, 72, or 96 h (Fig. 2g). When treated with PEG-IFN, 5-FU, or cisplatin, no significant differences were seen in proliferation between EpCAM(−) and EpCAM(+) cells (Fig. 2h).
qRT-PCR. qRT-PCR revealed AFP expression only in EpCAM(−) cells and K19 expression only in EpCAM (+) cells. The expression level of Alb in EpCAM(−) cells was 9.5 ± 3.2 times higher than that of EpCAM(+) cells. ABCG2 and CD133 were expressed on both EpCAM(+) and EpCAM(−) cells. CD34, CD56, and c-kit were not expressed on both EpCAM(+) and EpCAM(−) cells. All experiments were performed three times. There was no apparent difference in other HSPC markers. 416
We re-evaluated the original tumor of KMCH-1 histopathologically using several markers, including HSPC and hepatocyte markers. These findings suggested that the original tumor corresponded to CHC, classical type, in the latest WHO classification. EpCAM, K7, and K19 expressions were observed in both the original tumor and KMCH-1. Moreover, these markers predominantly showed positive in the ChC component of the original tumor. The immunoprofiles we showed here were consistent with our previous report on the immunophenotypes of human CHCs.17 Thus, we performed further investigation focusing on EpCAM in KMCH-1, which is one of the representative HSPC markers. EpCAM is associated with cell adhesion, cell migration, cell differentiation, cell proliferation, and embryogenesis.18 In human adult liver, the expression of EpCAM is found in cholangiocytes, but not in hepatocytes. In embryonic liver, the majority of hepatocytes have the expression of EpCAM.19 The clonally expanded
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Figure 2 Immunohistochemical staining of EpCAM(−) (the upper row) and EpCAM(+) cells (the lower row). (a,b) There is no morphologic difference between EpCAM(−) and EpCAM(+) cells with hematoxylin and eosin (HE) stain. (c,d) Both EpCAM(+) and EpCAM(−) cells express ABCG2. The number of ABCG2-positive cells is higher in EpCAM(−) than EpCAM(+) cells. (e,f) K19 expression is detected only in EpCAM(+) cells. Proliferation and drug resistance in vitro. (g) Proliferation rates between EpCAM(−) and EpCAM(+) cells at 24, 48, 72, or 96 h were not different. ●, EpCAM(+) ■; &, EpCAM(–). (h) Pegylated interferon α-2b (PEG-IFN), 5-fluorouracil (5-FU), cisplatin, and sorafenib did not show significant differences in proliferation between EpCAM(−) and EpCAM(+) cells. Expression of EpCAM after isolation of EpCAM(−) and EpCAM(+) cells. , EpCAM−; ■, EpCAM+. (i) Immediately after isolation by EpCAM antibody (red line) in KMCH-1, cell fractions isolated as EpCAM(−) and EpCAM(+) cells were composed of EpCAM(−) and EpCAM(+) cells respectively. Black line in the figures indicates EpCAM-positive cell fraction before isolation. Isolated EpCAM(−) and EpCAM(+) cells were cultured separately up to 7 weeks. Staining with control antibody or EpCAM antibody is shown by black or red lines, respectively. EpCAM(−) cells sequentially generated only EpCAM(−) cells (upper figures), but EpCAM(+) cells generated both EpCAM(−) and EpCAM(+) cells during observation period. EpCAM(+) cell rates at the 1st- and 7th week were 68.8% and 73.5%, respectively (lower figures).
Table 2
Soft agar colony formation and sphere formation assay
Soft agar Colony formation Sphere Formation
EpCAM (−)
EpCAM (+)
1215.0 ± 184.5†
1278.2 ± 284.2‡
7.8 ± 4.9§
24.5 ± 9.3¶
† versus ‡: not significant. § versus ¶: P < 0.05. The number of colony and sphere per dish and well, respectively.
cells from isolated EpCAM(+) cells exhibited unlimited proliferation and bi-directional differentiations toward cholangiocytes and hepatocytes.20 Recently, it has been suggested that various liver cancers originate from HSPCs.1,21–26 Of these, CHC is regarded as the best example of an HSPC-derived tumor. In the present study, EpCAM(+) KMCH-1 cells showed a higher proliferative rate in vivo compared with EpCAM(−) cells. EpCAM(+) cells initially had characteristics of ChC, and lacked those of HCC immunophenotypically and morphologically. EpCAM(+) cells generated both EpCAM(−) and EpCAM(+) cells in vitro and in vivo. Furthermore, tumors obtained from EpCAM(+) cells in vivo resembled the original tumor of KMCH-1 morphologically
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Figure 3 Panoramic views of a tumor obtained from EpCAM(−) cells inoculated in an non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse are shown in the upper row. The area indicated by a rectangle of the upper row corresponds to the lower row. (a,b) Tumor derived from EpCAM(−) shows HCC-like morphological features (hematoxylin and eosin [HE] stain). (c,d) IHC shows negative reaction for EpCAM. (e–h) IHC shows positive reaction for HepPar-1 (e,f) and albumin (g,h).
Figure 4 Panoramic views of a tumor obtained from EpCAM(+) cells inoculated in an non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse are shown in the upper row. The area indicated by a rectangle in 1 and 2 of the upper row corresponds to the middle row (HCC-like component) and the lower row (ChC-like component), respectively. Tumors obtained from EpCAM(+) show typical features of CHC composed of HCC-like and ChC-like components. HCC-like component (a-rectangle-1,e) has HepPar-1 (c,g) expression, and lacks EpCAM (b,f) and K19 (d,h) expression. ChC component (a-rectangle-2,i) shows positivity for EpCAM (b,j) and K19 (d,i). ChC component is negative for HepPar-1 (c,k).
