AbstractCholangiocarcinoma (CCA) is a highly lethal hepatobiliary malignancy, with prognosis is influenced by anatomical subtypes and etiological factors. This study successfully established three CCA cell lines: KKU-097, KKU-466, and KKU-610, from the primary tumors of patients in liver fluke-endemic areas. These cells represent the perihilar CCA (pCCA) and intrahepatic CCA (iCCA) subtypes. Comprehensive analyses, including histopathology, molecular profiling, biomarkers, cancer phenotype characterization, and drug sensitivity testing with standard chemotherapeutics, were conducted. Whole-exome sequencing was performed to explore genetic alterations. All three cell lines exhibited adherent growth with an epithelial morphology and positive expression of the bile duct epithelial markers CK-7 and CK-19. Cytogenetic analysis revealed highly complex hypertriploid karyotypes with multiple chromosomal aberrations. Among the cell lines, KKU-610 demonstrated higher growth and invasion rates, whereas KKU-466 and KKU-097 cells exhibited less aggressive phenotypes. Drug sensitivity testing demonstrated relative resistance to gemcitabine as a monotherapy and in combination with cisplatin in all three cells. Genomic profiling identified targetable mutations, highlighting these new cell lines as valuable models for investigating the pathogenesis of CCA and evaluating therapeutic strategies.
IntroductionCholangiocarcinoma (CCA) is a highly lethal and heterogeneous group of hepatobiliary malignancies that arise from epithelial cells lining the biliary tree. Based on anatomical classification, CCA is categorized into intrahepatic CCA (iCCA) and extrahepatic CCA (eCCA), with the latter further subdivided into perihilar (pCCA) and distal CCA (dCCA)1. The incidence of iCCA has increased worldwide (0.3 to 6/100,000) and varies by geography. Certain regions, particularly, Thailand, Laos, Cambodia, South China, and Korea, report high incidences of CCA (more than 6/100,000)2,3. Liver fluke infection, particularly with Opisthorchis viverrini (Ov) and Clonorchis sinensis has been associated with carcinogenesis in some Southeast Asian countries. In contrast, in Western countries, primary sclerosing cholangitis (PSC) is the most common risk factor for CCA4,5.Over the past decade, high-throughput next-generation sequencing technology has unveiled genomic and epigenetic alterations in CCA, revealing that genomic heterogeneity is closely related to tumor location and underlying risk factors driving cholangiocarcinogenesis6. The mutational landscape differs between Ov-associated CCA and non-Ov-associated CCA. Ov-associated CCA exhibits high mutational frequencies in TP53 and ARID1A, whereas non-Ov-associated CCA is characterized by high frequencies of mutations in BAP1, IDH1/2, and FGFR2 fusions. Notably, KRAS mutations are common in both groups7,8,9. These findings suggest that actionable target genes may differ based on the etiology of CCA. This highlights the importance of patient-derived cell lines and organoids research tools for developing treatment strategies across different CCA subtypes.Cancer cell lines are valuable tools for investigating the molecular mechanism of carcinogenesis and devising effective treatment strategies. Several patient-derived cell lines from CCA tumor tissues have been established, encompassing a range of gross pathological and histological subtypes. Approximately 75 CCA cell lines have been reported worldwide10. In northeast Thailand, where Ov infestation is endemic, we established cell lines, including KKU-100, KKU-213, KKU-023, and KKU-452 from CCA tumors collected from CCA patients11,12,13. Patient-derived CCA cell lines provide simple models yet representative the malignant characteristics of their tumor origin. To advance the understanding of CCA carcinogenesis and their distinct molecular and phenotypic characteristics, more CCA cell lines must be established as robust research tools for refining effective therapeutics for CCA treatment.ResultsEstablishment of human CCA cell lines from surgical specimensThree CCA cell lines were successfully established from pathologically proven CCA patients. The pathological profile and characteristics of the CCA patients are shown in Table 1. All patients were residence in the endemic area of liver fluke infection (Ov) in Northeast Thailand. Primary tumor tissues obtained during the surgical procedure from a 53-year-old female with pCCA were used to establish cultured cells and designated as KKU-097; cultured cells from a 62-year-old male with pCCA designated as KKU-466; and cultured cells from a 63-year-old female with iCCA designated as KKU-610 (Fig. 1A–C). A pathological analysis of the original tumor was conducted. H&E staining of KKU-097, KKU-466, and KKU-610 revealed that the cells were well-differentiated adenocarcinomas, intraductal papillary neoplasms of the bile duct (IPNB) with high-grade dysplasia, and poorly differentiated adenocarcinomas, respectively (Fig. 1D–F). Patient serum tumor marker levels were evaluated. The serum alpha-fetoprotein (AFP) levels of all patients were well within normal ranges, whereas most patients presented marked increases in carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) levels, with the exception of KKU-466, which presented normal CA 19-9 levels.Table 1 Pathology profile of CCA patients and characteristics of established cell lines.Full size tableFig. 