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Carcinogenesis, Vol. 20, No. 12, 2335-2340, December 1999
© 1999 Oxford University Press

Short Communications

Establishment of a novel rat cholangiocarcinoma cell culture model

Guan-Hua Lai and Alphonse E. Sirica1

Department of Pathology, Medical College of Virginia Campus of Virginia Commonwealth University, PO Box 980297, Richmond, VA 23298-297, USA


   Abstract
Top
 Abstract
Introduction
 References
 
Furan cholangiocarcinogenesis in rat liver is proving to be a unique and useful animal model for investigating important aspects of the cellular and molecular pathogenesis of cholangiocarcinoma potentially relevant to the human disease. We now describe the first culture model of rat cholangiocarcinoma cells derived from a transplantable cholangiocarcinoma originally induced in the liver of a furan-treated rat. An epithelial cell isolate highly enriched in viable cholangiocarcinoma cells was consistently obtained from transplantable cholangiocarcinoma tissue utilizing a similar procedure to that recently developed by us to establish a new rat hyperplastic bile ductular epithelial cell culture model characterized by the appearance of polarized bile ducts in vitro. Primary cholangiocarcinoma cell cultures could be readily established with these isolated cells and, in addition, we established from one such culture a novel rat cholangiocarcinoma cell line designated C611B. Cultured C611B cholangiocarcinoma cells retained a number of important characteristic features of the carcinoma cells of the parent tumor, including marked expression of the tyrosine kinase growth factor receptor proteins c-Met and c-Neu. Under basal culture conditions, the C611B cell line exhibited a cell doubling time of ~24 h and was aneuploid, with a predominant chromosomal count of 43. Moreover, C611B cells on collagen gels were 100% tumorigenic when transplanted into inguinal fat pads of syngeneic rats. All tumors formed at the transplantation site were cytokeratin 19-positive, mucin-producing tubular adenocarcinomas whose histological and phenotypic features closely resembled those of the furan-induced parent transplantable rat cholangiocarcinoma. Based on our findings, we believe that this novel rat cholangiocarcinoma cell culture model can serve as a valuable resource for investigating aberrant growth properties and tumor progression in biliary cancer.

Abbreviations: ChC, cholangiocarcinoma; HBDE, hyperplastic bile ductular epithelial cell; DMEM, Dulbecco's modified Eagle's medium.


   Introduction
Top
 Abstract
 Introduction
References
 
Cholangiocarcinoma (ChC) is a primary liver cancer characterized by high morbidity and mortality and for which there is no effective treatment (1,2). The development of novel cell culture models of ChC could provide important research tools with which to investigate relevant cellular alterations that may be pertinent to the human disease. In addition, such models can serve as the basis for the preclinical testing of new therapeutic strategies. However, while a few human ChC cell lines now exist (3–9), in general their availability has been limited, they have been subjected to long-term passage in vitro and have been shown to be markedly aneuploid (3–7). With respect to experimental animal ChC culture models, to our knowledge there has been only one published report to date describing the establishment of cell lines from liver fluke-associated ChC induced in a hamster model (10) and no reports on the development of rat ChC culture models.

During the past several years, our laboratory has focused on elucidating cellular and molecular alterations associated with the development of ChC in furan-treated rats (11–16). The tumors induced in this unique animal model of chemical cholangiocarcinogenesis closely resemble in their morphology and phenotypic features mucin-producing tubular ChC of human liver (11,12,14).

Most recently, we have demonstrated co-overexpression of the tyrosine kinase growth factor receptors c-Met and c-Neu to be a prominent feature of the neoplastic epithelium of furan-induced primary and derived transplantable rat ChC (13,16). Likewise, c-Met (17) and c-Neu (18,19) have each been reported to be overexpressed in the neoplastic epithelial cells of a significant percentage of human ChC examined.

The aim of the research described in the current paper was to establish for the first time a rat ChC cell culture model derived from a furan-induced transplantable tumor that retained important phenotypic properties of the parent tumor in vitro, including prominent expression of c-Met and c-Neu. In this regard, we have adapted a cell isolation procedure recently developed by us to isolate hyperplastic bile ductular epithelial cells (HBDE) from selected liver lobes with massive bile ductular hyperplasia (20) to isolate from transplantable rat ChC a viable cell fraction highly enriched in tumorigenic cells suitable for cell culturing and in vivo cell transplantation. In addition to readily establishing these cells in primary cultures, we also describe the development and initial characterization of a novel rat ChC cell line, which we designated C611B.

