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Transplant Division, Department of Surgery, Indiana University School of Medicine, Indianapolis, IndianaDepartment of General Surgery, Yeditepe University Faculty of Medicine, Istanbul, Turkey
Transplant Division, Department of Surgery, Indiana University School of Medicine, Indianapolis, IndianaDivision of Transplantation & Hepatobiliary Surgery, Department of Surgery, University of Florida College of Medicine, Gainesville, Florida
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IndianaDivision of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IndianaDivision of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IndianaDivision of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
Division of Gastroenterology and Hepatology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IndianaDivision of Research, Richard L. Roudebush VA Medical Center, Indianapolis, Indiana
Address correspondence to Burcin Ekser, M.D., Ph.D., Transplant Division, Department of Surgery, Indiana University School of Medicine, 550 University Blvd., Room 4601, Indianapolis, IN 46202.
Cholangiocarcinoma (CCA) is the second most common primary liver tumor and has been associated with a late diagnosis, limited treatment options, and a 5-year survival rate of around 30%. CCA cell lines were first established in 1971, and since then, only 70 to 80 CCA cell lines have been established. These cell lines have been essential in basic and translational research to understand and identify novel mechanistic pathways, biomarkers, and disease-specific genes. Each CCA cell line has unique characteristics, reflecting a specific genotype, sex-related properties, and patient-related signatures, making them scientifically and commercially valuable. CCA cell lines are crucial in the use of novel technologies, such as three-dimensional organoid models, which help to model the tumor microenvironment and cell-to-cell crosstalk between tumor-neighboring cells. This review highlights crucial information on CCA cell lines, including: i) type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) chemical digestion method (eg, trypsin or collagenase), iv) cell-sorting method (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or Dulbecco's modified Eagle's medium), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.
Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies originating from the biliary tree.
CCA accounts for approximately 3% of all malignancies of the gastrointestinal system and is the second most common primary hepatic tumor, after hepatocellular carcinoma.
The prevalence of CCA varies broadly by region; while the prevalence is 1.6 per 100,000 people in the United States, it could be as high as 85 to 90 per 100,000 people in northeastern Thailand, where infection with Opisthorchis viverrini, a CCA risk factor, is endemic.
Several additional risk factors for cholangiocarcinogenesis have been identified, such as Clonorchis sinensis infection, bile duct cyst, primary sclerosing cholangitis, hepatolithiasis, cholelithiasis, and inflammatory bowel disease.
Biliary cancer: intrahepatic cholangiocarcinoma vs. extrahepatic cholangiocarcinoma vs. gallbladder cancers: classification and therapeutic implications.
CCAs can also be subclassified by macroscopic growth pattern (mass forming, periductal infiltrating, intraductal, or mixed), cell of origin (cholangiocytes, goblet cells, hepatic stem cells, or biliary tree stem/progenitor cells), and microscopic features (adeno, squamous, adenosquamous, mucinous, undifferentiated, or sarcomatous).
Although the importance of the organ-specific or cancer-specific microenvironment has been recently highlighted due to cell-to-cell crosstalk, two-dimensional cancer cell lines are one of the best sources for investigating the respective cancer type.
CCA cell lines have been used for almost 50 years to: i) better understand CCA properties, ii) investigate treatment options, iii) model the disease in vitro, and iv) generate in vivo xenograft models.
Dr. Moore, oncologist, surgeon, accomplished scientist, and director of the RPMI, pioneered many cancer studies, established several types of cancer cell lines, and formulated RPMI 1640, a widely used cell-growth medium.
There is currently a growing interest in CCA-related basic and translational research, due to poor outcomes with the currently available treatment options. For recently developed novel experimental models [eg, three-dimensional (3D) organoids] and treatments, CCA cell lines have been used for the identification of novel mechanistic pathways, biomarkers, and disease-specific genes. Therefore, previously isolated human primary CCA cell lines, their isolation methods, and known essential characteristics are summarized herein to help researchers in their future studies.
Human Primary CCA Cell Lines
The first CCA cell line was established about a half-century ago, in 1971 in the United States
; more than half of currently available CCA cell lines were generated in Japan between 1975 and 1990. To date, researchers from Italy, Germany, China, South Korea, and Thailand have reported on the isolation of CCA cell lines and related experiments. In the past 2 decades, researchers have reported the isolation of multiple CCA cell lines in Thailand more than in any other country, possibly due to the high prevalence of the disease in that geographic region.