1Gross pathology and histopathology of the original tumors. (A–C) Gross pathology of the surgically resected specimen showing pCCA (KKU-097 and KKU-466) and iCCA (KKU-610). The arrows highlight the tumor parenchyma, and the asterisks indicate artifacts and sampling artifacts. (D–F) H&E staining revealed histopathology of KKU-097 cells with IPNB with high-grade dysplasia, KKU-466 cells with poorly differentiated adenocarcinoma, and KKU-610 cells with well-differentiated adenocarcinoma (scale bar = 200 μm).Full size imageMorphological features of CCA cellsAll new CCA cell lines grew as adherent monolayers. The cells were maintained in culture medium for more than 50 passages, indicating their stable genetics for long-term studies. KKU-097 and KKU-610 presented features of an irregular polygonal shape with multiple nuclei (Fig. 2A,C). KKU-466 had a smaller ovoid to cuboidal shape, forming a compact monolayer with multinuclear cells (Fig. 2B), suggesting its cellular heterogeneity. Immunocytochemical staining for the cholangiocyte markers CK-7 and CK-19 was positive in all the cell lines, suggesting that they originated from the bile duct epithelium (Fig. 2D–I). Mycoplasmas were not detected in the culture medium during the series of experiments. All the cell lines could be propagated under cryopreservation with no change in the characteristics of the cells at different passages.Fig. 2Morphology and immunocytochemistry of CK-7 and CK-19 in CCA cells. (A–C) Phase-contrast microscopy was used to observe the brightfield morphology of KKU-466, KKU-610, and KKU-097 cells in the 45th, 60th, and 66th passages (10X magnification, scale bar = 100 μM). (D–I) Immunocytochemistry of CK-7 and CK-19. All CCA cells were cultured in slide chambers for 24 h, and the cells were fixed and stained with CK-7 and CK-19 antibodies (20X magnification, scale bar = 100 μM).Full size imageKaryotype and short tandem repeat (STR) profiles of CCA cell linesNewly generated CCA cells were subjected to karyotype analysis. Numerical and structural aberrations were found in all the cell lines. KKU-097 cells were characterized as hypertriploid with a modal chromosome number of 74 (near-triploidy, 69 ±). The pattern of numerical abnormalities included nullisomy 22, monosomy 21, trisomy 1, 2, 3, 6, 8, 11, 13, and 17 and tetrasomy 9, 15, 16, and X. For structural abnormalities, the pattern included deletions of chromosomes 7q22, 7q32, and 10q22 and additional chromosomes of 12q24.3 and 14q32 (Fig. 3A). The KKU-466 cell line was a hypertriploid cell line with a modal chromosome number of 76, occurring in 20% of the cells. Monosomy 13 and iso(1)(p10p10) were present in all metaphases. Most cells had two X chromosomes (Fig. 3B). KKU-610 was a hypertriploid cell line with 70 modal chromosomes (near-triploidy, 69 ±). Trisomy 2, 4, 5, 10, 15, and 21; tetrasomy 11 and 12; and 3; and pentasomy 20 and X were presented. Structural alterations included the translocation of chromosomes 1q32 and 3p23, the translocations of 8q24.3 and 17p13, the additional chromosomes 1q25 and 13q10, and the isochromosomes of 14p10 and 17q10 (Fig. 3C).Fig. 3Representative karyotypes of CCA cell lines. (A) The karyotype of the KKU-097 strain was 74 < 3n > ,XXX, + X, − 4,5,del(7)(q22), + del(7)(q31), + 9,del(10)(q22), del(10)(q22), add(12)(q24.3), + add(12)(q24.3),add(14)(q32), + add(14)(q32), + 15, + 16, − 18, − 19, − 20, − 21, − 21, − 22, − 22, − 22, + 7mar. (B) KKU-466 cells were 76,XX, + 1, + 3, + 4, + 6, + 7, + 9, + 10, + 11, + 12, − 13, + 16, + 17,18, + 20, + 21 + iso(1)(p10p10), + mar, + min. (C) KKU-610 showed 70 < 3n > ,XXX, + X, + X, add(1)(q25),der(1)t(1;3)(q32;p23), + der(3)t(1;3)(q32;p23), − 7, − 8,der(8)t(8;17)(q24.3;p13), − 9, + 11, + 12, − 13,add(13)(q10),i(14)(p10), + 16, − 17,i(17)(q10), − 18, − 18, − 19, + 20, + 20, − 21, − 22, + 3mar.Full size imageThe STR profiles (24 loci) of the KKU-097, KKU-466, and KKU-610 cell lines from the two sources, the CCA cell lines and their original tissues, were analyzed to determine the identity of the cell lines. In the assessment of STR profiles, KKU-097 exhibited 88% similarity to the original tumor tissue across 24 loci, whereas KKU-610 displayed 70% concordance. Notably, while some loci demonstrated only one missing allele compared with the original tissue, others exhibited consistent allele retention (Supplementary Table S1). These findings indicate the presence of genetic heterogeneity of tumor tissues or genetic instability during the establishment of the cell lines. For KKU-466, although no tissue match was available, its STR profile was distinct and non-overlapping with KKU-097 and KKU-610, confirming its unique identity as a model for pCCA.Cell growth, migration, and invasion analysisThe three cell lines presented different growth rates, with doubling times of 32 h, 56 h, and 27 h for KKU-097, KKU-466, and KKU-610, respectively (Table 1, Fig. 4A). All the cell lines formed colonies, as shown by the clonogenic assay (Fig. 4B). The migration potential of CCA cells was evaluated via a wound healing and transwell migration assay. KKU-610 cells highly migrated by closing the scratch wound within 18 h. Meanwhile, the scratch wounds of KKU-466 and KKU-097 cells were closed after incubation for approximately 72 h and more, respectively (Fig. 4C–H). Similarly, in the transwell migration assay, KKU-610 cells exhibited a very high migratory capability, completing migration within 24 h. KKU-097 cells showed a moderate migration rate, while KKU-466 cells displayed the lowest migratory activity (Fig. 4I). Consistent with their ability to migrate, KKU-610 