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Alpha-fetoprotein producing cells act as cancer progenitor cells in human cholangiocarcinoma
Cancer Letters 294 (2010) 25–34
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Alpha-fetoprotein producing cells act as cancer progenitor cells in human cholangiocarcinoma Takamichi Ishii a,b,*, Kentaro Yasuchika b, Hirofumi Suemori a, Norio Nakatsuji c,d, Iwao Ikai b,e, Shinji Uemoto b a
Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Japan Department of Surgery, Graduate School of Medicine Kyoto University, Japan c Institute for Integrated Cell-Material Science, Kyoto University, Japan d Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Japan e Department of Surgery, Kyoto Medical Center, National Hospital Organization, Japan b
a r t i c l e
i n f o
Article history: Received 9 October 2009 Received in revised form 12 January 2010 Accepted 18 January 2010
Keywords: Cancer stem cell Cholangiocarcinoma AFP Notch ICC
a b s t r a c t We aimed to demonstrate that alpha-fetoprotein (AFP)-producing cells in cholangiocarcinomas possessed cancer stem cell (CSC)-like properties. AFP enhancer/promoter-driven EGFP gene was transfected into human cholangiocarcinoma cell lines. One cell line, RBE, expressed both AFP and EGFP. Clonal analyses revealed that one EGFP-positive cell generated both EGFP-positive and EGFP-negative cell fractions. However, one EGFP-negative cell never produced EGFP-positive cells. The EGFP-positive cells had a greater tumorigenic potential. Only the EGFP-positive cells expressed Notch1. AFP and Notch1 expression was observed in clinical intrahepatic cholangiocarcinomas. The AFP-producing cells were suggested to be CSCs. The Notch pathway might play an important role in maintaining the CSC characteristics. Ó 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Recent developments in stem cell biology have revealed the existence of cancer stem cells (CSCs) in various tumors such as leukemia [1], brain tumors [2], breast cancer [3] and hepatocellular carcinoma (HCC) [4]. These CSCs are thought to display a hierarchy in tumor tissues and generate tumor heterogeneity. Although the definition of CSCs has not yet been unequivocally established, CSCs should have both a self-renewal activity and multipotency like normal tissue stem cells [5]. Furthermore, CSCs have common features of normal tissue stem cells, especially the resistance to multiple drugs [6] and radiation [7]. Therefore, CSCs are supposed to survive even after anti-cancer
* Corresponding author. Address: Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. Tel.: +81 75 751 3821; fax: +81 75 751 3890. E-mail address: [email protected] (T. Ishii). 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.01.019
therapies and to generate therapy-refractory tumors. The elucidation of CSC biology might contribute not only to oncology in general, but also to the development of a new strategy for cancer therapy. Intrahepatic cholangiocarcinomas (ICCs) are the second most common cancer among the primary liver cancers, after HCC [8]. ICC has a poor prognosis and therefore, research regarding ICC is anticipated to improve the treatment outcomes in patients with ICC. During the normal hepatic development, hepatic stem/progenitor cells are thought to differentiate into hepatocytes and cholangiocytes [9]. Since alpha-fetoprotein (AFP) is one of the most common markers for hepatic progenitor cells in the fetal liver [10–12], AFP was focused on as a marker for CSCs in ICC. Whereas AFP is often used as a tumor marker for HCC, serum AFP is not usually observed in ICC patients. Therefore, if AFP-producing cells are present in ICCs, then they should have stem cell characteristics. In this study, a reporting vector that expressed enhanced green fluorescence protein (EGFP) under the
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control of human AFP enhancer/promoter was transfected into five human cholangiocarcinoma (CC) cell lines to characterize the AFP-producing cells as CSCs. The results showed that the Notch signaling pathway might play an important role in maintaining the self-renewal ability of the AFP-producing cells in the CC cell line. Moreover, the expression of AFP and Notch signaling protein in tumor cells was investigated utilizing surgically resected human ICC tumor specimens. 2. Materials and methods 2.1. Patients Twenty patients with ICC confirmed by pathologic analyses, who had undergone a hepatic resection at Kyoto University Hospital from June 2005 to December 2007, were included in this study. The patients included 12 men and 8 women, with a mean age at the surgery of 67.5 ± 7.9 years (range, 58–81 years). Written informed consent for the use of their resected tissues was obtained from all patients in accordance with the Declaration of Helsinki. The clinicopathological background is summarized in Supplementary Table S1.
the secondary antibodies were diluted at 1:500. The stained cells were covered with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). 2.4. Flow cytometry and cell sorting The cultured cells were prepared as described previously [11–13]. Dead cells were eliminated using 7-amino-actinomysin D (BD Biosciences, Franklin Lakes, NJ) staining. The cells were analyzed and isolated using a FACSVantage SE cell sorter (BD Biosciences). The isolated cells were cultured on plastic culture dishes in RPMI1640 supplemented with 10% FBS and penicillin/streptomycin in the presence or absence of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT, Sigma) dissolved in dimethyl sulfoxide (DMSO, Sigma). For the single-cell culture analyses, the individual isolated cells were sorted out into each well of 96-well culture plates using FACSVantage SE and the Clone-Cyt Plus software program (BD Biosciences). The wells containing only one cell were confirmed using a light microscope 10–16 h after cell sorting. 2.5. Cell proliferation assay, anchorage-independent cell growth assay, and sphere-forming assay
2.2. Cell cultures The human CC cell lines HuCCT-1 and TFK-1 were obtained from the Cell Resource Center for Biochemical Research, Tohoku University (Sendai, Japan); OZ was obtained from the Japan Health Science Foundation (Tokyo, Japan); and the SSP-25 and RBE cells were obtained from the RIKEN Bioresource Center (Tsukuba, Japan). These were cultured at 37 °C and in 5% CO2 in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Gibco). The transgene plasmid vectors that expressed EGFP under the control of human AFP enhancer/promoter were generated as described previously [11]. The transgenic vectors were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Stable transfected cells were selected in the presence of 100 lg/ml G418 (Sigma, St. Louis, MO) over a period of 30 days. The proper transgene insertion was confirmed by polymerase chain reactions. The stable transfectants were cultured in the presence of 100 lg/ml G418. 2.3. Immunohistochemistry of cultured cells The cultured cells were fixed and stained as previously described [11–13]. An anti-human cytokeratine 19 (CK19) mouse antibody (DakoCytomation, Glostrup, Denmark) diluted at 1:50, anti-human AFP rabbit antibody (DakoCytomation) diluted at 1:200 and 5 lg/ml anti-human Notch1 intracellular domain (NICD1) mouse antibody (R&D Systems, Minneapolis, MN) were used as the first antibodies. Alexa 555-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) for CK19 and NICD1 staining and Alexa 546-conjugated donkey anti-rabbit IgG (Molecular Probes) for AFP staining were used as the secondary antibodies. All
The isolated EGFP-positive and EGFP-negative cells were inoculated at a density of 1 103 per well, respectively, and then were allowed to grow for 8 days. The cell numbers were determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazorium, inner salt (MTS) assay (Cell Titer 96 Aqueous One Solution Reagent, Promega, Madison, WI), according to the manufacturer’s protocol. After 1 h of incubation, the absorbance value was measured using a plate reader at 490 nm. To examine the anchorage-independent growth, 1 104 EGFP-positive and EGFP-negative cells were suspended in 2.0 ml of 0.3% agar (Wako, Osaka, Japan) supplemented with the culture medium. The cell suspension was layered over the bottom layer of 2.0 ml of 0.6% agar. The colonies that contained more than 10 cells were counted 21 days after cell sorting. To investigate the ability to form cell spheres, the sorted EGFP-positive and EGFP-negative cells were seeded at a density of 1 105 cells/well in 6-well ultra low attachment plates (Corning Inc., Corning, NY) in serum-free PRMI1640 media. The spheres were observed 5 days after cell sorting. These assays were repeated for three times. 2.6. Reverse-transcription polymerase chain reaction (RTPCR) RT-PCR was performed as described previously [11]. Primers were generated for the following human genes: EGFP, AFP, GATA4, Foxa2, albumin, Notch1, Notch2, Notch3, Notch4, Delta-like 1 (Dll1), Jagged 1 (Jag1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Their sequences and PCR conditions are summarized in Supplementary Table S2.