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Table 3 The expression of EpEX and EpICD in human combined hepatocellular-cholangiocarcinoma EpEX
Case Case Case Case Case Case Case Case Case
1 2 3 4 5 6 7 8 9
EpICD
ChC
HCC
ChC
HCC
M
M
M
C
N
M
C
N
– + – + + – – – –
– + – – – – – – –
– – – – – – – +† +
– – – + + + + – +
– – – + + + + – –
– – – – – + – – +
– – – + + + + + +
– – – + + + + + –
†
Luminal expression at the glandular structure C, cytoplasmic expression; ChC, cholangiocarcinoma; EpEX, EpCAM extracellular domain; EpICD, EpCAM intracellular domain; HCC, hepatocellular carcinoma; M, membranous expression; N, nuclear expression.
and immunohistochemically. EpCAM(−) cells generated only EpCAM(−) cells in vitro and produced only HCC tumor in vivo. These features of EpCAM(+) cells overlapped with those of CSCs. Although some possible histogeneses of CHC-classical type are proposed,17 our results suggest that malignant transformed HSPC, which has EpCAM expression and ChC characteristics, differentiates to HCC. Our result indicated that loss of EpCAM expression might play a pivotal role in cell conversion or differentiation from ChC to HCC. These dramatic changes could be due to not only EpCAM but also to other molecules, including other HSPC markers. However, the precise mechanism by which this occurs remained poorly understood. Although our present study raises the possibility that some CHCs could be originated from EpCAM(+) HSPC, some limitations must be disclosed. First, our results were obtained from only one cell line. Therefore, in order to enforce our findings, investigation using another CHC cell line is ongoing in our laboratory. HSPCs are proposed to be rare populations. However, KMCH-1 showed high positive rate for EpCAM. This result raises the question whether EpCAM is universal marker of HSPC. Suzuki et al.27 suggests that it is unlikely that cells isolated with several surface markers, such as CD133, could form a large colony in single-cell culture. Moreover, these large colonies showed different gene expression, suggesting that even cells isolated with several markers might be heterologous. Thus, the cell population isolated with EpCAM in our present study might also be heterogeneous. Additional markers, such as K19 and/or K7, combined with EpCAM could be useful to identify to HSPC selectively. In addition to cell surface markers, functional assays, such as side population (SP) and aldehyde dehydrogenase 1 (ALDH1) activity, might be also useful.7,28,29 Therefore, we also performed SP assay and ALDH1 assay using KMCH-1 cells. SP cells had a tendency of high proliferation rate than non-SP cells. However, no apparent difference was observed in other assays, including proliferation, drug resistance (i.e. PEG-IFN, 5-FU, cisplatin, and sorafenib), sphere formation, and real-time PCR between SP and non-SP cells, or between ALDH1-positive and ALDH1-negative cells.
Several reports document CHC as an aggressive tumor with poorer outcomes than HCC.30–34 In addition, standard treatments for CHC are not well-established. Yamashita et al.35 found that EpCAM was a β-catenin transcription target, that EpCAM(+) cells showed higher proliferation than EpCAM-negative cells, and that proliferation of hepatocytes was suppressed when small interfering RNA was used to suppress EpCAM expression.5 These findings indicated that EpCAM could be a targeted therapy for liver CSCs. Efforts have been made to use monoclonal antibodies and immunotoxins to target EpCAM in malignant tumors expressing EpCAM,36–39 and it is hoped that therapies designed to suppress EpCAM signaling could prove to be effective. The results obtained from this present study suggest that molecular target therapy focusing on EpCAM might be effective in not only HCC but also in CHC. Further, the suppression of EpCAM expression may enhance cell conversion or differentiation to HCC. A few reports demonstrated that EpICD expression pattern is closely associated with patient prognosis in human malignancies.40,41 We found nuclear expression of EpICD in five of nine cases in human CHC. There was no apparent correlation between expression pattern and clinicopathologic findings. At present, its clinicopathologic significance in human CHC, classical type remains elusive. In conclusion, our results revealed that EpCAM(+) KMCH-1 cells show HSPC-like features and high tumorigenicity, and develop tumors with CHC features in vivo. It is speculated that some CHCs may be originated from EpCAM(+) neoplastic cells, and that these cells may play a role in malignant behavior and progression in such CHCs.
Acknowledgments We thank Ms Akemi Fujiyoshi for her excellent assistance in our experiments. This study has no financial support.