cells exhibited a high invasion rate. In contrast, a much lower degree of invasion was observed with KKU-466 and KKU-097 (Fig. 4J). These data suggest the highly aggressive ability of KKU-610 cells.Fig. 4Characterization of CCA cell lines. (A) Growth curve of CCA cells. (B) Representative images of the colony formation ability of each cell line. (C–E) Representative images of CCA cell migration were captured at 4X magnification (scale bar = 1000 μM). CCA cells were seeded onto 24-well plates. After the cells formed a confluent monolayer, a scratch line was made, and the distance of the scratch wound was measured after incubation until the wound closed. (F–H) Each bar represents the mean ± SD of wound closure. (I) Representative transwell migration and (J) invasion assay images were captured at 10X magnification (scale bar = 100 μM). For the migration assay, CCA cells were seeded in serum-free medium onto uncoated Transwell inserts. For the invasion assay, CCA cells were seeded onto Matrigel-coated Transwell inserts and incubated for 24 or 72 h. After incubation migrated and invaded cells were fixed, stained, and photographed.Full size imageSensitivity to standard chemotherapyThe combination of gemcitabine (Gem) and cisplatin (Cis), the current first-line chemotherapeutic drug for patients with advanced CCA14,15 was evaluated for the sensitivity in new CCA cells. The cells were treated with 0–25 μM Gem or 0–100 μM Cis for 24, 48, or 72 h. KKU-097 and KKU-610 cells were responsive toward Cis cytotoxicity, with the maximum killing (Emax) reaching nearly 100% in a concentration- and time-dependent manner. In contrast, KKU-466 cells were much less responsive (Fig. 5A–C,G). On the other hand, Gem showed low efficacy toward the CCA cells, as the estimated Emax values were less than 50% at the highest concentration of Gem (25 μM). However, the IC50 values were calculated for further experiments (Fig. 5D–G).Fig. 5Relative cytotoxicity of chemotherapeutic drugs in CCA cell lines. (A–F) Sensitivity of CCA cells to cisplatin (Cis) and gemcitabine (Gem). The cells were treated with Gem or Cis for 24, 48, or 72 h. Cell cytotoxicity was assessed via the MTT assay. Dose‒response curves are expressed as the mean ± SD of three independent experiments. (G) The IC50 values of single drug treatment at 24, 48, and 72 h are shown. (H–J) The cytotoxicities of Gem, Cis, and the combination of Gem and Cis at concentrations up to their IC50 values at 72 h were determined via the MTT assay. (K‒M) Drug synergism analysis was performed via a two-agent model in which the Bliss score was compared.Full size imageTo evaluate the effects of the interaction of the two drugs on cytotoxicity, various concentrations of Gem and Cis at around the IC50 values or lower for each drug were combined for 72 h. When these drugs were used in combination, the combination did not increase cell killing by more than 50%, as shown in the 3D surface plots (Fig. 5H‒J). The interactive effects of the drug combinations assessed by the Bliss synergy score revealed that most Gem/Cis combinations resulted in Bliss scores ranging from 0 to − 20, indicating additive to antagonistic effects (Fig. 5K‒M). These findings suggested that all the new CCA cell lines were insensitive to the combination of Gem and Cis.Comprehensive genomic sequencing of CCA cell linesWhole-exome sequencing (WES) was used to analyze gene mutations in 6 CCA cell lines, including KKU-097, KKU-466, KKU-610, and three previously established cell lines, namely, KKU-023, KKU-100, and KKU-45211,12. Among these, original tumor tissues for establishing four cell lines: KKU-023, KKU-097, KKU-452, and KKU-610, were available for genetic comparison. We found that the CCA cells harbored mutations in genes involved in CCA pathogenesis, including genes related to genome instability (e.g., TP53, BARD1, MSH3), epigenetic modifiers (e.g., ARID1A, KMT2C, IDH1), DNA damage and cell cycle control (e.g., BRCA1, CDKN1B, SMAD4), kinase RAS/RAF (e.g., KRAS, PIK3CA), and WNT signaling (e.g., AFF4, CTNNB1). We then investigated whether the characteristics of the original tissues were preserved in the cell lines. The established cell lines maintained the mutations identified in the corresponding original tissues at the same position in the six most recurrent mutated genes: TP53, KRAS, SMAD4, ARID1A, PIK3CA, and NCOR1 (Fig. 6A and Supplementary Table S2–S3). Notably, the KKU-023 cell line presented the highest number of somatic mutations concordant with its original tissue (Fig. 6A).Fig. 6Genomic sequencing of CCA cell lines (A) Comparison of mutations identified in CCA cells and their original samples. Genes harboring mutations were classified into 5 categories: genome instability, epigenetic modifiers, DNA damage and cell cycle control, kinase RAS/RAF, and WNT signaling. Frequencies of mutations identified as tissue-specific, CCA cell-specific, or both cell- and tissue-specific. (B) Coding variants with preclinical or clinical evidence of treatment according to the OncoKb database (May 23rd, 2024). Each column represents a cell line. Green: Level 1; Pink: Level 3; Light Blue: Level 4; Orange: Likely pathogenic. The symbols are as follows: circle, truncating mutation; triangle, missense mutation. (C) Annotated alterations in TP53 and KRAS were detected in CCA cell lines. (D) A representative image of the sequencing traces of amplified TP53 (NM_000546) and KRAS (NM_004985) in the KKU-100, KKU-466, KKU-610, and KKU-097 cell lines is shown. The red triangle and