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2.7. Cell transplantation procedures The sorted cells were transplanted into 6- to 8-weekold male non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice (CLEA Japan, Inc., Tokyo, Japan). The isolated EGFP-positive and EGFP-negative cells, ranging from 3 104 to 3 105 cells, were suspended in 200 ll Hank’s balanced salt solutions (Gibco). Under general anesthesia, they were engrafted intraperitoneally by means of splenic injection using a 29-gauge needle (BD Biosciences). The implanted mice were killed 10 weeks after the transplantation. All experimental procedures utilizing animals were performed in accordance with the Animal Protection Guidelines of Kyoto University. 2.8. Histology and immunohistology of tumor tissues The harvested tumors were fixed in 3.3% formaldehyde (Wako) for at least 1 week. After a thorough rinsing, the tissue was then embedded in paraffin and then sectioned into 3 lm thick sections. Hematoxylin–eosin (HE) staining was performed according to a standard protocol. Prior to immunohistology, the sections were incubated in HistoVT One (Nacalai Tesque, Kyoto, Japan) for 20 min at 90 °C for antigen retrieval. Immunofluorescent staining for AFP and NICD1 was performed as described above. In order to perform immunohistological staining for AFP, the deparaffinized and rehydrated sections were incubated in 3% hydrogen peroxide in methanol for 10 min and then were incubated with the diluted anti-human AFP antibodies at 4 °C for 16 h. Followed by thorough rinsing, they were incubated for 30 min with horseradish peroxidase-conjugated anti-rabbit IgG (Envision + Kit/horseradish peroxidase; DakoCytomation) and then for 1 min with 3,30 Diaminobenzidine (DAB) substrates (DakoCytomation). Counterstaining was performed using hematoxylin. To semi-quantitate the immunostaining results, the slides were independently scored by two evaluators (T.I. and K.Y.) for AFP staining. Visual assessment based on the frequency of immunoreactivity was classified into four categories: no staining (), focal staining (<5% of tumor cells were positive for AFP; +), moderate staining (<30%; ++) and diffuse staining (P30%; +++). 2.9. Statistical analysis All in vitro samples were collected from three to six independent trials. The results are presented as the means ± SD. The statistical analyses between two groups were performed using Student’s t-test. The tumorigenicity between the EGFP-positive and the EGFP-negative cell transplanted group was analyzed using Fisher’s exact test. A P value of <0.05 was considered to be statistically significant. All statistical tests were two-sided. 3. Results 3.1. AFP-producing cells in the RBE cell line RT-PCR revealed that AFP expression was observed in RBE and that weak expression was also detected in OZ (Fig. 1A). An immunostaining assay confirmed that all RBE cells were positive for CK19, a marker for
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cholangiocytes and that approximately 20% of the RBE cells were positive for AFP (Fig. 1B). The AFP–EGFP reporter vector was transfected into the five CC cell lines. Only the RBE cells expressed the EGFP fluorescence; the other four cell lines did not, despite the fact that they possessed the transgene in the genomic DNA (Fig. 1B and data not shown). The RBE cell line is derived from intrahepatic cholangiocarcinoma with moderately differentiated tubular adenocarcinoma [14]. A flow cytometric analysis showed that 23.6 ± 3.9% (n = 3) of the RBE cells expressed the EGFP fluorescence (Fig. 1C). There was no obvious difference in the morphology between the sorted EGFP-positive and EGFP-negative RBE cells (Fig. 1D). The sorted EGFP-positive cells expressed both EGFP and AFP, whereas the sorted EGFP-negative cells did not express either mRNA (Fig. 1E), thus indicating the EGFP-positive cells to correspond to the AFP-producing cells and that cell sorting could divide the RBE cells according to their AFP production. Therefore, a further investigation of the AFP-producing cells in RBE was conducted to determine whether or not they were cancer stem cells.
3.2. Single cell sort analyses of the AFP-producing and AFP-non-producing cells Since the RBE cells had a heterogeneous population, single-cell culture assays were performed in order to examine their self-renewal activity and multipotency. A single cell was sorted and cultured on 96-well culture plates. Following cell expansion by four passages, flow cytometric analyses were performed. A single EGFP-positive cell generated both the EGFP-positive and EGFP-negative cell fractions (Fig. 2A, B). One EGFP-negative cell, however, produced only the EGFP-negative fraction (Fig. 2C, D). These flow cytometric findings were reproducible in three trials (five EGFP-positive clones and three EGFP-negative clones), and the transgene was identified in the genomic DNA of all these clones (Supplementary Fig. S1). These results indicated that the EGFP-positive cells thus had a self-renewal activity and the ability to ‘differentiate’ into the EGFP-negative cells. They also suggested the progenitor-like characteristics of the AFP-producing cells.
3.3. Tumorigenic potential of the AFP-producing cells in vitro and in vivo To compare the tumorigenic abilities in vitro between the EGFP-positive and EGFP-negative cells, both cell proliferation and the anchorageindependent cell growth assays were performed. The EGFP-positive cells exhibited a higher proliferation activity than did the EGFP-negative cells, with a significant difference (P < 0.01, n = 3; Fig. 3A). Moreover, the EGFPpositive cells showed an approximately 4.5-fold higher ability to form colonies in soft agar than the EGFP-negative cells did (P < 0.001, n = 6; Fig. 3B). In addition, the sphere assays revealed that the EGFP-positive cells formed cell spheres 5 days after cell sorting, whereas the EGFP-negative cells did not (Fig. 3C). These findings suggested the EGFP-positive cells to therefore have stronger tumorigenic and malignant potentials in vitro than the EGFP-negative cells did. To examine the tumorigenicity in vivo, the EGFP-positive or EGFPnegative cells were transplanted into the abdominal cavities of NOD/SCID mice via their spleens. The transplantation of 3 104 EGFP-positive cells resulted in the development of tumors in one of three mice, 1 105 EGFPpositive cells resulted in tumors in seven of nine mice and 3 105 EGFPpositive cells did in two of two mice (Table 1). These tumors included one liver tumor (Fig. 3D; left panel), two cases of splenic tumors (Fig. 3D; right panel) and seven cases of peritoneal disseminated nodules. H&E staining revealed the disseminated nodules to consist of tumor cells with tube-like formations, occasional atypical multinucleated giant cells and stromal rich components (Fig. 3E). Immunostaining of the peritoneal dissemination foci showed that all of these tumor cells were positive for human CK19 (Fig. 3F; left panel) and that approximately 20% of the tumor cells expressed AFP (Fig. 3F; right panel). Some transplanted EGFP-positive AFP-producing cells generated the AFP-non-producing cells, thus resulting in the original proportion of AFP-producing cells. On the other hand, the engraftment of the 1 105 EGFP-negative cells resulted in only one tumor in six mice. The transplantation of the 3 104 or 3 105 EGFPnegative cells did not form any tumor in the NOD/SCID mice. These results indicated the EGFP-positive cells to have an increased tumorigenicity in vivo, while also demonstrating the characteristics of cancerinitiating cells.