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10 Holczbauer A, Factor VM, Andersen JB et al. Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types. Gastroenterology 2013; 145: 221–31. 11 Wittekind C, Fisher H, Ponchon T. WHO Classification of Tumours of the Digestive System. Lyon: IARC Press, 2000; 181. 12 Murakami T, Yano H, Maruiwa M, Sugihara S, Kojiro M. Establishment and characterization of a human combined hepatocholangiocarcinoma cell line and its heterologous transplantation in nude mice. Hepatology 1987; 7: 551–6. 13 Ogasawara S, Yano H, Iemura A, Hisaka T, Kojiro M. Expressions of basic fibroblast growth factor and its receptors and their relationship to proliferation of human hepatocellular carcinoma cell lines. Hepatology 1996; 24: 198–205. 14 Ueda K, Ogasawara S, Akiba J et al. Aldehyde dehydrogenase 1 identifies cells with cancer stem cell-like properties in a human renal cell carcinoma cell line. PLoS ONE 2013; 8: e75463. 15 Nakayama M, Ogasawara S, Akiba J et al. Side population cell fractions from hepatocellular carcinoma cell lines increased with tumor dedifferentiation, but lack characteristic features of cancer stem cells. J. Gastroenterol. Hepatol. 2014; 29: 1092–101. 16 Yano H, Iemura A, Haramaki M et al. Interferon alfa receptor expression and growth inhibition by interferon alfa in human liver cancer cell lines. Hepatology 1999; 29: 1708–17. 17 Akiba J, Nakashima O, Hattori S et al. Clinicopathologic analysis of combined hepatocellular-cholangiocarcinoma according to the latest WHO classification. Am. J. Surg. Pathol. 2013; 37: 496–505. 18 Trzpis M, McLaughlin PM, de Leij LM, Harmsen MC. Epithelial cell adhesion molecule: more than a carcinoma marker and adhesion molecule. Am. J. Pathol. 2007; 171: 386–95. 19 de Boer CJ, van Krieken JH, Janssen-van Rhijn CM, Litvinov SV. Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver. J. Pathol. 1999; 188: 201–6. 20 Okabe M, Tsukahara Y, Tanaka M et al. Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver. Development 2009; 136: 1951–60. 21 Theise ND, Yao JL, Harada K et al. Hepatic “stem cell” malignancies in adults: four cases. Histopathology 2003; 43: 263–71. 22 Nakanuma Y, Sasaki M, Ikeda H et al. Pathology of peripheral intrahepatic cholangiocarcinoma with reference to tumorigenesis. Hepatol. Res. 2008; 38: 325–34. 23 Fujii T, Zen Y, Harada K et al. Participation of liver cancer stem/progenitor cells in tumorigenesis of scirrhous hepatocellular carcinoma—human and cell culture study. Hum. Pathol. 2008; 39: 1185–96. 24 Kim H, Park C, Han KH et al. Primary liver carcinoma of intermediate (hepatocyte-cholangiocyte) phenotype. J. Hepatol. 2004; 40: 298–304. 25 Roskams T. Liver stem cells and their implication in hepatocellular and cholangiocarcinoma. Oncogene 2006; 25: 3818–22. 26 Kozaka K, Sasaki M, Fujii T et al. A subgroup of intrahepatic cholangiocarcinoma with an infiltrating replacement growth pattern
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and a resemblance to reactive proliferating bile ductules: “bile ductular carcinoma”. Histopathology 2007; 51: 390–400. Suzuki A, Sekiya S, Onishi M et al. Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver. Hepatology 2008; 48: 1964–78. Chiba T, Kita K, Zheng YW et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 2006; 44: 240–51. Ma S, Chan KW, Lee TK et al. Aldehyde dehydrogenase discriminates the CD133 liver cancer stem cell populations. Mol. Cancer Res. 2008; 6: 1146–53. Yano Y, Yamamoto J, Kosuge T et al. Combined hepatocellular and cholangiocarcinoma: a clinicopathologic study of 26 resected cases. Jpn. J. Clin. Oncol. 2003; 33: 283–7. Jarnagin WR, Weber S, Tickoo SK et al. Combined hepatocellular and cholangiocarcinoma: demographic, clinical, and prognostic factors. Cancer 2002; 94: 2040–6. Koh KC, Lee H, Choi MS et al. Clinicopathologic features and prognosis of combined hepatocellular cholangiocarcinoma. Am. J. Surg. 2005; 189: 120–5. Ng IO, Shek TW, Nicholls J, Ma LT. Combined hepatocellular-cholangiocarcinoma: a clinicopathological study. J. Gastroenterol. Hepatol. 1998; 13: 34–40. Lee CC, Wu CY, Chen JT, Chen GH. Comparing combined hepatocellular-cholangiocarcinoma and cholangiocarcinoma: a clinicopathological study. Hepatogastroenterology 2002; 49: 1487–90. Yamashita T, Budhu A, Forgues M, Wang XW. Activation of hepatic stem cell marker EpCAM by Wnt-beta-catenin signaling in hepatocellular carcinoma. Cancer Res. 2007; 67: 10831–9. Kurtz JE, Dufour P. Adecatumumab: an anti-EpCAM monoclonal antibody, from the bench to the bedside. Expert Opin. Biol. Ther. 2010; 10: 951–8. Gires O, Bauerle PA. EpCAM as a target in cancer therapy. J. Clin. Oncol. 2010; 28: e239–40. author reply e41–2. MacDonald GC, Rasamoelisolo M, Entwistle J et al. A phase I clinical study of VB4-845: weekly intratumoral administration of an anti-EpCAM recombinant fusion protein in patients with squamous cell carcinoma of the head and neck. Drug Des. Devel. Ther. 2009; 2: 105–14. Kowalski M, Guindon J, Brazas L et al. A phase II study of oportuzumab monatox: an immunotoxin therapy for patients with noninvasive urothelial carcinoma in situ previously treated with bacillus Calmette-Guerin. J. Urol. 2012; 188: 1712–8. Fong D, Moser P, Kasal A et al. Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer. Histopathology 2014; 64: 683–92. Ralhan R, Cao J, Lim T, Macmillan C, Freeman JL, Walfish PG. EpCAM nuclear localization identifies aggressive thyroid cancer and is a marker for poor prognosis. BMC Cancer 2010; 10: 331.