square represent point mutation and nucleotide insertion, respectively. (E) Principal component analysis (PCA) score plot of the mutational spectrum data showing the separation between the older set (yellow dots) and the new set (purple dots) of established CCA cell lines. (F) PCA loading plot highlighting the gene set contributing to the PC1 loading variable. The blue bars indicate the gene set contributing to the negative loading, which correlates with the older group of CCA cell lines; the red bars indicate the gene set contributing to the positive loading, which is associated with the newly established group of CCA cell lines.Full size imageMoreover, these established CCA cell lines presented many targetable mutations with preclinical and clinical evidence. According to the OncoKB database, a total of five actionable mutations were identified. KKU-023 harbored an actionable mutation in PIK3CAH1047R and ATRI774Nfs*3 (therapeutic level of evidence 1) and ARID1AD1850Gfs*4 mutations (level of evidence 4). KKU-466, KKU-100, KKU-097, and KKU-452 harbored mutations in PIK3CAR88Q (level 1), KRASG12D (level 3), KRASG13C (level 4), and ARID1AN1571* (level 4), respectively. BRCA1 genes found in KKU-023, KKU-097, and KKU-610 were assigned as likely oncogenic genes. For those genes, annotated alterations of BRCA1, ATR, and ARID1A were truncating mutations, suggesting loss of function, whereas PIK3CA and KRAS were missense mutations (Fig. 6B and Supplementary Table S2).TP53 and KRAS mutations are the most common mutated genes in CCA pathogenesis. We further evaluated and confirmed the mutation of these two genes in all new cell lines by using Sanger sequencing. KKU-100 was used as a reference cell line with KRAS and TP53 mutations11. In the TP53 mutation analysis, KKU-100, KKU-466, and KKU-097 cells presented missense mutations. Moreover, KKU-466 contained insertion nucleotide bases affecting the DNA-binding domain, leading to a frameshift mutation, and KKU-610 cells presented a truncating mutation. KRAS oncogenic mutations were identified in 33.3% (2/6) of our established cells. KKU-097 cells presented a missense mutation at codon G13C, a position different from that of KKU-100, which was mutated at codon G12D in exon 2 (Fig. 6C,D). This result was consistent with the WES analysis.A large set of CCA cells was recruited in principle component analysis of mutational variations to explore the heterogeneity of CCA cells. Notably, mutations in KRAS, PIK3R1, and FANCD2 were associated with the CCA cells, including KKU-100, KKU-055, KKU-213A, KKU-213B, and KKU-213C cells. While PTEN, STK11P, EGF, MSH3, AFF3, KMT2C, and POLI were observed in the group of CCA cells including KKU-097, KKU-466, KKU-610, KKU-023 and KKU-452 cells (Fig. 6E,F).DiscussionCCA is the second most common hepatobiliary malignancy and is characterized by a very poor prognosis and limited treatment options2,4. The evidence indicates that CCA is heterogeneously associated with various etiologies. Our previously published data demonstrated that genetic alterations in Ov-associated CCA differ from those in non-Ov-associated CCA9,16. Establishing more cell lines with diverse genetic variations can reveal the heterogeneity of human tumors, facilitating a comprehensive investigation into the complex molecular pathways underlying cancer progression, vulnerability pathways, and translational research.This study successfully established three new CCA cell lines, KKU-097, KKU-466, and KKU-610, derived from three CCA patients. These cell lines were genetically profiling via STR analysis and found to be concordant with primary tumor tissues. Characterization included chromosome analysis, determinations of doubling time, migration and invasion ability, drug sensitivity, and comprehensive genomic profiling.AFP and CA 19-9 are circulating tumor markers associated with hepatocellular carcinoma (HCC) and CCA, respectively, whereas CEA is a marker for CCA and many malignant tumors17,18,19. Increased levels of CA 19-9 and CEA were observed in all new CCA cell lines, except KKU-466, which presented normal CA 19-9 levels. However, the AFP level remained within the normal range. The growth kinetics of the three cell lines were correlated with the survival time of patients, with KKU-466 showing a slow growth rate, which is consistent with the very long survival time of the patient (r = 0.99, p = 0.049) from whom this cell line was developed.Karyotyping analysis revealed numerical and structural abnormalities, including monosomy, trisomy, and tetrasomy, across various chromosomes in all new cell lines. These chromosomal aberrations contribute to CCA pathogenesis and influence tumor behavior and the response to chemotherapeutic agents. For example, a hypertriploid or near-triploid karyotype, identified as a hallmark of aggressive cancer behavior, chemotherapy resistance, and poor prognosis20, was observed in all CCA cell lines with modal chromosome numbers ranging from 70 to 76. Previous research has reported that breast cancer cells with near-triploid karyotypes are resistant to paclitaxel and doxorubicin due to DNA damage mechanisms and increased Ki67 expression21.Migration and invasion are critical processes in cancer metastasis22,23. Understanding their underlying mechanisms is crucial for targeting and inhibiting cancer progression. In our study, KKU-610 cells exhibited highly aggressive behavior, characterized by high migration and invasion rates, followed by KKU-097 cells. The high aggressiveness of