Figure 1Go schematically depicts our procedure for routinely obtaining a viable ChC epithelial cell isolate from transplantable rat ChC originated in the liver of a furan-treated Fischer 344 male rat and serially propagated in the right inguinal fat pad of young adult Fischer 344 male rats (Harlan Sprague–Dawley, Indianapolis, IN) as previously described (11,13,16). The final ChC cell isolate was suspended in cell culture medium composed of Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 5 µg/ml transferrin, 0.1 µmol/l insulin, 100 U/ml penicillin and 100 µg/ml streptomycin (basal medium) plus 10% fetal bovine serum. Following a 30 min period of plating on plastic as a means of differentially removing contaminating fibroblasts, primary cultures were established by plating recovered viable ChC cells at a density of 4x106 cells in 4 ml of culture medium into 60 mm Biocoat collagen I-coated culture dishes (Becton Dickinson Labware, Bedford, MA) or onto type I rat tail collagen gels prepared as previously described (20,21). The C611B cell line was established from ChC epithelial cells initially isolated from a transplantable rat ChC at in vivo passage 12 by employing differential cell harvesting with 0.05% trypsin, 0.53 mM EDTA (Life Technologies) combined with subsequent serial propagation in vitro.



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  		Fig. 1. Schematic diagram of procedure for isolating and culturing malignant epithelial cells from a furan-induced subcutaneously transplanted rat ChC.

 
The mean ChC cell yield (± SD) obtained from 22 individual tumors was 3.05 ± 2.3x107, with a mean cell viability determined by trypan blue dye exclusion of 84.6 ± 8.3%. When plated for 4 h on type I rat tail collagen gel, freshly isolated rat ChC cells appeared under phase contrast in the form of attached single cells and small cell aggregates. However, by 4–7 days after initial cell plating, the cultured ChC cells typically formed into epithelial cell monolayers with obvious areas of cell piling up at the medium surface of the gels. Histological sections of the rat ChC cells in primary culture on collagen gel confirmed the cell piling up feature (Figure 2A and BGo), as well as revealing the presence of a few adenocarcinomatous-like structures, mostly formed within the collagen gel (Figure 2AGo), but also observed at the medium surface (Figure 2CGo). Phenotypically, the cultured rat ChC cells retained characteristic traits of their in vivo cell counterparts, including strong cytoplasmic immunoreactivity for biliary cytokeratin 19 (Figure 2BGo) and positive histochemical staining for mucin (Figure 2CGo). When transplanted into the inguinal fat pad (data not shown) or liver (Figure 2DGo) of recipient Fischer 344 rats, freshly isolated (n = 16) as well as primary rat ChC cells (n = 4) gave rise to a 100% incidence of mucin-producing adenocarcinoma whose morphological features were essentially identical to those of the parent tumor from which the tumorigenic ChC cells were isolated.



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  		Fig. 2. (A) Rat ChC cells in 4-day-old primary gel culture. Note that piling up of cells at the medium surface and an adenocarcinomatous glandular or tubular structure (arrowhead) formed inside the collagen gel substratum (hematoxylin and eosin, x33). (B) Rat ChC epithelial cells showing strongly positive cytoplasmic immunostaining for cytokeratin 19 in primary gel culture. Note that ChC epithelial cells are piled up at the medium surface of the gel (x132). (C) Positive histochemical staining for mucin (pink-red staining) shown by rat ChC epithelial cells in primary gel culture (mucicarmine, x132). (D) Positive histochemical staining for mucin in lumens of neoplastic glands within an adenocarcinoma formed in liver of a recipient rat after intrasplenic cell transplantation of 2x106 ChC cells freshly isolated from a furan-induced transplantable ChC (mucicarmine, x132).

 
Like primary rat ChC cells, the rat C611B ChC cell line was also found to be 100% tumorigenic when inoculated into the inguinal fat pad of syngeneic recipient rats (n = 4). Tumors formed at the cell transplantation site had a latency period of ~30–40 days and, as represented by the photomicrographs shown in Figure 3A–CGo, were determined to be cytokeratin 19-positive, mucin-producing tubular adenocarcinomas closely resembling in their histological and phenotypic features those of the parent tumor.



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  		Fig. 3. Histopathology of tumors developed in recipient rats following cell transplantation of the C611B rat ChC epithelial cell line. (A) Adenocarcinoma formed in the right inguinal fat pad of a recipient rat 40 days after implantation of C611B rat ChC cells on type I rat tail collagen gel (hematoxylin and eosin, x132). (B) Histological section of adenocarcinoma formed in the inguinal fat pad of a rat following cell transplantation of C611B cells demonstrating strongly positive cytoplasmic immunoreactivity for biliary cytokeratin 19 in the neoplastic epithelial glands (x132). (C) Positive histochemical staining for mucin in neoplastic glands within an adenocarcinoma formed in the inguinal fat pad of a recipient rat following cell transplantation of C611B cells (mucicarmine, x132).