Journal of Gastroenterology and Hepatology 30 (2015) 413–420 © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
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doi:10.1111/jgh.12692
H E PAT O L O G Y
Epithelial cell adhesion molecule-positive human hepatic neoplastic cells: development of combined hepatocellular-cholangiocarcinoma in mice Sachiko Ogasawara,* Jun Akiba,* Masamichi Nakayama,* Osamu Nakashima,† Takuji Torimura‡ and Hirohisa Yano* *Department of Pathology, Kurume University School of Medicine, †Department of Clinical Laboratory Medicine, Kurume University Hospital, and ‡ Division of Gastroenterology, Department of Medicine, Kurume University, Kurume, Japan
Key words combined hepatocellular-cholangiocarcinoma, epithelial cell adhesion molecule, hepatic stem/progenitor cell. Accepted for publication 14 July 2014. Correspondence Dr Sachiko Ogasawara, Department of Pathology, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Email: [email protected]
Abstract Background and Aim: Human combined hepatocellular-cholangiocarcinoma (CHC) expresses several hepatic stem/progenitor cell (HSPC) markers, suggesting this neoplasm originates from HSPCs. We examined the significance of HSPC marker in CHC using a human CHC cell line. Methods: We used a human CHC cell line (KMCH-1) previously established in our laboratory. The original tumor was classified as CHC, showing areas of typical hepatocellular carcinoma (HCC) and cholangiocarcinoma (ChC). We examined the expression of HSPC markers and hepatocyte markers in KMCH-1 by flow cytometry (FCM) and quantitative real-time polymerase chain reaction. EpCAM(+) and EpCAM(−) KMCH-1 cells were isolated. Subsequently, their morphological features, HSPC marker expression, and biological characteristics were examined in vitro and in vivo. Results: FCM showed expression of EpCAM, K7, K19, and ABCG2 in KMCH-1, with various degrees. EpCAM(+) cells expressed K19 mRNA, but did not express α-fetoprotein (AFP). In contrast, EpCAM(−) cells expressed AFP mRNA, but did not express K19. EpCAM(+) cells produced both EpCAM(+) and EpCAM(−) cells, but EpCAM(−) cells produced only EpCAM(−) cells in vitro. EpCAM(+) cells showed higher tumorigenicity and formed larger tumors than EpCAM(−) cells. Inoculation of EpCAM(+) and EpCAM(−) cells produced both ChC and HCC-like component and HCC-like component only, respectively. Conclusion: It is speculated that some CHCs may originate from EpCAM(+) neoplastic cells, and that these cells may affect malignant behavior and progression in such CHCs.
Introduction Combined hepatocellular-cholangiocarcinoma (CHC) is defined as a tumor containing unequivocal, intimately mixed elements of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (ChC). ChC also known as mixed tumor of the liver. According to the latest World Health Organization (WHO) classification (2010)1, CHC is classified into two major categories, that is classical type and subtypes with stem cell features. The latter often express hepatic stem/progenitor cell (HSPC) markers; therefore, HSPC origin of this tumor is suspected. It is known that HSPC (also referred to as oval cells in animal models) can differentiate into both hepatocytes and bile duct epithelial cells. No pathological or immunological methods have yet been established to identify these HSPC, but they are found near the canals of Hering where interlobular bile ducts and hepatocytes intersect. They are thought to be small cells similar to canals of Hering or cholangiole
cells.2 When liver damage occurs in the mature liver, it is hypothesized that hepatocytes located near the canals of Hering are activated and become oval cells, which then help to regenerate the damaged liver tissue. In animal models, if severe liver injury is introduced when hepatocyte proliferation is suppressed, oval cells positive for albumin (Alb), α-fetoprotein (AFP), as well as biliary type keratin (K) appear in the portal region and are considered to possess the features of both hepatocytes and bile duct cells.3 Previous reports have shown that c-kit, epithelial cell adhesion molecule (EpCAM), neural cell adhesion molecule (NCAM, CD56), CD133, and CXCR4 are HSPC markers.4,5 With the recent advance of molecular biology, the cancer stem cell (CSC) theory is now widely accepted not only in hematopoietic but also solid neoplasms. According to CSC theory, tumor cells develop from a morphologically and functionally heterogeneous subset of tumor cells that include CSCs with features and characteristics similar to those of ordinary stem cells. These
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tumor-initiating cells initiate a hierarchical organization of cancer cells and are thought to be deeply involved in carcinogenesis, malignancy, metastases, and recurrence. They also play a role in resistance to chemotherapy and radiotherapy and evasion of immunosurveillance, and are therefore suggested to be an important target for cancer therapy.6–8 Marquardt et al.9 have identified high efficiency self-renewal, differentiation along at least two independent lineages, resistance to traditional genotoxic therapy, and capacity to establish and recapitulate the original tumor as criteria for recognition of CSCs. In HCC, HSPC and cancer stem markers have been observed to overlap. Human CHC is thought to be the best example of an HSPCderived tumor.10 Moreover, while CHC is reported to be biologically more malignant than HCC,11 no studies have been done to examine expression of HSPC markers or CSC characteristics in CHC. In the present study we examined studied the expression of HSPC markers in a KMCH-1 cell line developed at our department, and focused particularly on EpCAM as a means of evaluating the biological significance of these markers.