KKU-610 cells may be attributed to their histopathological classification as poorly differentiated adenocarcinoma. Conversely, KKU-466 cells demonstrated a less metastatic phenotype, were derived from intraductal papillary neoplasm of the bile ducts (IPNB), a histology type associated with a generally good prognosis 24,25.The Gem/Cis regimen is the standard of care for patients with unresectable or metastatic CCA14,15. Despite its clinical efficacy, the overall survival of patients remains unsatisfactory. In cisplatin study, KKU-097 cells appeared to be the most sensitive, whereas KKU-466 cells were the least sensitive. The IC50 value and Emax of KKU-097 are comparable to those of KKU-055, another CCA cell line. For Gem, all three cells have very limited efficacy, as the Emax was less than 50%, similar to KKU-055 cells, which demonstrated Emax of approximately 50%26. Moreover, other cells derived from iCCA, including RBE, ICC-X1, and ICC-X2 cells, are relatively resistant to Gem27,28. The present study investigated whether a combination of Gem and Cis could improve the efficacy of cell killing in these three resistant cell lines. The overall effect of the combined drugs of Gem/Cis was unsatisfactory. Some combinations even showed antagonistic effects by decrease of cytotoxicity. Resistance to Gem may be an innate resistance mechanism, increasing the ability to repair drug-induced DNA alterations or overactivation of an oncogenic signaling pathway. The present study highlights the limited efficacy of current Gem/Cis treatments, which results in a poor therapeutic response29. These findings suggest that novel chemotherapy strategies are needed to achieve an optimal therapeutic response. Furthermore, genetic alterations such as ARID1A mutation, PTEN deficiency, and BAP1 deficiency, especially SNPs in PTEN, contribute to Gem-based chemotherapeutic response in CCA30,31,32. Therefore, understanding genetic alterations is essential for predicting chemotherapeutic responses and providing therapeutic options for chemoresistant CCA patients.It was well-documented that the pathogenesis of CCA was different depending on geographical regions and etiological factors, including liver fluke infestation. Our limitation of samples, only 6 CCA cell lines, may not reflect the whole subsets of CCA. However, our result revealed a mutational pattern consistent with Ov-associated CCAs characterized by mutations enriched in ARID1A, KRAS, and TP53. In contrast, BAP1 and FGFRs alterations were more frequently found in non–Ov associated CCA1,2, but was not detected in our newly established cell lines. This suggested our established CCA cells reflected the different etiological factors regarding the pathogenesis of CCA7. Long-term culture of cells as 2D monolayers can lead to the acquisition of additional mutations, which is a limitation of our study. However, to provide the relevance of the cell lines, we compared their genomic profiles with those of the original tumor tissues. We identified common mutated genes of CCA in both the cell lines and matched tumor tissues, including TP53, KRAS, and ARID1A. These shared mutations support the validity of using these cell lines as models for CCA research.In this study, six established CCA cell lines, three cells from the present study and another three cells from previous studies, presented many targetable mutations with preclinical and clinical evidence. At least five actionable mutations (ARID1A, BRCA1, PIK3CA, KRAS, and ATR mutations) were identified. ARID1A genes usually have inactive mutations that render ARID1A loss of expression33,34, our KKU-023 and KKU-452 cell lines also harbor truncating mutations in ARID1A. Various treatment regimens targeting ARID1A mutations are in phase II clinical trials35. KRAS is one of the most common oncogenic genes in cancer, and many efforts have been made to develop covalent allele-specific inhibitors that trap KRASG12C in its inactive conformation36 and effectively in suppressing tumor growth in cancer patients37,38. At least two KRAS inhibitors have been approved by the U.S. FDA, with several others in clinical trials39. Our cell lines harboring KRAS mutants may be valuable tools for developing KRAS-directed therapeutics, particularly in KRAS-driven CCA. BRCA1 is a tumor suppressor gene involved in DNA damage repair (DDR) via homologous recombination. Dysfunction of BRCA causes genomic instability, promoting tumor progression40. Previous studies demonstrated synthetic lethality between BRCA gene defects and DDR inhibition, leading to increased double-strand breaks and cancer cell death41. BRCA-deficient tumors exhibit high sensitivity to DDR inhibitors due to synthetic lethality mechanisms. Several PARP inhibitors (olaparib, niraparib, talazoparib, and rucaparib) have been approved for treating BRCA-mutant cancers42. KKU-023, KKU-610, and KKU-097 represent BRCA1 truncation mutations, which are valuable for researching and developing new targeted drugs for a subgroup of CCAs. Interestingly, comutations in KRAS and TP53, which are observed in KKU-097 cells, are linked to DDR deficiency, leading to an increased mutation load in cancer cells43. Targeting the DDR mechanism is crucial for cancer treatment, and DDR-targeted drugs have been shown to be effective in different cancer types. This finding could provide potential benefits for use as a valuable tool for drug testing and understanding cholangiocarcinogenesis in a subset of CCAs.MethodsChemicals and reagentsCollagenase type IV, mouse