Each CCA cell line is unique in reflecting a specific genotype, sex-related properties, and patient-related signatures, making them especially valuable for scientific and commercial applications. It would undoubtedly be helpful to have an international and collective cell bank that includes cell-identification analysis from each study to appropriately define the CCA properties and prevent any misidentification of the existing cell lines.
For instance, the ETK-1 CCA cell line was retracted in 2004 after a short tandem repeat polymorphism analysis revealed that the ETK-1 and SSP-25 cell lines were identical.
However, later, it was understood that SSP-25 should have been retracted instead of ETK-1 because the SSP-25 and RBE cell lines were isolated from the same patient, but their short tandem repeat profiling results did not match (https://cell.brc.riken.jp/en/rcb/rbessp-25_announce, last accessed May 5, 2022). As a result, the ETK-1 cells have been distributed under the name of SSP-25 since 2004, but it is unclear when that cell line was initially misidentified. It is also unclear what happened to the SSP-25 cell line. Similarly, the M156, M213, and M214 cell lines were regarded as having been isolated from three separate patients; however, results from recent short tandem repeat profiling showed that they were isolated from the same patient.
Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.
and might initiate inaccurate data chains. Various levels of the cell-culturing process have been blamed for cross-contamination, including: i) accidental inoculation, ii) mislabeling, iii) confusion in freeze-thawing, iv) work with more than one cell line in a biosafety cabinet at the same time, and v) contamination of the stock bottle of media with cells.
Additionally, cancer cells may carry a greater risk for cross-contamination due to their greater capacity for proliferation. Even a minimal amount of inoculation of cancer cell lines could suppress other cell lines in the culture and take their place over time. Therefore, the preservation and maintenance of the cell line are as important and challenging as is the establishment of one.
In a different case, CHGS was mentioned as a CCA cell line in 2015 by Zach et al
and is listed in the Cellosaurus database (accession number CVCL_M272; https://web.expasy.org/cellosaurus/CVCL_M272, last accessed May 5, 2022). However, the first mention of CHGS, in 1988 by Katoh et al,
indicates that CHGS is a CCA tissue line passaged in mice, and that cancer cells have not been isolated from this tissue line. Similarly, the CC-CL-1 cell line was studied as a CCA cell line in a research study referred to in previously published studies; however, none of the references cited in the research study contained any information regarding the CC-CL-1 cell line, generating doubt about the credibility of the cell line.
In addition to primary CCA cell lines, derivative CCA cell lines have been established for the study of drug resistance. QBC939/ADM is doxorubicin resistant; HuCCT1-G100, YSCCC-G100, RTFK-1, KKU-M139/GEM, KKU-213B/GEM, MT-CHC01R1.5, and SNU-1196/GR are gemcitabine resistant; and KKU-M055/46 and KKU-213B/246 are 5-fluorouracil–resistant CCA cell lines.
Tables 1 and 2 summarize the CCA cell lines available between 1971 and 2000, and between 2001 from 2021, respectively, from the English and non-English published literature. Tables 1 and 2 include: i) the type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) tissue-digestion method (eg, trypsin or collagenase), iv) cell-sorting and -purification methods (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or DMEM), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.
Table 1Human Primary Cholangiocarcinoma Cell Lines Established between 1971 and 2000
Colony isolation by trypsin and EDTA-soaked filter; other cells removed by enzymatic and mechanical treatments
Williams' E + 10% newborn calf serum + P/S
Large nucleus with 1 to 2 nucleoli; dark cytoplasm; high nucleus/cytoplasm ratio; pavement-like proliferation. Abundant production of gel-like substance
Fibroblasts gradually decreased with each passage and disappeared in a month
Ham's F12 + P/S
Monolayer, adherent, polygonal shaped epithelial cells with occasionally multiple, round to oval nuclei; granule-filled cytoplasms; piling up of cells and occasional gland-like appearances
Polymorphic epithelial-like cells; one or more large, irregular, round to oval nuclei with a few prominent nucleoli; relatively poor, round to polygonal cytoplasm; pavement-like proliferation; tubular formation
A human combined hepatocellular and cholangiocarcinoma cell line (KMCH-2) that shows the features of hepatocellular carcinoma or cholangiocarcinoma under different growth conditions.
Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.
Polygonal or spindle-shaped polymorphic cells; occasional large vacuoles in the cytoplasm; forming small clusters or clumps; one or more large, irregular, round or oval nuclei with a few prominent nucleoli; pavement-like proliferation
Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.
Fibroblasts removed with a cell scraper and by differential trypsinization
Ham's F12 + 20% FBS + P/S
Compact, polygonal to spindle-shaped cells; floating or clumping in a confluent monolayer; large nucleus containing two to five nucleoli and a clear cytoplasm
Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.
Monolayer, adherent cells; pleomorphic appearance with multiple cytoplasmic processes; numerous cytoplasmic vacuoles in some cells; some multinucleated cells
72 hours
9
SNU-1196
EHCCA
Monolayer, adherent cells; trabecular pattern consisted of spindle to polygonal shaped cells having vesicular nuclei and multiple small nucleoli
Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment.
The general principles of the CCA cell–isolation protocol are summarized in Figure 1. CCA cell isolation requires either a solid sample (eg, primary tumor or xenograft) or a liquid sample (eg, ascites or pleural effusion). Samples can come from autopsy, surgery, paracentesis, or animal harvesting. Samples could be transferred into Ham's F12 culture medium at 4°C.
It is better to process the samples as soon as possible to avoid ischemia-related problems in cells. If the CCA sample needs to be frozen, freeze-thawing the tissue samples in 10% dimethyl sulfoxide in culture medium is a viable option for tissue preservation.
Figure 1CCA cell isolation method. CCA cell lines can be isolated from either fresh samples (eg, HuCCA-1 cell line) or frozen-thawed samples (eg, huh-28 cell line) from primary tumors or from xenografts, which can be kept in different mediums. After manual dissociation, plated with or without digestion.
Herein, the general perspective of isolation protocols is briefly summarized. All tissue processing should be performed under aseptic conditions in a biological safety cabinet with sterile or disposable surgical instruments. The first and the most essential step of CCA cell isolation from solid samples is cutting the tissue into small pieces (>1 mm in diameter).
After that, the protocol could be continued either with or without enzymatic digestion. For the protocols without enzymatic digestion, a stainless-steel mesh is helpful for releasing CCA cells.
Chemical digestion usually proceeds at 37°C, and the digestion time is dependent on the concentration and type of the enzyme used (Table 1). Several types of collagenases (types I, II, and IV) and trypsin have successfully been used for chemical digestion (Table 1). Plasminase to digest fibrin clumps and DNase I to lyse DNA leaked into cell suspension could be included in the dissociation protocol (Figure 1).
; the pellet can then be resuspended in a variety of media conditions [RPMI, Dulbecco's modified Eagle medium, minimum essential medium, William's, and Ham's F12].
After cell attachment, CCA cells can be purified using scraping of the contaminating cells, colony isolation, and/or passaging cells until all contaminating cells have been removed (Figure 1).
Cell lines provide indispensable in vitro model systems for the assessment of features of cancer cells, based on the properties that make the cells direct descendants of the primary tumors.
These features have allowed the establishment of thousands of cell lines from various malignancies, of which only 70 to 80 belong to the CCA family after >50 years of research (Tables 1 and 2). While each new cell line has not necessarily been superior to previous lines, collectively, they have been important for the understanding of common and differing features of CCA, including: i) antigenic variations, ii) new genotypes, and iii) enzymes that sustain tumorigenic growth to drug sensitivities or resistances.
One reason that CCA remains difficult to cure is the lack of a precise understanding of oncogenesis.
The established CCA cell lines can be utilized to identify the properties of cells that can help in predicting positive or negative responses to antigen- or pathway-targeted therapies. CCA cell lines could be powerful in determining many aspects of CCA phenotypes, especially when used in a 3D microenvironment in the presence of other liver cells to study the impact of adjacent cells in the tumor milieu.
In a recent study, an established IHCCA cell line was used for understanding the impact of RA190, a proteasome subunit ADRM1 inhibitor that induces cell apoptosis, in a pathway-targeted therapy model.