Methods Cell line and reagents. We used a CHC cell line, KMCH-1, which had been established in our department.12 Detailed information on the cell line, patient records, and pathological features of the original tumor were reported previously. Expression of HSPC and hepatocyte markers in the original tumor. We performed immunohistochemistry (IHC) to examine the expression of HSPC markers, such as CD34, CD56, CD133, K 7, K19, c-kit, and EpCAM, and hepatocyte markers, such as hepatocyte paraffin (HepPar)-1. IHC staining was performed using the streptavidin-biotin-peroxidase method. Detailed information regarding the primary antibodies used in this study was listed in Table 1. Alcian blue staining was also performed to assess mucin production.
Table 1
Primary antibodies
Primary
Clone
Source
Antibody ABCG2 Albumin α-fetoprotein CD34 CD56 CD133 CD133 K7 K19 c-kit
BXP-21 polyclonal polyclonal QB-END/10 1B6 AC133 293C3 OV-TL 12/30 BA17 K963
EpCAM (EpEX) EpCAM (EpICD) EpCAM HepPar-1 MUC1
HEA125 E144 EBA-1 OCH1E5 Ma695
EDM Millipore Co. DAKO DAKO Leica Microsystem Leica Microsystem Miltenyi Biotec K.K. Miltenyi Biotec K.K. DAKO DAKO Immuno-Biological Laboratories Co., Ltd. Abcam Abcam Becton Dickinson DAKO Leica Microsystem
Dilution IHC
FCM
1:25 1:500 1:100 1:25 1:50 1:50 — 1:100 1:50 1:200
1:50 — — — 1:50 — 1:10 1:50 1:5 1:50
1:20 1:350 — 1:200 1:100
— 1:1 — —
—, indicate not performed; EpEX, EpCAM extracellular domain; EpICD, EpCAM intracellular domain; FCM, flow cytometry; IHC, immunohistochemistry.
Soft agar colony formation assay. Fifty thousand EpCAM(−) or EpCAM(+) cells were plated in 2 ml of medium containing 0.36% (w/v) top agar layered over a basal layer of 0.72% (w/v) agar in 35 mm dishes (n = 5) and allowed to grow for 2 weeks. Number of colonies was counted using light microscopy.
Sphere formation assay. Sphere formation assay of EpCAM(−) or EpCAM(+) cells was performed according to our previous reports14,15 with minor modification.
Flow cytometric (FCM) analysis and cell isolation. FCM analyses for HSPC markers in KMCH-1 were performed as described in our previous report.13 KMCH-1 cells were stained with antibody for ATP-binding cassette transporter G2 (ABCG2), CD56, CD133, K7, K19, c-kit, and PE conjugated mouse anti-EpCAM (Table 1). In addition, EpCAM-negative (EpCAM[−]) and EpCAM-positive (EpCAM[+]) KMCH-1 cells were isolated with FACS Aria II (Becton Dickinson, San Jose, CA, USA).
Proliferation and drug resistance. Growth speed of EpCAM(−) or EpCAM(+) cells was examined with colorimetry using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kits (Chemicon, Temecula, CA, USA) as described elsewhere.16 In order to examine drug resistance, the cells were seeded and cultured for 24 h, and the medium was changed to medium containing pegylated interferon α-2b (PEG-IFN) (2000 or 4000 IU/ mL; MSD Co., Ltd, Tokyo, Japan), 5-fluorouracil (5-FU) (0.5 or 1 μM; Kyowa Hakko Kirin Co., Ltd, Tokyo, Japan), cisplatin (1.25, 2.5, or 5 μg/mL; Nippon Kayaku Co., Ltd, Tokyo, Japan), or sorafenib (1.25 or 2.5 μM; Cell Signaling Technology., Inc., Danvers, MA, USA). After 72 h of culture, the number of viable cells was examined.
Morphological features and IHC. EpCAM(−) or EpCAM(+) cells were seeded in Lab-Tek chamber slides, cultured for a few days until achieving subconfluency, fixed, and stained with HE, or fixed and IHC for ABCG2 and K19 (Table 1) was performed using CSA II kit (DAKO, Carpinteria, CA, USA).
Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The expression of HSPC markers at the mRNA level of EpCAM(−) or EpCAM(+) cells was examined as described previously.14 qRT-PCR was carried out with TaqMan technology using the ABI 7500
Morphologic features of KMCH-1 cell line. The cells were cultured on Lab-Tek chamber slides (Nunc, Inc., Roskilde, Denmark), then fixed in Carnoy’s solution for 20 min, and stained with hematoxylin and eosin (HE) or Alcian blue.
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Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Gene expression assays primer and probe mixes were used for ABCG2 (Hs00184979_m1), AFP (Hs00173490_ m1), Alb (Hs00609411_m1), c-kit (Hs00174029_m1), CD34 (Hs00156373_m1), CD56 (Hs00169851_m1), CD133 (Hs00195682_m1), K19 (Hs00761767_m1), and β-actin (Hs99999903_m1). β-actin was used for normalization. Relative quantitation was carried out using the ΔΔCt method. Expression of EpCAM after isolation. EpCAM(−) or EpCAM(+) cells were plated at 5 × 104 cells/10 cm in dishes and EpCAM expression was examined weekly from week 1 to week 7 by FCM. To facilitate continued observation of EpCAM expression, every week, the plating process was repeated with the remaining cells at 5 × 104 cells/dish, and chronological changes in EpCAM expression were observed through the end of week 7. Tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. EpCAM(−) or EpCAM(+) cells (106 cells/mouse) were subcutaneously injected into the left and right backs, respectively, of 4-week-old female NOD/SCID mice. The NOD/SCID mice were sacrificed after 16 weeks. Resected tumors were weighed and
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subjected to morphological studies (e.g. HE staining and IHC). The expressions of AFP, Alb, K19, EpCAM (extracellular domain (EpEX) and intracellular domain [EpICD]), HepPar-1, and MUC1 (Table 1) were examined. All procedures were approved by the Ethics Review Committee for Animal Experimentation of Kurume University School of Medicine. EpCAM expressions of human CHC tissues. The expression of EpCAM was examined using two antibodies for its EpEX and EpICD (Table 1). Nine cases previously diagnosed as CHC, classical type were used in this study. Statistical analysis. Data are expressed as the mean ± SD. Comparisons between groups were performed using Student’s t-test. Differences were considered significant at P < 0.05. This study was approved by the ethical committee of Kurume University (approved #10229 and #10294).