monoclonal antibody against cytokeratin-7 (CK-7), and MTT reagent were purchased from Sigma‒Aldrich (St. Louis, MO, USA). Mouse monoclonal antibody against cytokeratin-19 (CK-19) was obtained from Dako (Santa Clara, CA, USA). Ham’s F12 nutrient mixture, MEM nonessential amino acids, fetal bovine serum (FBS), 0.25% trypan blue dye, and 0.25% trypsin–EDTA were purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). The ROCK inhibitor was purchased from Santa Cruz Biotechnology (San Diego, CA, USA). Bovine serum albumin (BSA) was obtained from Capricorn Scientific (Hesse, Germany).Patient backgroundThree donor patients were pathologically confirmed to have bile duct cancer. They were residents of the northeastern region of Thailand, in an area with liver fluke infections. Human samples were handled and processed under institutional guidelines and protocols approved by the Human Research Ethics Committee of Khon Kaen University (HE571283 and HE631599). The studies were conducted in accordance with the Declaration of Helsinki. All procedures were performed at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand, with informed written consent from patients. The primary tumor from a 53-year-old female with pCCA, encoded as H097 (later named KKU-097), a 62-year-old male with pCCA, encoded as 2KU140466 (KKU-466), and a 63-year-old female with iCCA, encoded as 2KU150610 (KKU-610), were obtained postsurgery. The patients were evaluated for serum AFP, CEA, and CA 19-9 tumor markers.Establishment of human CCA cell linesThe CCA cells were derived from surgically resected liver specimens via protocols described in previous reports11. Briefly, tumor samples were processed under aseptic conditions by immersion in sterile Hank’s balanced salt solution (HBSS) and subsequently minced into small fragments via scalpels. CCA tumor pieces were dissociated using 1 mg/ml collagenase type IV for 30–45 min at 37 °C. Then, the cell suspensions were washed with HBSS and resuspended in Ham’s F12 nutrient mixture supplemented with 10% (v/v) FBS, 12.5 mM HEPES, MEM nonessential amino acids, an antibiotic mixture (50 μg/mL cefazolin, 10 μg/mL ciprofloxacin, and 2.5 μg/mL amphotericin B), and 5 μM ROCK inhibitor. The supernatant was then collected and filtered through a cell restrainer with a 70-mesh filter, followed by centrifugation at 2000 rpm for 5 min and culture in T25 cell culture flasks under an atmosphere of 5% CO2 at 37 °C. The mixed fibroblasts were mechanically removed under a microscope during primary cell culture to purify the epithelial cells. Cultured cells were initially subpassaged every week until a stable growth rate was reached and then subpassaged when the cells reached 50–70% confluence using 0.25% trypsin–EDTA. Following a few passages of primary cultures, the ROCK inhibitor was removed from the medium mixture. The cell lines were tested for mycoplasma contamination via MycoAlert™ Plus Mycoplasma® (Lonza, Rockland, ME, USA) following the manufacturer’s instructions.ImmunocytochemistryCCA cells were cultured in a chamber with 2 × 104 cells/well for adherent growth. Following culture, the cells were rinsed twice with PBS, fixed with cold methanol for 20 min, and then air-dried before they were incubated with 5% BSA for 2 h. After that, the cells were incubated overnight with mouse monoclonal antibodies against CK-7 (1:500) and CK-19 (ready-to-use), followed by the secondary antibody of the standard horseradish peroxidase (HRP) method. The staining signal was assessed according to the intensity from no staining: − = negative, + = < 25% tumor cells stained, + + = 25–50% tumor cells stained, and + + + = > 50% tumor cells stained.Chromosome analysisCCA cell lines were subjected to chromosome analysis upon achieving stability in culture. The established cells were treated with 1 µg/mL colcemid, followed by a hypotonic solution, and harvested according to standard cytogenetic procedures. The cells were fixed, dropped on slides, and subjected to trypsin‒Giemsa banding to identify individual metaphase chromosomes. The representative chromosome was photographed for karyotype analysis with GenASIs Cytogenetics Suite Systems (Applied Spectral Imaging (ASI), Carlsbad, CA, USA). Three cytogeneticists (Y.C., M.T., and P.W.) analyzed the karyotypes of the CCA cells on the basis of the International System for Human Cytogenetic Nomenclature (ISCN) criteria44.Short tandem repeat analysisDNA from CCA cells and original tissue was extracted using a genomic DNA extraction kit (Qiagen, Hilden, Germany). A standard STR profile was obtained with 23 loci and a sex marker (amelogenin). The obtained data were analyzed via GeneMapper™ ID-X software version 1.6 (Applied Biosystems, Waltham, MA, USA).Morphological examination and growth kineticsThe cultured cells were regularly monitored and imaged via phase-contrast microscopy (Nikon Instruments, Tokyo, Japan). For population doubling time assessment, cells were seeded onto 6-well plates at a density of 1 × 104 cells, and the culture medium was changed every three days. The cells were detached from the wells with 0.25% trypsin–EDTA and stained with 0.25% trypan blue dye. The average number of viable cells was counted daily in a hemocytometer over 10 days. The growth curve was plotted, and the doubling time of the cell population was calculated during the log phase via the formula Td = t × Lg2/Lg(N1/N0), where Td is the doubling time, t is the time interval, N1 is the endpoint cell number, and N0 is the initial cell number.Colony