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Fig. 1. The human cholangiocarcinoma cell lines that expressed EGFP under the control of the human AFP enhancer/promoter. (A) An RT-PCR analysis of the five CC cell lines. (B) Immunocytological assay for RBE. The RBE cells expressed AFP (red fluorescence) with the proportion of approximately 20% (left upper panel) and all of them expressed CK19 (red; right upper panel). Blue fluorescence indicates DAPI. The stable transfectants of RBE cells expressed EGFP florescence (left lower panel). The figure of the right lower panel showed the phase contrast image of the same visual field of the left lower panel. (C) A flow cytometric analysis revealed the RBE cells to express EGFP in the proportion of 23.6 ± 3.9%. The longitudinal axis indicates the blank fluorescence and the horizontal one does the intensity of EGFP. (D) The sorted EGFP-positive cells (left panel) were morphologically similar to the EGFP-negative cells (right panel). The small inserts showed the fluorescent microscopy images. Green fluorescence indicates EGFP. (E) The sorted out EGFP-positive cells expressed mRNA both of EGFP and of AFP (left lane), whereas the sorted EGFP-negative cells expressed neither EGFP nor AFP mRNA (right lane). GAPDH was used as an internal control and RT served as a negative control. Original magnifications: (B, D) 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. The involvement of Notch signaling pathway in CSC maintenance In order to investigate the difference between CSCs and non-CSCs, the expressions of previously reported genes related with cancer or hepatic stem cells were compared between the EGFP-positive and EGFP-negative cells and RT-PCR was performed to compare the genetic expression between the two groups (Fig. 4A). There was no difference in the endoderm-related genes except for AFP (Fig. 1D). However, mRNA of Notch1 was expressed specifically by the EGFPpositive cells. The expression of neither the other three Notch receptors nor the Notch ligands showed any difference between the two cell groups. To examine the effect of the Notch signaling pathway on CSCs, immunostaining for NICD1 was performed. The NICD1 was found in both the cytoplasm and nuclei of the isolated EGFP-positive cells (Fig. 4B), whereas the EGFP-negative cells did not express NICD1 (Fig. 4C), thus indicating the Notch signaling to be activated only in the EGFP-positive cell fraction. In addition, the purified EGFP-positive cells were cultured in the presence of DAPT. DAPT acts as a c-secretase inhibitor and prevents the Notch intercellular domain from releasing from the cell membrane, thereby blocking the Notch signaling pathway [15]. After a 7 day culture, a portion of the EGFP-positive cells in the control group ceased to express EGFP spontaneously and showed 78.9 ± 1.4% of the EGFP-positive proportion (Fig. 4D). However, the EGFP-positive cells that were cultured in the presence of 50 lM DAPT showed a significant decrease in the proportion of EGFP-positive cells with 63.4 ± 1.0% (P = 0.001, n = 6; Fig. 4D, E). The inhibition of the Notch signaling pathway thus caused a decrease of the EGFP-
positive cells. These results suggested the Notch signaling pathway to possibly play a crucial role in the maintenance of the stemness of the EGFP-positive AFP-producing cells. 3.5. The expression of AFP and Notch1 in the patients with ICC To examine the AFP and NICD1 expression in human clinical samples of ICC, immunohistological assays were performed utilizing 20 surgical resected ICC tumors (Supplementary Table S3). No AFP-positive cell was observed in 11 patients (Fig. 5A). Six of the twenty patients with ICC exhibited a focal staining pattern for AFP (Fig. 5B, C). A small portion of tumor cells (less than 5%) was positive for AFP. Two tumors displayed a moderate AFP staining pattern, meaning that 5–30% of tumor cells expressed AFP (Fig 5D). A diffuse staining pattern was observed in one case (Fig. 5E). The preoperative serum AFP values did not correlate with the AFP staining degree in the tumor tissue specimens. The tumor cells that co-expressed AFP and NICD1 were found in seven of the nine patients with the AFP-producing ICC cells. In all seven cases, however, they only made up an extremely small proportion of the tumor tissues, namely comprising less than 1% of all tumor cells. No AFP-negative cell expressed NICD1.
4. Discussion Intrahepatic cholangiocarcinoma is one of the carcinomas with the poorest prognosis and it is also the second
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Fig. 2. Single cell sort analyses of the AFP-producing EGFP-positive cells and AFP-non-producing EGFP-negative cells. (A) The individual EGFP-positive cells were sorted into each well of 96-well culture plates. The single cell indicated by the bold dot generated both the EGFP-positive and EGFP-negative cell fractions (B). On the other hand, the single EGFP-negative cell indicated by the bold dot in (C) produced only the EGFP-negative cell fraction (D).
most common primary hepatic carcinoma, following HCC [8]. Cancer stem cell biology concerning CC has not been investigated until now, although several articles have reported the existence of CSCs in its counterpart, HCC [4,16]. Both hepatocytes and cholangiocytes are descendants of hepatic stem/progenitor cells in the normal process of hepatic development [9]. CSCs show similarities with their normal tissue stem cells. The present study focused on AFP as a CSC-marker because AFP is one of the most common markers for fetal hepatoblasts or hepatic stem/progenitor cells. CSCs should have at least three characteristics; a selfrenewal ability, multipotency and tumorigenicity [5]. The
single-cell culture analyses showed that only the AFP– EGFP-positive cell fraction had self-renewal ability and differentiation potency. One AFP–EGFP-positive cell had the ability to generate not only the AFP–EGFP-positive cells but also the AFP–EGFP-negative cells. These findings indicated that the AFP-producing cells showed functional similarities to lineage-committed progenitor cells, thus indicating the AFP-producing cells to indeed be cancer progenitor cells. On the other hand, one EGFP-negative cell generated the only EGFP-negative fraction, thus indicating it to only have a self-renewal activity. This also suggested the EGFP-negative cells to have decreased stem-like characteristics. In this study, however, it was impossible to
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Fig. 3. Tumorigenic potential of the AFP-producing cells in vitro and in vivo. (A) The MTS assays between the two cell populations were tested using repeated-measures ANOVA. P < 0.01. (B) The graph indicates the colony numbers of the EGFP-positive and EGFP-negative cells in soft agar. P < 0.001 (Student’s t-test). The results are presented as the means ± SD. (C) The EGFP-positive cells formed cell spheres in suspension cultures (left panel), whereas the EGFP-negative cells did not (right panel). The small insert showed the fluorescent microscopy image. Green fluorescence indicates EGFP. Tumorigenicity in vivo was shown by xenotransplantation into NOD/SCID mice. One liver tumor was detected in one mouse (D; left panel) and splenic tumors were detected in two mice (D; right panel) in the EGFP-positive cell engrafted group. The arrowheads indicate the tumors. Peritoneal disseminations were found in seven mice of the EGFP-positive cell engrafted group. Figure E shows the H&E staining of the disseminated nodules. Figure F indicate the merged immunostaining assay for human CK19 (left panel; red fluorescence) and AFP (right panel; red fluorescence). Blue fluorescence indicates DAPI. Original magnifications: (C), 100; (D), 10; (E, F), 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
demonstrate both fractions of these cells to differentiate into a mature cholangiocyte lineage. Furthermore, the higher in vitro malignant potency of the AFP–EGFP-positive cells was observed in the MTS assay, anchorage-indepen-
dent growth assay, and sphere-forming assay. Xenotransplantation experiments showed that the AFP-producing cells had a markedly greater tumorigenicity than the AFP-non-producing cells did. These results indicated that
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T. Ishii et al. / Cancer Letters 294 (2010) 25–34 Table 1 The tumorigenicity of the isolated EGFP-positive and EGFP-negative cells in NOD/SCID mice. Cell numbers for injection
EGFP-positive cells
EGFP-negative cells
3 104 1 105 3 105
1/3 7/9* 2/2
0/3 1/6* 0/3
The table shows the tumor incidence in RBE cell-injected mice. The left row indicates the cell numbers of transplantation per mouse. The numerators indicate the number of mice in which tumors were detected, and the denominators indicate the number of mice that received the xenotransplantation. * P < 0.05 by Fisher’s exact test.