Results Morphology and IHC. The original KMCH-1 tumor was composed of both HCC (Fig. 1a), with cancer nests of tumor cells
Figure 1 Histological findings of original tumor. (a) The hepatocellular carcinoma (HCC) portion showing a trabecular proliferative pattern (hematoxylin and eosin staining). (b) The hepatocellular-cholangiocarcinoma (ChC) portion showing gland formation. (c) Transition between HCC and ChC. (d) Mucin production is observed inside the rim of the glands in the ChC portion (Alcian blue staining). (e) K19 expression is found in the glands of the ChC component. (f) The expression of EpCAM is observed in both the HCC and ChC components although the intensity is stronger in ChC components than HCC components. Histological findings of KMCH-1 cells. (g) Hematoxylin and eosin staining. (h) Mucin production is observed. (i) Flow cytometric analyses revealed 65.3%, 28.7%, 29.4%, and 25.1% of KMCH-1 cells were positive for EpCAM, K7, K19, and ABCG2, respectively. CD56, CD133, and c-kit were not expressed. Staining with control antibody or specific antibodies is shown by black line or red line, respectively.
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in a trabecular arrangement, and ChC (Fig. 1b), with glandular structures accompanied by abundant fibrous stroma. A transitional area was observed between them (Fig. 1c). Mucin production was confirmed in the rim of the glandular structure of ChC component by Alcian blue stain (Fig. 1d). K7, K19, and EpCAM were positive for tumor cells (Fig. 1e,f), but the expression of CD34, CD56, CD133, c-kit, and HepPar-1 was negative. The expression of EpCAM was stronger in ChC component than in HCC component. These findings indicated that the original tumor corresponded to CHC, classical type, in the latest WHO classification.1
Morphologic features and HSPC marker expression in KMCH-1. KMCH-1 cells proliferated in a pave stone arrangement. They had large, round nuclei containing several nucleoli (Fig. 1g). Mucin production was found in KMCH-1 by Alcian blue stain (Fig. 1h).
FCM analysis. FCM analyses revealed that EpCAM, K7, K19, and ABCG2 were expressed on KMCH-1 cells, and their positive cell rates were 65.3%, 28.7%, 29.4%, and 25.1%, respectively (Fig. 1i). CD56, CD133, and c-kit were not expressed.
Morphologic features and HSPC marker expressions of EpCAM(−) and EpCAM(+) cells. There was no morphologic difference between EpCAM(−) and EpCAM(+) cells with HE stain (Fig. 2a,b). Both EpCAM(+) and EpCAM(−) cells expressed ABCG2; however, the number of ABCG2 positive cell was higher in EpCAM(+) cells than in EpCAM(−) cells (Fig. 2c,d). K19 (Fig. 2e,f) expression was detected only in EpCAM(+) cells.
Soft agar colony formation and sphere formation assay. The average numbers of soft agar colony formation in EpCAM(−) and EpCAM(+) cells were 1215.0 ± 184.5 and 1278.2 ± 284.2 colonies, respectively. There was no significant difference between them (Table 2). Sphere formation was significantly higher in EpCAM(+) cells (24.5 ± 9.3) than EpCAM(−) cells (7.8 ± 4.9) (P < 0.05, Table 2).
Expression of EpCAM after isolation. EpCAM(−) cells generated only EpCAM(−) cells through weeks 1 to 7. EpCAM(+) cells generated both EpCAM(−) and EpCAM(+) cells through all 7 weeks. EpCAM(+) cells rates at the 1st- and 7thweek were 68.8% and 73.5%, respectively (Fig. 2i). Tumorigenicity in NOD/SCID mice. One of five mice inoculated with EpCAM(−) cells developed a tumor that was 169 mg in weight. This tumor showed HCC-like morphological features and lacked a ChC-like component (Fig. 3a,b). Immunohistochemically, the tumor lacked EpCAM expression (Fig. 3c,d), but had HepPar-1 (Fig. 3e,f), Alb (Fig. 3g,h), and AFP (data not shown) expression. The expression of K19 was equivocal (data not shown). In contrast, four of five mice inoculated with EpCAM(+) cells generated tumors, and the average tumor weight was 1758 ± 223 mg. These tumors morphologically mimicked the original tumor of KMCH-1. Tumors were composed of HCC-like and ChC-like components. HCC-like component (Fig. 4arectangle-1, e) lacked EpCAM (Fig. 4b,f) and K19 (Fig. 4d,h) expression, and had HepPar-1 (Fig. 4c,g), Alb, and AFP (data not shown) expression. ChC-like component (Fig. 4a-rectangle-2, i) showed positivity for EpCAM (Fig. 4b,j), K19 (Fig. 4d,l), and MUC-1 (data not shown). Mucin production was found by mucicarmine stain. ChC component was negative for HepPar-1 (Fig. 4c,k). EpEX and EpICD expression tumor obtained from NOD/SCID mice and human CHC tissues. The expression of EpICD was found only in ChC component of tumors obtained from EpCAM(+) cells. The expression of EpICD in ChC component of tumors obtained from EpCAM(+) cells was in accordance with cell membrane, and no expression was observed in cytoplasm or nucleus. EpICD expression was not found in HCC component of tumors obtained from EpCAM(+) cells and a tumor from EpCAM(−) cells (data not shown). The expression pattern of EpEX and EpICD in human CHCs was described in Table 3. There was no apparent correlation between expression patterns and clinicopathologic findings, including microvascular permeation and patient prognosis.