formation assayCCA cells at a density of 600 cells/well were seeded in a 6-well plate and incubated overnight under 5% CO2 at 37 °C for 14–21 days in complete medium, with medium renewal every three days. Then, the colonies of more than 50 cells were fixed with absolute methanol at 4 °C for 30 min, stained with 0.5% crystal violet in 2% ethanol for 1 h, washed with tap water to remove excess dye, and air-dried overnight. The colonies were captured via a ChemiDocMP imaging system (Bio-Rad, Hercules, CA, USA).Wound healing assayCCA cells at a density of 2 × 105 cells were seeded in a 24-well plate and allowed to adhere overnight. A wound was created in the cell monolayer by using a 200 μL sterile pipette tip, followed by washing with PBS to remove detached cells. The cells were then incubated in medium containing 10% FBS. Images of the wound gap were captured regularly until complete healing occurred. Cell migration, as measured by the closure of the wound gap, was analyzed via Image-Pro Plus software (Media Cybernetics Inc., Rockville, MD, USA).Transwell migration and invasion assayThe migration assay was performed using a Transwell chamber (Corning, NY, USA). For the invasion assay, transwell chamber was briefly pre-coated with a 1:24 dilution of Matrigel solution (Corning, NY, USA) in coating buffer overnight. The excess gel was removed before 200 μL of cells (2 × 104 cells/well) in medium without 10% (v/v) FBS was seeded into the chamber. At the same time, 700 μL of medium supplemented with 10% (v/v) FBS was added to the lower chamber of the Transwell system. After incubation at 37 °C, the invading cells were fixed with absolute methanol for 30 min and stained with 0.5% crystal violet in 0.5% methanol for 10 min. Images of the invading cells were captured with a Nikon ECLIPSE Ni-U upright microscope (Nikon Instruments, Japan).Drug sensitivity analysisThe chemotherapeutic sensitivity of CCA cells was evaluated by the MTT assay. Briefly, CCA cells were seeded onto 96-well plates at a density of 7.5 × 103 cells/well for KKU-466 and 5 × 103 cells/well for KKU-610 and KKU-097. After that, the cells were treated with various concentrations of Gem, Cis, or a combination for 24–72 h. For the assay, the plates containing treated cells were incubated with 100 µL of MTT in culture media without FBS (1:10 dilution) for 3 h. Following incubation, the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The absorbance was measured using a microplate reader at a wavelength of 540 nm.Potential drug synergy in drug combinations was evaluated via SynergyFinder, a web application that uses a Bliss-independent model to quantify the degree of drug interaction. The synergy score is interpreted as the average excess response due to drug interactions, whereas scores of ≤ − 10, − 10 to 10, and ≥ 10 are likely to be antagonistic, additive and synergistic, respectively45,46. A 3D plot of the drug combination and Bliss score was created via SigmaPlot 10.0.Whole exome sequencing and data analysisWES was used to analyze gene mutations in 6 CCA cell lines. This analysis included KKU-097, KKU-466, KKU-610, and three previously established cell lines, including KKU-023 (JCRB1778), KKU-100 (JCRB1568), and KKU-452 (JCRB1772)11,12. DNA extraction from CCA cell lines was performed by using a QIAamp® blood mini kit (#51106, Qiagen, Hilden, Germany). Sequencing and data analysis were performed at Molecular Genomics (Molecular Genomics, Pte Ltd., Panda Loop, Singapore). Briefly, 100 ng of each DNA sample was quantified on a Qubit reader via a broad-range DNA assay kit (#Q32851, Thermo Fisher Scientific, USA). The assay libraries were subsequently prepared via SureSelect XT HS v2 (Agilent, Santa Clara, CA, USA) following the manufacturer’s protocol. The Human All Exon V8 SureSelect bait was used for capturing exonic regions. Libraries were sequenced on a NovaSeq 6000 platform with 2 × 150 bp paired-end sequences. The raw sequencing data (fastq files) were processed and analyzed using Agilent SureCall analysis software. The sequencing reads were aligned to the reference human genome (hg38). Variants were identified via SureCall’s SNP caller with the following parameters: a variant score threshold of 0.3, minimum base quality of 30, variant call quality threshold of 100, and minimum allele frequency of 0.1. The criteria for selecting mutations included those that are clinically relevant, supported by evidence of pathogenicity, and related to the COSMIC, GWAS, and NCBI SNP databases. Furthermore, mutations were considered only if they included stop codons, exon deletions that maintained the reading frame, mutations in the start codon, missense mutations that occurred at stop codons, or insertions or deletions that caused a shift in the reading frame.Four cell lines (KKU-023, KKU-097, KKU-452, and KKU-610) that matched tumor-adjacent normal tissue samples were available. Tissues were extracted from fresh frozen samples via the QIAamp® DNA Mini Kit (#51304, Qiagen, Hilden, Germany). The somatic mutations from paired tumor-normal tissues were examined via the Genome Analysis Toolkit (GATK) pipeline. The raw reads were aligned via BWA-0.7.175347. The BAM files were generated through samtools-1.9 and then further processed through duplicate marking, base quality score recalibration, and variant quality score recalibration by GATK-4.1.0.05448. Somatic mutations were called by MuTect2 and annotated with ANNOVAR49. The resulting somatic variants were further filtered via GATK’s