Fig. 4. The Notch signaling pathway plays an important role in maintaining the undifferentiated state of the AFP-producing cells. (A) An RT-PCR analysis of the RNA samples extracted from the freshly isolated EGFP-positive and EGFP-negative cells. Dll1, Delta-like 1; Jag1, Jagged 1 and RT indicates a negative control. The immunostaining analyses for NICD1 of the isolated EGFP-positive (B) and EGFP-negative cells (C). The red fluorescence indicates NICD1 and the blue fluorescence does DAPI. Original magnifications: (B, C), 400. (D) Flow cytometric plots of the cultured EGFP-positive cells in the presence of DAPT (left panel) and the control group (right panel). The EGFP-positive cells that were cultured in the presence of 0.1% DMSO instead of DAPT were used as the control group. The longitudinal axes indicate the blank fluorescence and the horizontal ones indicate the intensity of EGFP. (E) The proportion of EGFPpositive cells decreased in the DAPT added group with statistical significance. The results are presented as the means ± SD. P = 0.001 by Student’s t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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the AFP-producing cells were cancer-initiating cells both in vitro and in vivo. Taken together with the findings that the AFP-producing cells were cancer progenitor cells and cancer-initiating cells, they were strongly suggested to be cancer stem cells. The Notch signaling pathway affects cell fates during development and regeneration by controlling cell survival, cell differentiation and the self-renewal activity of multipotent cells. Furthermore, its dysregulation results in
malignancies in some tissues [17]. The Notch signaling pathway also plays an important role in the maintenance of hepatic progenitor cells [18], the differentiation into cholangiocytes [19] and the development of CC [20]. The mRNA of Notch1 was specifically expressed in the AFP-producing cells. The immunostaining assay revealed that NICD1 was observed only in the EGFP-positive cell fraction, thus suggesting the Notch signaling pathway to be activated only in the EGFP–AFP-positive cells. The inhibition
Fig. 5. The immunohistological analyses of human ICC tissues. The figures A–E were obtained from different patients. The degree of AFP staining was classified based on the frequency of the AFP-positive cells: (A) no staining (), (B, C) focal staining (<5% of the tumor cells were positive for AFP; +), (D) moderate staining (<30%; ++) and (E) diffuse staining (P30%; +++). The left row indicates H&E staining of tumor tissue; the center two rows do immunostaining for AFP. The right row shows immunostaining for AFP, NICD1 and DAPI. The green fluorescent shows AFP, the red fluorescence indicates NICD1 and the blue fluorescence does DAPI. Arrowheads indicate the cells that express both AFP and NICD1. Original magnifications: the left two rows, 100; the right two rows, 400. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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of the Notch pathway using DAPT resulted in a decreased proportion of the sorted EGFP-positive cells. Taken together, the Notch signaling inhibition was therefore suggested to decrease the stem-like characteristics of the EGFP-positive cells. No other specific gene expression for the EGFP-positive cells was observed regarding the genes associated with CSCs, including Oct-3/4, the polycomb group gene Bmi1, the sonic hedgehog-signaling pathway, and the Wnt/catenin pathway (Supplementary Fig. S2). The serum AFP is not usually detected in patients with ICC. Therefore, five CC cell lines were examined to clarify whether they expressed AFP. RT-PCR revealed mRNA of AFP to be observed in two of these cell lines, namely OZ and RBE. However, no EGFP fluorescence was detected in the OZ cells that were transfected with the AFP enhancer/promoter-driven EGFP gene. This was probably because of the extremely weak expression of AFP in OZ, which was shown by immunostaining assay (data not shown). Therefore, in this case, the detection of EGFP fluorescence might display a lower sensitivity than the detection of the AFP mRNA by RT-PCR. On the other hand, nine of the 20 patients with ICC expressed AFP in the clinical samples. Among most cases, the AFP-producing cells were a minor population among the tumor cells. Therefore, this might explain the fact that the serum AFP values did not correlate with the degree of tumor tissue AFP immunoreactivity. Furthermore, AFP–NICD1-coexpressing cells were observed in seven of the nine patients with the AFP-positive tumor cells. These double positive cells existed in an extremely small number of the entire tumor cells in all seven tumors. These findings indicated that the AFP-producing cells existed and the Notch signaling pathway was activated not only in a CC cell line but also in clinical samples, thus supporting the hypothesis that the AFP-producing cells were CSCs in ICC. The fact that not all CC cell lines and ICC patients expressed AFP suggested that either AFPproducing CSCs are present in a minority of ICCs, or that the majority of the AFP-negative tumor samples and cell lines do not have stem-like components. Further study would therefore be required to elucidate the relationship between the AFP-producing cells and CSCs, because this study utilized only one transgenic cell line as an in vitro model of CSCs in CC. In the present study, the identification of the AFP-producing cells required genetic manipulation. Therefore, specific surface markers were needed in order to isolate viable CSCs from clinical samples. Recent studies demonstrated the CSCs in HCC to be identified using several cell-surface antigens including CD90 [21], or as a side population [4]. In particular, CD133-positive cells have been assumed to be CSCs in HCC cell lines and clinical samples [16,22]. Additionally, CD133 has been reported to be a CSC-marker for brain tumors [2] and colon cancer [23,24]. CD34 has also been referred to as a leukemia initiating cell marker [1] and CD44 as a CSC-marker for breast cancer [3], prostate cancer [25] and pancreatic cancer [26]. Regarding mouse hepatic development, the hepatic progenitor cells were characterized as CD49f+/lowCD45CD117 (c-kit) cells [27]. Oval cells, which are considered to be hepatic stem/ progenitor cells in adult livers, express CD34, CD90 and CD117 [28,29]. In this study, however, these surface anti-
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gens previously used for CSCs and hepatic stem/progenitor cells could not be applied to the selection of the AFP-producing RBE cells as CSCs (Supplementary Fig. S3). In conclusion, AFP-producing cells were demonstrated to be cancer progenitor cells and cancer-initiating cells. These findings strongly supported the hypothesis that AFP-producing cells in CC are CSCs. Furthermore, the activation of Notch was suggested to play an essential role in maintaining the stem-like characteristics of the AFP-producing cells. The present study may thus bring new insights into CSCs in ICC, while also opening the possibility of developing novel therapeutic approaches targeting CSCs in ICC treatment. 5. Conflict of interest The authors indicate no potential conflict of interest. Acknowledgements This work was supported in part by grants from the Scientific Research Fund of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2010. 01.019. References [1] D. Bonnet, J. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nature Medicine 3 (1997) 730–737. [2] S. Singh, I. Clarke, M. Terasaki, V. Bonn, C. Hawkins, J. Squire, P. Dirks, Identification of a cancer stem cell in human brain tumors, Cancer Research 63 (2003) 5821–5828. [3] M. Al-Hajj, M. Wicha, A. Benito-Hernandez, S. Morrison, M. Clarke, Prospective identification of tumorigenic breast cancer cells, Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 3983–3988. [4] T. Chiba, K. Kita, Y. Zheng, O. Yokosuka, H. Saisho, A. Iwama, H. Nakauchi, H. Taniguchi, Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties, Hepatology 44 (2006) 240–251. [5] E. Lagasse, Cancer stem cells with genetic instability: the best vehicle with the best engine for cancer, Gene Therapy 15 (2008) 136–142. [6] M. Dean, T. Fojo, S. Bates, Tumour stem cells and drug resistance, Nature Reviews Cancer 5 (2005) 275–284. [7] T. Phillips, W. McBride, F. Pajonk, The response of CD24(/low)/ CD44+ breast cancer-initiating cells to radiation, Journal of the National Cancer Institute 98 (2006) 1777–1785. [8] G. Gores, Cholangiocarcinoma: current concepts and insights, Hepatology 37 (2003) 961–969. [9] N. Fausto, Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells, Hepatology 39 (2004) 1477–1487. [10] B. Spear, Alpha-fetoprotein gene regulation: lessons from transgenic mice, Seminars in Cancer Biology 9 (1999) 109–116. [11] T. Ishii, K. Fukumitsu, K. Yasuchika, K. Adachi, E. Kawase, H. Suemori, N. Nakatsuji, I. Ikai, S. Uemoto, Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells, American Journal of Physiology Gastrointestinal and Liver Physiology 295 (2008) G313–G321. [12] T. Ishii, K. Yasuchika, H. Fujii, T. Hoppo, S. Baba, M. Naito, T. Machimoto, N. Kamo, H. Suemori, N. Nakatsuji, I. Ikai, In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes, Experimental Cell Research 309 (2005) 68–77.
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[13] T. Ishii, K. Yasuchika, T. Machimoto, N. Kamo, J. Komori, S. Konishi, H. Suemori, N. Nakatsuji, M. Saito, K. Kohno, S. Uemoto, I. Ikai, Transplantation of embryonic stem cell-derived endodermal cells into mice with induced lethal liver damage, Stem Cells 25 (2007) 3252–3260. [14] M. Enjoji, H. Sakai, H. Nawata, K. Kajiyama, M. Tsuneyoshi, Sarcomatous and adenocarcinoma cell lines from the same nodule of cholangiocarcinoma, In Vitro Cellular and Developmental Biology – Animal 33 (1997) 681–683. [15] B. Nickoloff, B. Osborne, L. Miele, Notch signaling as a therapeutic target in cancer: a new approach to the development of cell fate modifying agents, Oncogene 22 (2003) 6598–6608. [16] S. Ma, K. Chan, L. Hu, T. Lee, J. Wo, I. Ng, B. Zheng, X. Guan, Identification and characterization of tumorigenic liver cancer stem/ progenitor cells, Gastroenterology 132 (2007) 2542–2556. [17] M. Jang, A. Zlobin, W. Kast, L. Miele, Notch signaling as a target in multimodality cancer therapy, Current Opinion in Molecular Therapeutics 2 (2000) 55–65. [18] S. Ochsner, H. Strick-Marchand, Q. Qiu, S. Venable, A. Dean, M. Wilde, M. Weiss, G. Darlington, Transcriptional profiling of bipotential embryonic liver cells to identify liver progenitor cell surface markers, Stem Cells 25 (2007) 2476–2487. [19] N. Tanimizu, A. Miyajima, Notch signaling controls hepatoblast differentiation by altering the expression of liver-enriched transcription factors, Journal of Cell Science 117 (2004) 3165–3174. [20] N. Ishimura, S. Bronk, G. Gores, Inducible nitric oxide synthase upregulates Notch-1 in mouse cholangiocytes: implications for carcinogenesis, Gastroenterology 128 (2005) 1354–1368. [21] Z. Yang, D. Ho, M. Ng, C. Lau, W. Yu, P. Ngai, P. Chu, C. Lam, R. Poon, S. Fan, Significance of CD90+ cancer stem cells in human liver cancer, Cancer Cell 13 (2008) 153–166.