Discussion Proliferation and drug resistance in vitro. No difference was observed in proliferation rates between EpCAM(−) and EpCAM(+) cells at 24, 48, 72, or 96 h (Fig. 2g). When treated with PEG-IFN, 5-FU, or cisplatin, no significant differences were seen in proliferation between EpCAM(−) and EpCAM(+) cells (Fig. 2h).
qRT-PCR. qRT-PCR revealed AFP expression only in EpCAM(−) cells and K19 expression only in EpCAM (+) cells. The expression level of Alb in EpCAM(−) cells was 9.5 ± 3.2 times higher than that of EpCAM(+) cells. ABCG2 and CD133 were expressed on both EpCAM(+) and EpCAM(−) cells. CD34, CD56, and c-kit were not expressed on both EpCAM(+) and EpCAM(−) cells. All experiments were performed three times. There was no apparent difference in other HSPC markers. 416
We re-evaluated the original tumor of KMCH-1 histopathologically using several markers, including HSPC and hepatocyte markers. These findings suggested that the original tumor corresponded to CHC, classical type, in the latest WHO classification. EpCAM, K7, and K19 expressions were observed in both the original tumor and KMCH-1. Moreover, these markers predominantly showed positive in the ChC component of the original tumor. The immunoprofiles we showed here were consistent with our previous report on the immunophenotypes of human CHCs.17 Thus, we performed further investigation focusing on EpCAM in KMCH-1, which is one of the representative HSPC markers. EpCAM is associated with cell adhesion, cell migration, cell differentiation, cell proliferation, and embryogenesis.18 In human adult liver, the expression of EpCAM is found in cholangiocytes, but not in hepatocytes. In embryonic liver, the majority of hepatocytes have the expression of EpCAM.19 The clonally expanded
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Figure 2 Immunohistochemical staining of EpCAM(−) (the upper row) and EpCAM(+) cells (the lower row). (a,b) There is no morphologic difference between EpCAM(−) and EpCAM(+) cells with hematoxylin and eosin (HE) stain. (c,d) Both EpCAM(+) and EpCAM(−) cells express ABCG2. The number of ABCG2-positive cells is higher in EpCAM(−) than EpCAM(+) cells. (e,f) K19 expression is detected only in EpCAM(+) cells. Proliferation and drug resistance in vitro. (g) Proliferation rates between EpCAM(−) and EpCAM(+) cells at 24, 48, 72, or 96 h were not different. ●, EpCAM(+) ■; &, EpCAM(–). (h) Pegylated interferon α-2b (PEG-IFN), 5-fluorouracil (5-FU), cisplatin, and sorafenib did not show significant differences in proliferation between EpCAM(−) and EpCAM(+) cells. Expression of EpCAM after isolation of EpCAM(−) and EpCAM(+) cells. , EpCAM−; ■, EpCAM+. (i) Immediately after isolation by EpCAM antibody (red line) in KMCH-1, cell fractions isolated as EpCAM(−) and EpCAM(+) cells were composed of EpCAM(−) and EpCAM(+) cells respectively. Black line in the figures indicates EpCAM-positive cell fraction before isolation. Isolated EpCAM(−) and EpCAM(+) cells were cultured separately up to 7 weeks. Staining with control antibody or EpCAM antibody is shown by black or red lines, respectively. EpCAM(−) cells sequentially generated only EpCAM(−) cells (upper figures), but EpCAM(+) cells generated both EpCAM(−) and EpCAM(+) cells during observation period. EpCAM(+) cell rates at the 1st- and 7th week were 68.8% and 73.5%, respectively (lower figures).
Table 2
Soft agar colony formation and sphere formation assay
Soft agar Colony formation Sphere Formation
EpCAM (−)
EpCAM (+)
1215.0 ± 184.5†
1278.2 ± 284.2‡
7.8 ± 4.9§
24.5 ± 9.3¶
† versus ‡: not significant. § versus ¶: P < 0.05. The number of colony and sphere per dish and well, respectively.
cells from isolated EpCAM(+) cells exhibited unlimited proliferation and bi-directional differentiations toward cholangiocytes and hepatocytes.20 Recently, it has been suggested that various liver cancers originate from HSPCs.1,21–26 Of these, CHC is regarded as the best example of an HSPC-derived tumor. In the present study, EpCAM(+) KMCH-1 cells showed a higher proliferative rate in vivo compared with EpCAM(−) cells. EpCAM(+) cells initially had characteristics of ChC, and lacked those of HCC immunophenotypically and morphologically. EpCAM(+) cells generated both EpCAM(−) and EpCAM(+) cells in vitro and in vivo. Furthermore, tumors obtained from EpCAM(+) cells in vivo resembled the original tumor of KMCH-1 morphologically
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Figure 3 Panoramic views of a tumor obtained from EpCAM(−) cells inoculated in an non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse are shown in the upper row. The area indicated by a rectangle of the upper row corresponds to the lower row. (a,b) Tumor derived from EpCAM(−) shows HCC-like morphological features (hematoxylin and eosin [HE] stain). (c,d) IHC shows negative reaction for EpCAM. (e–h) IHC shows positive reaction for HepPar-1 (e,f) and albumin (g,h).