 
In culture, the C611B cell line tested negative for mycoplasma and was characterized by rapidly growing pleomorphic epithelial cells. The cell growth curve shown in Figure 4Go is for C611B cells cultured at in vitro passage 15 on collagen-coated plastic in DMEM medium containing 10% fetal bovine serum. Under these conditions, a cell population doubling line of ~24 h was determined. This is comparable with the cell doubling time previously reported for a hamster ChC cell line derived from an allotransplanted tumor that originated from a liver fluke-associated ChC (10). In comparison, the population doubling times for some human ChC cell lines cultured under similar conditions were found to range from ~50–180 h (3,4,6,7). The cell population doubling time exhibited by the C611B cells is also estimated to be at least twice that previously determined by us from measurements of total cell protein for a rat HBDE cell line at in vitro passage 4, which originated from a bile duct-ligated rat and which was maintained under similar culture conditions as those used in the current study (21).



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  		Fig. 4. (A) Cell growth curve for rat C611B ChC cells cultured in DMEM medium containing 10% fetal bovine serum. Cells cultured on rat type I collagen-coated plastic. Each point value represents the mean ± SD of three to six separate determinations. (B) Aneuploidy analysis of cultured rat C611B ChC cells. The total number of C611B metaphase chromosome spreads analyzed was 72.

 
Chromosome analysis of cultured C611B cells at in vitro passage 14 demonstrated aneuploid chromosome counts, with 72% of the analyzed metaphase spreads having chromosome counts ranging in number from 43 to 46 (Figure 4BGo). In comparison, rat HBDE cells in primary culture were determined to be diploid (data not shown). In this regard, it is interesting that the majority of C611B metaphase spreads analyzed deviated from the normal diploid count by an increase of only a single chromosome. Human ChC have been previously shown to be characterized by abnormal chromosome numbers and typically have been found to be highly aneuploid, with modal chromosome numbers usually ranging between 60 and 70 (3–5,7). However, a hypodiploid human ChC cell line has also been described with modal and median chromosome numbers of 40 and 41, respectively (7). In addition to numerical chromosome deviations, human ChC cell lines have been further shown to exhibit numerous structural abnormalities (5,7). We are now intending to perform a much more extensive cytogenetic analysis of C611B rat ChC cells at both early and late in vitro passages, as well as of derived tumorigenic cell clones, in order to identify possible non-random numerical alterations and potentially relevant structural rearrangements that may be linked to the tumorigenic phenotype of these malignant cells.

Figure 5Go demonstrates another salient characteristic of the C611B ChC cells, namely their prominent expression of c-Met and c-Neu when compared with cultured HBDE (20). These data are highly consistent with our recently published in vivo findings indicating that c-Neu and c-Met are concordantly overexpressed in neoplastic glandular epithelia of furan-induced primary and transplantable rat ChC when compared with normal and hyperplastic intrahepatic biliary epithelia (16). The rat C611B ChC cell line has now undergone >=26 in vitro passages, having been maintained for ~1 year in culture. However, viable C611B cells have been cryopreserved at almost every in vitro passage time, from which new cultures may be established. In addition, we now have preliminary data demonstrating that C611B cells can be used to establish clonal epithelial cell cultures that strongly express c-Met and c-Neu and which also give rise to mucin-producing tubular adenocarcinoma when transplanted into right inguinal fat pads of syngeneic rats (G.-H.Lai and A.E.Sirica, unpublished data). Overall, our results support a novel rat ChC model that can serve as a valuable resource for investigating aberrant growth properties and other critical alterations associated with biliary tumor progression.



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  		Fig. 5. (A) Cultured C611B ChC cells strongly positive for c-Met immunoreactivity. (B) Negative immunostaining control for (A) in which primary anti-Met antibody had been neutralized by pretreatment with the immunizing peptide. (C) Cultured C611B ChC cells demonstrating positive cell surface immunoreactivity for c-Neu. (D) Negative immunostaining control for (C) in which primary anti-Neu antibody had been neutralized by pretreatment with the immunizing peptide (A–D, x132). Western blots demonstrate enhanced expression of p185Neu and both p170Met and p140Met by C611B ChC cells cultured for 4 days on rat type I collagen-coated plastic compared with rat HBDE in 4 day primary culture.

 


   Acknowledgments
 
We wish to thank Ms Dung Pham and Ms Tracy Lamb for their valuable technical assistance. We also wish to thank Ms Sharon Beard for typing the final draft of the manuscript. This work was supported by grant 5 R01 CA 39225 to A.E.S. from the National Cancer Institute, NIH. Presented in part at Experimental Biology 99 held in Washington, DC, April 17–21, 1999, and published in abstract form (22).


   Notes
 
1 To whom correspondence should be addressed Email: asirica{at}hsc.vcu.edu. Back


   References
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 Introduction
 References
 

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Received April 23, 1999; revised July 30, 1999; accepted August 9, 1999.


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