[22] S. Yin, J. Li, C. Hu, X. Chen, M. Yao, M. Yan, G. Jiang, C. Ge, H. Xie, D. Wan, S. Yang, S. Zheng, J. Gu, CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity, International Journal of Cancer 120 (2007) 1444–1450. [23] C. O’Brien, A. Pollett, S. Gallinger, J. Dick, A human colon cancer cell capable of initiating tumour growth in immunodeficient mice, Nature 445 (2007) 106–110. [24] L. Ricci-Vitiani, D. Lombardi, E. Pilozzi, M. Biffoni, M. Todaro, C. Peschle, R. De Maria, Identification and expansion of human coloncancer-initiating cells, Nature 445 (2007) 111–115. [25] A. Collins, P. Berry, C. Hyde, M. Stower, N. Maitland, Prospective identification of tumorigenic prostate cancer stem cells, Cancer Research 65 (2005) 10946–10951. [26] C. Li, D. Heidt, P. Dalerba, C. Burant, L. Zhang, V. Adsay, M. Wicha, M. Clarke, D. Simeone, Identification of pancreatic cancer stem cells, Cancer Research 67 (2007) 1030–1037. [27] T. Hoppo, H. Fujii, T. Hirose, K. Yasuchika, H. Azuma, S. Baba, M. Naito, T. Machimoto, I. Ikai, Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver, Hepatology 39 (2004) 1362–1370. [28] H. Crosby, D. Kelly, A. Strain, Human hepatic stem-like cells isolated using c-kit or CD34 can differentiate into biliary epithelium, Gastroenterology 120 (2001) 534–544. [29] B. Petersen, B. Grossbard, H. Hatch, L. Pi, J. Deng, E. Scott, Mouse A6positive hepatic oval cells also express several hematopoietic stem cell markers, Hepatology 37 (2003) 632–640.
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Alpha-fetoprotein producing cells act as cancer progenitor cells in human cholangiocarcinoma Takamichi Ishii a,b,*, Kentaro Yasuchika b, Hirofumi Suemori a, Norio Nakatsuji c,d, Iwao Ikai b,e, Shinji Uemoto b a
Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Japan Department of Surgery, Graduate School of Medicine Kyoto University, Japan c Institute for Integrated Cell-Material Science, Kyoto University, Japan d Department of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Japan e Department of Surgery, Kyoto Medical Center, National Hospital Organization, Japan b
a r t i c l e
i n f o
Article history: Received 9 October 2009 Received in revised form 12 January 2010 Accepted 18 January 2010
Keywords: Cancer stem cell Cholangiocarcinoma AFP Notch ICC
a b s t r a c t We aimed to demonstrate that alpha-fetoprotein (AFP)-producing cells in cholangiocarcinomas possessed cancer stem cell (CSC)-like properties. AFP enhancer/promoter-driven EGFP gene was transfected into human cholangiocarcinoma cell lines. One cell line, RBE, expressed both AFP and EGFP. Clonal analyses revealed that one EGFP-positive cell generated both EGFP-positive and EGFP-negative cell fractions. However, one EGFP-negative cell never produced EGFP-positive cells. The EGFP-positive cells had a greater tumorigenic potential. Only the EGFP-positive cells expressed Notch1. AFP and Notch1 expression was observed in clinical intrahepatic cholangiocarcinomas. The AFP-producing cells were suggested to be CSCs. The Notch pathway might play an important role in maintaining the CSC characteristics. Ó 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Recent developments in stem cell biology have revealed the existence of cancer stem cells (CSCs) in various tumors such as leukemia [1], brain tumors [2], breast cancer [3] and hepatocellular carcinoma (HCC) [4]. These CSCs are thought to display a hierarchy in tumor tissues and generate tumor heterogeneity. Although the definition of CSCs has not yet been unequivocally established, CSCs should have both a self-renewal activity and multipotency like normal tissue stem cells [5]. Furthermore, CSCs have common features of normal tissue stem cells, especially the resistance to multiple drugs [6] and radiation [7]. Therefore, CSCs are supposed to survive even after anti-cancer
* Corresponding author. Address: Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. Tel.: +81 75 751 3821; fax: +81 75 751 3890. E-mail address: [email protected] (T. Ishii). 0304-3835/$ - see front matter Ó 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2010.01.019
therapies and to generate therapy-refractory tumors. The elucidation of CSC biology might contribute not only to oncology in general, but also to the development of a new strategy for cancer therapy. Intrahepatic cholangiocarcinomas (ICCs) are the second most common cancer among the primary liver cancers, after HCC [8]. ICC has a poor prognosis and therefore, research regarding ICC is anticipated to improve the treatment outcomes in patients with ICC. During the normal hepatic development, hepatic stem/progenitor cells are thought to differentiate into hepatocytes and cholangiocytes [9]. Since alpha-fetoprotein (AFP) is one of the most common markers for hepatic progenitor cells in the fetal liver [10–12], AFP was focused on as a marker for CSCs in ICC. Whereas AFP is often used as a tumor marker for HCC, serum AFP is not usually observed in ICC patients. Therefore, if AFP-producing cells are present in ICCs, then they should have stem cell characteristics. In this study, a reporting vector that expressed enhanced green fluorescence protein (EGFP) under the
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control of human AFP enhancer/promoter was transfected into five human cholangiocarcinoma (CC) cell lines to characterize the AFP-producing cells as CSCs. The results showed that the Notch signaling pathway might play an important role in maintaining the self-renewal ability of the AFP-producing cells in the CC cell line. Moreover, the expression of AFP and Notch signaling protein in tumor cells was investigated utilizing surgically resected human ICC tumor specimens. 2. Materials and methods 2.1. Patients Twenty patients with ICC confirmed by pathologic analyses, who had undergone a hepatic resection at Kyoto University Hospital from June 2005 to December 2007, were included in this study. The patients included 12 men and 8 women, with a mean age at the surgery of 67.5 ± 7.9 years (range, 58–81 years). Written informed consent for the use of their resected tissues was obtained from all patients in accordance with the Declaration of Helsinki. The clinicopathological background is summarized in Supplementary Table S1.
the secondary antibodies were diluted at 1:500. The stained cells were covered with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). 2.4. Flow cytometry and cell sorting The cultured cells were prepared as described previously [11–13]. Dead cells were eliminated using 7-amino-actinomysin D (BD Biosciences, Franklin Lakes, NJ) staining. The cells were analyzed and isolated using a FACSVantage SE cell sorter (BD Biosciences). The isolated cells were cultured on plastic culture dishes in RPMI1640 supplemented with 10% FBS and penicillin/streptomycin in the presence or absence of N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT, Sigma) dissolved in dimethyl sulfoxide (DMSO, Sigma). For the single-cell culture analyses, the individual isolated cells were sorted out into each well of 96-well culture plates using FACSVantage SE and the Clone-Cyt Plus software program (BD Biosciences). The wells containing only one cell were confirmed using a light microscope 10–16 h after cell sorting. 2.5. Cell proliferation assay, anchorage-independent cell growth assay, and sphere-forming assay
2.2. Cell cultures The human CC cell lines HuCCT-1 and TFK-1 were obtained from the Cell Resource Center for Biochemical Research, Tohoku University (Sendai, Japan); OZ was obtained from the Japan Health Science Foundation (Tokyo, Japan); and the SSP-25 and RBE cells were obtained from the RIKEN Bioresource Center (Tsukuba, Japan). These were cultured at 37 °C and in 5% CO2 in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Gibco). The transgene plasmid vectors that expressed EGFP under the control of human AFP enhancer/promoter were generated as described previously [11]. The transgenic vectors were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Stable transfected cells were selected in the presence of 100 lg/ml G418 (Sigma, St. Louis, MO) over a period of 30 days. The proper transgene insertion was confirmed by polymerase chain reactions. The stable transfectants were cultured in the presence of 100 lg/ml G418. 2.3. Immunohistochemistry of cultured cells The cultured cells were fixed and stained as previously described [11–13]. An anti-human cytokeratine 19 (CK19) mouse antibody (DakoCytomation, Glostrup, Denmark) diluted at 1:50, anti-human AFP rabbit antibody (DakoCytomation) diluted at 1:200 and 5 lg/ml anti-human Notch1 intracellular domain (NICD1) mouse antibody (R&D Systems, Minneapolis, MN) were used as the first antibodies. Alexa 555-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) for CK19 and NICD1 staining and Alexa 546-conjugated donkey anti-rabbit IgG (Molecular Probes) for AFP staining were used as the secondary antibodies. All
The isolated EGFP-positive and EGFP-negative cells were inoculated at a density of 1 103 per well, respectively, and then were allowed to grow for 8 days. The cell numbers were determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazorium, inner salt (MTS) assay (Cell Titer 96 Aqueous One Solution Reagent, Promega, Madison, WI), according to the manufacturer’s protocol. After 1 h of incubation, the absorbance value was measured using a plate reader at 490 nm. To examine the anchorage-independent growth, 1 104 EGFP-positive and EGFP-negative cells were suspended in 2.0 ml of 0.3% agar (Wako, Osaka, Japan) supplemented with the culture medium. The cell suspension was layered over the bottom layer of 2.0 ml of 0.6% agar. The colonies that contained more than 10 cells were counted 21 days after cell sorting. To investigate the ability to form cell spheres, the sorted EGFP-positive and EGFP-negative cells were seeded at a density of 1 105 cells/well in 6-well ultra low attachment plates (Corning Inc., Corning, NY) in serum-free PRMI1640 media. The spheres were observed 5 days after cell sorting. These assays were repeated for three times. 2.6. Reverse-transcription polymerase chain reaction (RTPCR) RT-PCR was performed as described previously [11]. Primers were generated for the following human genes: EGFP, AFP, GATA4, Foxa2, albumin, Notch1, Notch2, Notch3, Notch4, Delta-like 1 (Dll1), Jagged 1 (Jag1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Their sequences and PCR conditions are summarized in Supplementary Table S2.