Although the outcomes of that in vitro study were encouraging, the study did not evaluate the roles of the tumor microenvironment and other CCA cell lines to determine whether a genotypic difference within the CCA lines may have modulated (increased or decreased) the response and sensitivity to RA190.
A counterexample is evidenced by the recent discovery in Italy of a novel IHCCA cell line characterized by the presence of genes encoding resistance to fluorouracil, carboplatin, and oxaliplatin—drugs actively and frequently used in CCA treatment.
The establishment of new, well-defined and well-characterized CCA cell lines is crucial in enhancing the understanding of this aggressive disease. The establishment of oncogenesis through the cancer cells forms their existence, the drug-sensitivity and -resistance mechanism, and helps to identify possible novel drug targets. The inclusion of tumor stromal cells, such as hepatic stellate cells, liver sinusoidal endothelial cells, and tumor-associated macrophages, in the CCA cell lines makes these culture models more representative of the CCA tumor microenvironment (ie, 3D tumor organoids)
(Figure 2). Therefore, the inclusion of multiple other cell types will help to determine the ways in which CCA cells interact with other neighboring cells and alter their genotypes, phenotypes, and transcriptomic profiles.
Figure 2CCA organoids with multiple cell lines. Three-dimensional (3D) CCA organoids include multiple cell lines, such as CCA cell lines (cholangiocytes; CHOL), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) (representative). Other liver cells, such as hepatocytes and Kupffer cells, can also be added, if needed. These organoids can be made scaffold-free without any biomaterial (eg, Matrigel) in a low-binding plate using culture mediums and can be kept for 14 to 28 days. There is good evidence that CCA cells express cytokeratin (CK)-7, a cholangiocyte marker, and organoids can be stained with other cellular markers (eg, vascular endothelial cadherin; VE-Cad) that are present in organoids. CCA organoids can be exposed to immune cells. An example is seen at the bottom right. Bright-field microscopy shows a 3D CCA organoid exposed to mast cells. Scale bar = 500 μm. Original magnification, ×10.
The establishment of well-characterized CCA cell lines with a close resemblance to primary tumors, especially in a 3D microenvironment, will provide an infinite capacity for replicability, limiting the use of small animal in vivo studies and making them prime materials for CCA research.
Author Contributions
A.I. drafted the manuscript. A.Y., D.S., and E.A. helped in the writing and reviewing the manuscript. B.E. developed the concept and helped in the writing and critically reviewing the manuscript. All of the authors critically reviewed and participated in the writing of the manuscript, and approved the final version.
Biliary cancer: intrahepatic cholangiocarcinoma vs. extrahepatic cholangiocarcinoma vs. gallbladder cancers: classification and therapeutic implications.
Functional and genetic characterization of three cell lines derived from a single tumor of an Opisthorchis viverrini-associated cholangiocarcinoma patient.
A human combined hepatocellular and cholangiocarcinoma cell line (KMCH-2) that shows the features of hepatocellular carcinoma or cholangiocarcinoma under different growth conditions.
Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.
Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment.
Partially supported by an ASTS Faculty Development grant (B.E.); Indiana University Health Values Fund for Research award VFR-457-Ekser (B.E.); an Indiana University Health Foundation Jerome A. Josephs Fund for Transplant Innovation grant (B.E.); a Hickam Endowed Chair, Gastroenterology, Medicine, Indiana University grant (G.A.); an Indiana University Health – Indiana University School of Medicine Strategic Research Initiative grant (G.A.); Senior Career Scientist award IK6 BX004601 (G.A.); VA merit award 5I01BX000574 (G.A.); US Department of Veteran's Affairs Career Scientist Award IK6BX005226 (H.F.); VA merit award 1I01BX003031 (H.F.); Biomedical Laboratory Research and Development Service NIH grants DK108959 (H.F.), DK119421 (H.F.), DK054811 (G.A. and S.G.), DK115184 (G.A. and S.G.), DK076898 (G.A. and S.G.), DK107310 (G.A. and S.G.), DK110035 (G.A. and S.G.), DK062975 (G.A. and S.G.), and AA028711 (G.A. and S.G.); a Partners Seeking a Cure grant (G.A.); CPRIT grant RP210213 (S.C.); and the Richard L. Roudebush VA Medical Center (Indianapolis, IN). The views expressed in this article are those of the authors and do not necessarily represent the views of the US Department of Veterans Affairs.
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