FilterMutectCalls to retain high-confidence somatic mutations. Finally, the validated somatic mutations were functionally annotated for potential clinical actionability via databases such as OncoKB and classified on the basis of their likely impact on protein function and involvement in oncogenic pathways.Sanger sequencing for TP53 and KRAS mutationsTo investigate TP53 and KRAS mutations in new CCA cell lines, KKU-100 was used as a positive control. Genomic DNA was extracted to analyze KRAS (NM_004985) mutations in exon 2. For the TP53 (NM_000546) mutation, RNA was extracted from each cell line and subsequently converted to cDNA following the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). As previously reported, PCR amplification was performed using 100 ng of DNA template and specific PCR primer sequences for TP53 and KRAS11. The quality of the PCR amplicons was assessed via agarose gel electrophoresis. For Sanger sequencing, the PCR products were purified via the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA) and then subjected to nucleotide sequencing by the First BASE Laboratories (Selangor, Malaysia). Sequencing traces of the amplified products were aligned to reference sequences via Lasergene 10.1 (DNASTAR) and analyzed via visual inspection.Comparison of mutational spectra in CCA cellsPrincipal component analysis (PCA) was used to examine differential mutational patterns among CCA cell lines. The analysis was performed via Python (version 3.12.3) with the sklearn.decomposition package. The mutational data from 10 CCA cells (KKU-097, KKU-466, KKU-610, KKU-023, KKU-452 cells, KKU-100, KKU-055, KKU-213A, KKU-213B, and KKU-213C cells) were encoded in a binary format, with mutations represented as ‘1’ and nonmutations as ‘0’. This method was implemented to reduce the degree of technical variability among different WES platforms and ensure consistent data analysis across samples.
Data availability
Sequence data that support the findings of this study have been deposited in the NCBI’s SRA database with the primary accession code PRJNA1144247.
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Download referencesFundingS.K. was supported by NSRF under the Basic Research Fund of Khon Kaen University and the Faculty of Medicine, Khon Kaen University, Thailand (Grant Number IN67050). R.J. was supported by Research Fund for Supporting Lecturer to Admit High Potential Student to Study and Research on His Expert Program Year 2022, Graduate School, Khon Kaen University (651H220). B.T.T., P.G., and J.H.H. were supported by the 3rd A*STAR-AMED Joint Grant (R23I2IR070). B.T.T. was supported by the National Medical Research Council Singapore Translational Research Investigator Award (NMRC MOH-000248-00), the Tan Yew Oo Professorship in Pathology, the Verdant Foundation, and the NCC Cancer Fund.Author informationAuthors and AffiliationsDepartment of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandRattanaporn Jaidee, Piman Pocasap, Veerapol Kukongviriyapan, Laddawan Senggunprai, Auemduan Prawan & Sarinya KongpetchCholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, ThailandRattanaporn Jaidee, Apinya Jusakul, Piman Pocasap, Veerapol Kukongviriyapan, Laddawan Senggunprai, Auemduan Prawan, Watcharin Loilome, Attapol Titapun, Apiwat Jareanrat, Vasin Thanasukarn, Natcha Khuntikeo, Nisana Namwat & Sarinya KongpetchCentre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, ThailandApinya JusakulDepartment of Systems Biosciences and Computational Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandWatcharin Loilome & Nisana NamwatDepartment of Surgery, Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandAttapol Titapun, Apiwat Jareanrat, Vasin Thanasukarn & Natcha KhuntikeoDepartment of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, ThailandYaovalux Chamgramol, Malinee Thanee, Phongsathorn Wichian & Chawalit PairojkulCancer and Stem Cell Biology Program, Duke-NUS Medical School, Singapore, SingaporeJing Han Hong & Bin Tean TehGenome Institute of Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, SingaporePeiyong Guan & Bin Tean TehLaboratory of Cancer Epigenome, Division of Medical Science, National Cancer Center Singapore, Singapore, SingaporeHong Lee Heng & Bin Tean TehInstitute of Molecular and Cell Biology, Agency for Science Technology and Research (A*STAR), Singapore, SingaporeBin Tean TehAuthorsRattanaporn JaideeView author publicationsYou can also search for this author in
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PubMed Google ScholarContributionsR.J. performed the experiments, analyzed data, and wrote the manuscript. A.J. designed the study, analyzed data and wrote the manuscript. P.P. designed the study and performed a comparison of mutational spectra analysis. V.K. wrote, reviewed, and revised the manuscript. L.S. and A.P. provided material and technical support. W.L. and N.N. provided patient samples. A.T., Ap.J., V.T., and N.K. contributed to clinical diagnosis and sample collection. Y.C., M.T., and P.W. performed karyotypes analysis. J.H.H., P.G., and H.L.H. performed WES analysis. C.P. performed histological diagnosis and provided material specimens. B.T.T. provided WES analysis and suggestions. S.K. conceived, designed, supervised the research, wrote and revised the manuscript. All authors have read and approved the final manuscript.Corresponding authorCorrespondence to
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KeywordsCholangiocarcinomaCCA
Opisthorchis viverrini
Cell lineLiver fluke