Figure 4 Panoramic views of a tumor obtained from EpCAM(+) cells inoculated in an non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse are shown in the upper row. The area indicated by a rectangle in 1 and 2 of the upper row corresponds to the middle row (HCC-like component) and the lower row (ChC-like component), respectively. Tumors obtained from EpCAM(+) show typical features of CHC composed of HCC-like and ChC-like components. HCC-like component (a-rectangle-1,e) has HepPar-1 (c,g) expression, and lacks EpCAM (b,f) and K19 (d,h) expression. ChC component (a-rectangle-2,i) shows positivity for EpCAM (b,j) and K19 (d,i). ChC component is negative for HepPar-1 (c,k).
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Table 3 The expression of EpEX and EpICD in human combined hepatocellular-cholangiocarcinoma EpEX
Case Case Case Case Case Case Case Case Case
1 2 3 4 5 6 7 8 9
EpICD
ChC
HCC
ChC
HCC
M
M
M
C
N
M
C
N
– + – + + – – – –
– + – – – – – – –
– – – – – – – +† +
– – – + + + + – +
– – – + + + + – –
– – – – – + – – +
– – – + + + + + +
– – – + + + + + –
†
Luminal expression at the glandular structure C, cytoplasmic expression; ChC, cholangiocarcinoma; EpEX, EpCAM extracellular domain; EpICD, EpCAM intracellular domain; HCC, hepatocellular carcinoma; M, membranous expression; N, nuclear expression.
and immunohistochemically. EpCAM(−) cells generated only EpCAM(−) cells in vitro and produced only HCC tumor in vivo. These features of EpCAM(+) cells overlapped with those of CSCs. Although some possible histogeneses of CHC-classical type are proposed,17 our results suggest that malignant transformed HSPC, which has EpCAM expression and ChC characteristics, differentiates to HCC. Our result indicated that loss of EpCAM expression might play a pivotal role in cell conversion or differentiation from ChC to HCC. These dramatic changes could be due to not only EpCAM but also to other molecules, including other HSPC markers. However, the precise mechanism by which this occurs remained poorly understood. Although our present study raises the possibility that some CHCs could be originated from EpCAM(+) HSPC, some limitations must be disclosed. First, our results were obtained from only one cell line. Therefore, in order to enforce our findings, investigation using another CHC cell line is ongoing in our laboratory. HSPCs are proposed to be rare populations. However, KMCH-1 showed high positive rate for EpCAM. This result raises the question whether EpCAM is universal marker of HSPC. Suzuki et al.27 suggests that it is unlikely that cells isolated with several surface markers, such as CD133, could form a large colony in single-cell culture. Moreover, these large colonies showed different gene expression, suggesting that even cells isolated with several markers might be heterologous. Thus, the cell population isolated with EpCAM in our present study might also be heterogeneous. Additional markers, such as K19 and/or K7, combined with EpCAM could be useful to identify to HSPC selectively. In addition to cell surface markers, functional assays, such as side population (SP) and aldehyde dehydrogenase 1 (ALDH1) activity, might be also useful.7,28,29 Therefore, we also performed SP assay and ALDH1 assay using KMCH-1 cells. SP cells had a tendency of high proliferation rate than non-SP cells. However, no apparent difference was observed in other assays, including proliferation, drug resistance (i.e. PEG-IFN, 5-FU, cisplatin, and sorafenib), sphere formation, and real-time PCR between SP and non-SP cells, or between ALDH1-positive and ALDH1-negative cells.
Several reports document CHC as an aggressive tumor with poorer outcomes than HCC.30–34 In addition, standard treatments for CHC are not well-established. Yamashita et al.35 found that EpCAM was a β-catenin transcription target, that EpCAM(+) cells showed higher proliferation than EpCAM-negative cells, and that proliferation of hepatocytes was suppressed when small interfering RNA was used to suppress EpCAM expression.5 These findings indicated that EpCAM could be a targeted therapy for liver CSCs. Efforts have been made to use monoclonal antibodies and immunotoxins to target EpCAM in malignant tumors expressing EpCAM,36–39 and it is hoped that therapies designed to suppress EpCAM signaling could prove to be effective. The results obtained from this present study suggest that molecular target therapy focusing on EpCAM might be effective in not only HCC but also in CHC. Further, the suppression of EpCAM expression may enhance cell conversion or differentiation to HCC. A few reports demonstrated that EpICD expression pattern is closely associated with patient prognosis in human malignancies.40,41 We found nuclear expression of EpICD in five of nine cases in human CHC. There was no apparent correlation between expression pattern and clinicopathologic findings. At present, its clinicopathologic significance in human CHC, classical type remains elusive. In conclusion, our results revealed that EpCAM(+) KMCH-1 cells show HSPC-like features and high tumorigenicity, and develop tumors with CHC features in vivo. It is speculated that some CHCs may be originated from EpCAM(+) neoplastic cells, and that these cells may play a role in malignant behavior and progression in such CHCs.
Acknowledgments We thank Ms Akemi Fujiyoshi for her excellent assistance in our experiments. This study has no financial support.
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