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2.7. Cell transplantation procedures The sorted cells were transplanted into 6- to 8-weekold male non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice (CLEA Japan, Inc., Tokyo, Japan). The isolated EGFP-positive and EGFP-negative cells, ranging from 3 104 to 3 105 cells, were suspended in 200 ll Hank’s balanced salt solutions (Gibco). Under general anesthesia, they were engrafted intraperitoneally by means of splenic injection using a 29-gauge needle (BD Biosciences). The implanted mice were killed 10 weeks after the transplantation. All experimental procedures utilizing animals were performed in accordance with the Animal Protection Guidelines of Kyoto University. 2.8. Histology and immunohistology of tumor tissues The harvested tumors were fixed in 3.3% formaldehyde (Wako) for at least 1 week. After a thorough rinsing, the tissue was then embedded in paraffin and then sectioned into 3 lm thick sections. Hematoxylin–eosin (HE) staining was performed according to a standard protocol. Prior to immunohistology, the sections were incubated in HistoVT One (Nacalai Tesque, Kyoto, Japan) for 20 min at 90 °C for antigen retrieval. Immunofluorescent staining for AFP and NICD1 was performed as described above. In order to perform immunohistological staining for AFP, the deparaffinized and rehydrated sections were incubated in 3% hydrogen peroxide in methanol for 10 min and then were incubated with the diluted anti-human AFP antibodies at 4 °C for 16 h. Followed by thorough rinsing, they were incubated for 30 min with horseradish peroxidase-conjugated anti-rabbit IgG (Envision + Kit/horseradish peroxidase; DakoCytomation) and then for 1 min with 3,30 Diaminobenzidine (DAB) substrates (DakoCytomation). Counterstaining was performed using hematoxylin. To semi-quantitate the immunostaining results, the slides were independently scored by two evaluators (T.I. and K.Y.) for AFP staining. Visual assessment based on the frequency of immunoreactivity was classified into four categories: no staining (), focal staining (<5% of tumor cells were positive for AFP; +), moderate staining (<30%; ++) and diffuse staining (P30%; +++). 2.9. Statistical analysis All in vitro samples were collected from three to six independent trials. The results are presented as the means ± SD. The statistical analyses between two groups were performed using Student’s t-test. The tumorigenicity between the EGFP-positive and the EGFP-negative cell transplanted group was analyzed using Fisher’s exact test. A P value of <0.05 was considered to be statistically significant. All statistical tests were two-sided. 3. Results 3.1. AFP-producing cells in the RBE cell line RT-PCR revealed that AFP expression was observed in RBE and that weak expression was also detected in OZ (Fig. 1A). An immunostaining assay confirmed that all RBE cells were positive for CK19, a marker for
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cholangiocytes and that approximately 20% of the RBE cells were positive for AFP (Fig. 1B). The AFP–EGFP reporter vector was transfected into the five CC cell lines. Only the RBE cells expressed the EGFP fluorescence; the other four cell lines did not, despite the fact that they possessed the transgene in the genomic DNA (Fig. 1B and data not shown). The RBE cell line is derived from intrahepatic cholangiocarcinoma with moderately differentiated tubular adenocarcinoma [14]. A flow cytometric analysis showed that 23.6 ± 3.9% (n = 3) of the RBE cells expressed the EGFP fluorescence (Fig. 1C). There was no obvious difference in the morphology between the sorted EGFP-positive and EGFP-negative RBE cells (Fig. 1D). The sorted EGFP-positive cells expressed both EGFP and AFP, whereas the sorted EGFP-negative cells did not express either mRNA (Fig. 1E), thus indicating the EGFP-positive cells to correspond to the AFP-producing cells and that cell sorting could divide the RBE cells according to their AFP production. Therefore, a further investigation of the AFP-producing cells in RBE was conducted to determine whether or not they were cancer stem cells.
3.2. Single cell sort analyses of the AFP-producing and AFP-non-producing cells Since the RBE cells had a heterogeneous population, single-cell culture assays were performed in order to examine their self-renewal activity and multipotency. A single cell was sorted and cultured on 96-well culture plates. Following cell expansion by four passages, flow cytometric analyses were performed. A single EGFP-positive cell generated both the EGFP-positive and EGFP-negative cell fractions (Fig. 2A, B). One EGFP-negative cell, however, produced only the EGFP-negative fraction (Fig. 2C, D). These flow cytometric findings were reproducible in three trials (five EGFP-positive clones and three EGFP-negative clones), and the transgene was identified in the genomic DNA of all these clones (Supplementary Fig. S1). These results indicated that the EGFP-positive cells thus had a self-renewal activity and the ability to ‘differentiate’ into the EGFP-negative cells. They also suggested the progenitor-like characteristics of the AFP-producing cells.
3.3. Tumorigenic potential of the AFP-producing cells in vitro and in vivo To compare the tumorigenic abilities in vitro between the EGFP-positive and EGFP-negative cells, both cell proliferation and the anchorageindependent cell growth assays were performed. The EGFP-positive cells exhibited a higher proliferation activity than did the EGFP-negative cells, with a significant difference (P < 0.01, n = 3; Fig. 3A). Moreover, the EGFPpositive cells showed an approximately 4.5-fold higher ability to form colonies in soft agar than the EGFP-negative cells did (P < 0.001, n = 6; Fig. 3B). In addition, the sphere assays revealed that the EGFP-positive cells formed cell spheres 5 days after cell sorting, whereas the EGFP-negative cells did not (Fig. 3C). These findings suggested the EGFP-positive cells to therefore have stronger tumorigenic and malignant potentials in vitro than the EGFP-negative cells did. To examine the tumorigenicity in vivo, the EGFP-positive or EGFPnegative cells were transplanted into the abdominal cavities of NOD/SCID mice via their spleens. The transplantation of 3 104 EGFP-positive cells resulted in the development of tumors in one of three mice, 1 105 EGFPpositive cells resulted in tumors in seven of nine mice and 3 105 EGFPpositive cells did in two of two mice (Table 1). These tumors included one liver tumor (Fig. 3D; left panel), two cases of splenic tumors (Fig. 3D; right panel) and seven cases of peritoneal disseminated nodules. H&E staining revealed the disseminated nodules to consist of tumor cells with tube-like formations, occasional atypical multinucleated giant cells and stromal rich components (Fig. 3E). Immunostaining of the peritoneal dissemination foci showed that all of these tumor cells were positive for human CK19 (Fig. 3F; left panel) and that approximately 20% of the tumor cells expressed AFP (Fig. 3F; right panel). Some transplanted EGFP-positive AFP-producing cells generated the AFP-non-producing cells, thus resulting in the original proportion of AFP-producing cells. On the other hand, the engraftment of the 1 105 EGFP-negative cells resulted in only one tumor in six mice. The transplantation of the 3 104 or 3 105 EGFPnegative cells did not form any tumor in the NOD/SCID mice. These results indicated the EGFP-positive cells to have an increased tumorigenicity in vivo, while also demonstrating the characteristics of cancerinitiating cells.
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Fig. 1. The human cholangiocarcinoma cell lines that expressed EGFP under the control of the human AFP enhancer/promoter. (A) An RT-PCR analysis of the five CC cell lines. (B) Immunocytological assay for RBE. The RBE cells expressed AFP (red fluorescence) with the proportion of approximately 20% (left upper panel) and all of them expressed CK19 (red; right upper panel). Blue fluorescence indicates DAPI. The stable transfectants of RBE cells expressed EGFP florescence (left lower panel). The figure of the right lower panel showed the phase contrast image of the same visual field of the left lower panel. (C) A flow cytometric analysis revealed the RBE cells to express EGFP in the proportion of 23.6 ± 3.9%. The longitudinal axis indicates the blank fluorescence and the horizontal one does the intensity of EGFP. (D) The sorted EGFP-positive cells (left panel) were morphologically similar to the EGFP-negative cells (right panel). The small inserts showed the fluorescent microscopy images. Green fluorescence indicates EGFP. (E) The sorted out EGFP-positive cells expressed mRNA both of EGFP and of AFP (left lane), whereas the sorted EGFP-negative cells expressed neither EGFP nor AFP mRNA (right lane). GAPDH was used as an internal control and RT served as a negative control. Original magnifications: (B, D) 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. The involvement of Notch signaling pathway in CSC maintenance In order to investigate the difference between CSCs and non-CSCs, the expressions of previously reported genes related with cancer or hepatic stem cells were compared between the EGFP-positive and EGFP-negative cells and RT-PCR was performed to compare the genetic expression between the two groups (Fig. 4A). There was no difference in the endoderm-related genes except for AFP (Fig. 1D). However, mRNA of Notch1 was expressed specifically by the EGFPpositive cells. The expression of neither the other three Notch receptors nor the Notch ligands showed any difference between the two cell groups. To examine the effect of the Notch signaling pathway on CSCs, immunostaining for NICD1 was performed. The NICD1 was found in both the cytoplasm and nuclei of the isolated EGFP-positive cells (Fig. 4B), whereas the EGFP-negative cells did not express NICD1 (Fig. 4C), thus indicating the Notch signaling to be activated only in the EGFP-positive cell fraction. In addition, the purified EGFP-positive cells were cultured in the presence of DAPT. DAPT acts as a c-secretase inhibitor and prevents the Notch intercellular domain from releasing from the cell membrane, thereby blocking the Notch signaling pathway [15]. After a 7 day culture, a portion of the EGFP-positive cells in the control group ceased to express EGFP spontaneously and showed 78.9 ± 1.4% of the EGFP-positive proportion (Fig. 4D). However, the EGFP-positive cells that were cultured in the presence of 50 lM DAPT showed a significant decrease in the proportion of EGFP-positive cells with 63.4 ± 1.0% (P = 0.001, n = 6; Fig. 4D, E). The inhibition of the Notch signaling pathway thus caused a decrease of the EGFP-
positive cells. These results suggested the Notch signaling pathway to possibly play a crucial role in the maintenance of the stemness of the EGFP-positive AFP-producing cells. 3.5. The expression of AFP and Notch1 in the patients with ICC To examine the AFP and NICD1 expression in human clinical samples of ICC, immunohistological assays were performed utilizing 20 surgical resected ICC tumors (Supplementary Table S3). No AFP-positive cell was observed in 11 patients (Fig. 5A). Six of the twenty patients with ICC exhibited a focal staining pattern for AFP (Fig. 5B, C). A small portion of tumor cells (less than 5%) was positive for AFP. Two tumors displayed a moderate AFP staining pattern, meaning that 5–30% of tumor cells expressed AFP (Fig 5D). A diffuse staining pattern was observed in one case (Fig. 5E). The preoperative serum AFP values did not correlate with the AFP staining degree in the tumor tissue specimens. The tumor cells that co-expressed AFP and NICD1 were found in seven of the nine patients with the AFP-producing ICC cells. In all seven cases, however, they only made up an extremely small proportion of the tumor tissues, namely comprising less than 1% of all tumor cells. No AFP-negative cell expressed NICD1.
4. Discussion Intrahepatic cholangiocarcinoma is one of the carcinomas with the poorest prognosis and it is also the second
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Fig. 2. Single cell sort analyses of the AFP-producing EGFP-positive cells and AFP-non-producing EGFP-negative cells. (A) The individual EGFP-positive cells were sorted into each well of 96-well culture plates. The single cell indicated by the bold dot generated both the EGFP-positive and EGFP-negative cell fractions (B). On the other hand, the single EGFP-negative cell indicated by the bold dot in (C) produced only the EGFP-negative cell fraction (D).
most common primary hepatic carcinoma, following HCC [8]. Cancer stem cell biology concerning CC has not been investigated until now, although several articles have reported the existence of CSCs in its counterpart, HCC [4,16]. Both hepatocytes and cholangiocytes are descendants of hepatic stem/progenitor cells in the normal process of hepatic development [9]. CSCs show similarities with their normal tissue stem cells. The present study focused on AFP as a CSC-marker because AFP is one of the most common markers for fetal hepatoblasts or hepatic stem/progenitor cells. CSCs should have at least three characteristics; a selfrenewal ability, multipotency and tumorigenicity [5]. The
single-cell culture analyses showed that only the AFP– EGFP-positive cell fraction had self-renewal ability and differentiation potency. One AFP–EGFP-positive cell had the ability to generate not only the AFP–EGFP-positive cells but also the AFP–EGFP-negative cells. These findings indicated that the AFP-producing cells showed functional similarities to lineage-committed progenitor cells, thus indicating the AFP-producing cells to indeed be cancer progenitor cells. On the other hand, one EGFP-negative cell generated the only EGFP-negative fraction, thus indicating it to only have a self-renewal activity. This also suggested the EGFP-negative cells to have decreased stem-like characteristics. In this study, however, it was impossible to
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Fig. 3. Tumorigenic potential of the AFP-producing cells in vitro and in vivo. (A) The MTS assays between the two cell populations were tested using repeated-measures ANOVA. P < 0.01. (B) The graph indicates the colony numbers of the EGFP-positive and EGFP-negative cells in soft agar. P < 0.001 (Student’s t-test). The results are presented as the means ± SD. (C) The EGFP-positive cells formed cell spheres in suspension cultures (left panel), whereas the EGFP-negative cells did not (right panel). The small insert showed the fluorescent microscopy image. Green fluorescence indicates EGFP. Tumorigenicity in vivo was shown by xenotransplantation into NOD/SCID mice. One liver tumor was detected in one mouse (D; left panel) and splenic tumors were detected in two mice (D; right panel) in the EGFP-positive cell engrafted group. The arrowheads indicate the tumors. Peritoneal disseminations were found in seven mice of the EGFP-positive cell engrafted group. Figure E shows the H&E staining of the disseminated nodules. Figure F indicate the merged immunostaining assay for human CK19 (left panel; red fluorescence) and AFP (right panel; red fluorescence). Blue fluorescence indicates DAPI. Original magnifications: (C), 100; (D), 10; (E, F), 200. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
demonstrate both fractions of these cells to differentiate into a mature cholangiocyte lineage. Furthermore, the higher in vitro malignant potency of the AFP–EGFP-positive cells was observed in the MTS assay, anchorage-indepen-
dent growth assay, and sphere-forming assay. Xenotransplantation experiments showed that the AFP-producing cells had a markedly greater tumorigenicity than the AFP-non-producing cells did. These results indicated that
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T. Ishii et al. / Cancer Letters 294 (2010) 25–34 Table 1 The tumorigenicity of the isolated EGFP-positive and EGFP-negative cells in NOD/SCID mice. Cell numbers for injection
EGFP-positive cells
EGFP-negative cells
3 104 1 105 3 105
1/3 7/9* 2/2
0/3 1/6* 0/3
The table shows the tumor incidence in RBE cell-injected mice. The left row indicates the cell numbers of transplantation per mouse. The numerators indicate the number of mice in which tumors were detected, and the denominators indicate the number of mice that received the xenotransplantation. * P < 0.05 by Fisher’s exact test.
Fig. 4. The Notch signaling pathway plays an important role in maintaining the undifferentiated state of the AFP-producing cells. (A) An RT-PCR analysis of the RNA samples extracted from the freshly isolated EGFP-positive and EGFP-negative cells. Dll1, Delta-like 1; Jag1, Jagged 1 and RT indicates a negative control. The immunostaining analyses for NICD1 of the isolated EGFP-positive (B) and EGFP-negative cells (C). The red fluorescence indicates NICD1 and the blue fluorescence does DAPI. Original magnifications: (B, C), 400. (D) Flow cytometric plots of the cultured EGFP-positive cells in the presence of DAPT (left panel) and the control group (right panel). The EGFP-positive cells that were cultured in the presence of 0.1% DMSO instead of DAPT were used as the control group. The longitudinal axes indicate the blank fluorescence and the horizontal ones indicate the intensity of EGFP. (E) The proportion of EGFPpositive cells decreased in the DAPT added group with statistical significance. The results are presented as the means ± SD. P = 0.001 by Student’s t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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the AFP-producing cells were cancer-initiating cells both in vitro and in vivo. Taken together with the findings that the AFP-producing cells were cancer progenitor cells and cancer-initiating cells, they were strongly suggested to be cancer stem cells. The Notch signaling pathway affects cell fates during development and regeneration by controlling cell survival, cell differentiation and the self-renewal activity of multipotent cells. Furthermore, its dysregulation results in
malignancies in some tissues [17]. The Notch signaling pathway also plays an important role in the maintenance of hepatic progenitor cells [18], the differentiation into cholangiocytes [19] and the development of CC [20]. The mRNA of Notch1 was specifically expressed in the AFP-producing cells. The immunostaining assay revealed that NICD1 was observed only in the EGFP-positive cell fraction, thus suggesting the Notch signaling pathway to be activated only in the EGFP–AFP-positive cells. The inhibition
Fig. 5. The immunohistological analyses of human ICC tissues. The figures A–E were obtained from different patients. The degree of AFP staining was classified based on the frequency of the AFP-positive cells: (A) no staining (), (B, C) focal staining (<5% of the tumor cells were positive for AFP; +), (D) moderate staining (<30%; ++) and (E) diffuse staining (P30%; +++). The left row indicates H&E staining of tumor tissue; the center two rows do immunostaining for AFP. The right row shows immunostaining for AFP, NICD1 and DAPI. The green fluorescent shows AFP, the red fluorescence indicates NICD1 and the blue fluorescence does DAPI. Arrowheads indicate the cells that express both AFP and NICD1. Original magnifications: the left two rows, 100; the right two rows, 400. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
T. Ishii et al. / Cancer Letters 294 (2010) 25–34
of the Notch pathway using DAPT resulted in a decreased proportion of the sorted EGFP-positive cells. Taken together, the Notch signaling inhibition was therefore suggested to decrease the stem-like characteristics of the EGFP-positive cells. No other specific gene expression for the EGFP-positive cells was observed regarding the genes associated with CSCs, including Oct-3/4, the polycomb group gene Bmi1, the sonic hedgehog-signaling pathway, and the Wnt/catenin pathway (Supplementary Fig. S2). The serum AFP is not usually detected in patients with ICC. Therefore, five CC cell lines were examined to clarify whether they expressed AFP. RT-PCR revealed mRNA of AFP to be observed in two of these cell lines, namely OZ and RBE. However, no EGFP fluorescence was detected in the OZ cells that were transfected with the AFP enhancer/promoter-driven EGFP gene. This was probably because of the extremely weak expression of AFP in OZ, which was shown by immunostaining assay (data not shown). Therefore, in this case, the detection of EGFP fluorescence might display a lower sensitivity than the detection of the AFP mRNA by RT-PCR. On the other hand, nine of the 20 patients with ICC expressed AFP in the clinical samples. Among most cases, the AFP-producing cells were a minor population among the tumor cells. Therefore, this might explain the fact that the serum AFP values did not correlate with the degree of tumor tissue AFP immunoreactivity. Furthermore, AFP–NICD1-coexpressing cells were observed in seven of the nine patients with the AFP-positive tumor cells. These double positive cells existed in an extremely small number of the entire tumor cells in all seven tumors. These findings indicated that the AFP-producing cells existed and the Notch signaling pathway was activated not only in a CC cell line but also in clinical samples, thus supporting the hypothesis that the AFP-producing cells were CSCs in ICC. The fact that not all CC cell lines and ICC patients expressed AFP suggested that either AFPproducing CSCs are present in a minority of ICCs, or that the majority of the AFP-negative tumor samples and cell lines do not have stem-like components. Further study would therefore be required to elucidate the relationship between the AFP-producing cells and CSCs, because this study utilized only one transgenic cell line as an in vitro model of CSCs in CC. In the present study, the identification of the AFP-producing cells required genetic manipulation. Therefore, specific surface markers were needed in order to isolate viable CSCs from clinical samples. Recent studies demonstrated the CSCs in HCC to be identified using several cell-surface antigens including CD90 [21], or as a side population [4]. In particular, CD133-positive cells have been assumed to be CSCs in HCC cell lines and clinical samples [16,22]. Additionally, CD133 has been reported to be a CSC-marker for brain tumors [2] and colon cancer [23,24]. CD34 has also been referred to as a leukemia initiating cell marker [1] and CD44 as a CSC-marker for breast cancer [3], prostate cancer [25] and pancreatic cancer [26]. Regarding mouse hepatic development, the hepatic progenitor cells were characterized as CD49f+/lowCD45CD117 (c-kit) cells [27]. Oval cells, which are considered to be hepatic stem/ progenitor cells in adult livers, express CD34, CD90 and CD117 [28,29]. In this study, however, these surface anti-
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gens previously used for CSCs and hepatic stem/progenitor cells could not be applied to the selection of the AFP-producing RBE cells as CSCs (Supplementary Fig. S3). In conclusion, AFP-producing cells were demonstrated to be cancer progenitor cells and cancer-initiating cells. These findings strongly supported the hypothesis that AFP-producing cells in CC are CSCs. Furthermore, the activation of Notch was suggested to play an essential role in maintaining the stem-like characteristics of the AFP-producing cells. The present study may thus bring new insights into CSCs in ICC, while also opening the possibility of developing novel therapeutic approaches targeting CSCs in ICC treatment. 5. Conflict of interest The authors indicate no potential conflict of interest. Acknowledgements This work was supported in part by grants from the Scientific Research Fund of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2010. 01.019. References [1] D. Bonnet, J. Dick, Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell, Nature Medicine 3 (1997) 730–737. [2] S. Singh, I. Clarke, M. Terasaki, V. Bonn, C. Hawkins, J. Squire, P. Dirks, Identification of a cancer stem cell in human brain tumors, Cancer Research 63 (2003) 5821–5828. [3] M. Al-Hajj, M. Wicha, A. Benito-Hernandez, S. Morrison, M. Clarke, Prospective identification of tumorigenic breast cancer cells, Proceedings of the National Academy of Sciences of the United States of America 100 (2003) 3983–3988. [4] T. Chiba, K. Kita, Y. Zheng, O. Yokosuka, H. Saisho, A. Iwama, H. Nakauchi, H. Taniguchi, Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties, Hepatology 44 (2006) 240–251. [5] E. Lagasse, Cancer stem cells with genetic instability: the best vehicle with the best engine for cancer, Gene Therapy 15 (2008) 136–142. [6] M. Dean, T. Fojo, S. Bates, Tumour stem cells and drug resistance, Nature Reviews Cancer 5 (2005) 275–284. [7] T. Phillips, W. McBride, F. Pajonk, The response of CD24(/low)/ CD44+ breast cancer-initiating cells to radiation, Journal of the National Cancer Institute 98 (2006) 1777–1785. [8] G. Gores, Cholangiocarcinoma: current concepts and insights, Hepatology 37 (2003) 961–969. [9] N. Fausto, Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells, Hepatology 39 (2004) 1477–1487. [10] B. Spear, Alpha-fetoprotein gene regulation: lessons from transgenic mice, Seminars in Cancer Biology 9 (1999) 109–116. [11] T. Ishii, K. Fukumitsu, K. Yasuchika, K. Adachi, E. Kawase, H. Suemori, N. Nakatsuji, I. Ikai, S. Uemoto, Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells, American Journal of Physiology Gastrointestinal and Liver Physiology 295 (2008) G313–G321. [12] T. Ishii, K. Yasuchika, H. Fujii, T. Hoppo, S. Baba, M. Naito, T. Machimoto, N. Kamo, H. Suemori, N. Nakatsuji, I. Ikai, In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes, Experimental Cell Research 309 (2005) 68–77.
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