A 62-year-old female, was admitted to the First Hospital of Lanzhou University in July 2022 with intermittent pain in her right upper abdomen and lower back. The results of physical examination were unremarkable, except for slight pressure pain in the upper right abdomen. Preoperative computed tomography and magnetic resonance imaging revealed the dilatation of the common bile duct and intrahepatic bile duct. This was accompanied by a soft tissue shadow in the upper part of the common bile duct showing arterial phase enhancement, indicative of bile duct cancer (Figures 1A and B; arrow). Radical surgery was performed under general anesthesia without any treatment prior to surgery. Intraoperative frozen section confirms negative margins. However, the postoperative pathological diagnosis revealed cholangiocarcinoma sarcoma of the porta hepatis, comprising 90% adenocarcinoma and 10% chondrosarcoma. The American Joint Committee on Cancer staging system of the tumor was T2aN2Mx (Figures 1C and G; arrow). The tumor markers were within the normal range, with alpha-fetoprotein at 2.1 U/mL, carbohydrate antigen 19-9 (CA19-9) at 9.4 U/mL, and carcinoembryonic antigen (CEA) at 0.7 U/mL (Figure 1G). Hematoxylin and eosin (HE) staining revealed microscopic epithelial heterogeneous hyperplasia with striated, nested, and glandular arrangements; increased cell volume; large deep-stained nuclei with pathological nuclear abnormalities; localized mucus and mucous cartilage; and infiltrative growth of cancer cells (Figures 1D and E; arrow).

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Figure 1 Clinical and pathological profile of CBC2T-2. A and B: Contrast-enhanced computed tomography of the abdomen showing a high-grade occupying lesion of the common bile duct with high level obstruction of the common bile duct (orange arrows); C: Specimen of postoperative carcinosarcoma of the extrahepatic bile duct with thickened duct wall and polyp-like protrusion into the lumen; D and E: Hematoxylin and eosin and immunohistochemistry staining of primary tumor tissue. Scale bars, 200 μm; F: Immunohistochemistry staining of primary tumor tissue. Antibodies against Vimentin, Scale bars, 200 μm; G: Clinicopathological characteristics of patients. AFP: Alpha-fetoprotein; CEA: Carcinoembryonic antigen; CA199: Carbohydrate antigen199.

This study was approved by the Ethics Committee of the First Hospital of Lanzhou University (LDYYLL-2022-489) and informed consent was obtained from all the patients. This study adhered to the 1964 Declaration of Helsinki and its subsequent amendments and similar ethical standards[14].

The specimens were collected under the guidance of a pathologist to ensure that the diagnosis in the pathology report was unaffected. The tumor specimens were rinsed thrice with phosphate buffer saline (PBS; BI), cut into 1-2 mm³ pieces using a sterile blade, and then digested with collagenase type IV (0.1 mg/mL; Gibco) for 10 min in a 37 °C incubator. The cell pellet was maintained in DMEM/F-12 (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). It was cultured at 37 °C under humidified air containing 5% CO₂. For continuous culture, the cells were periodically passaged and frozen in liquid nitrogen at regular intervals. No contamination with other cells or foreign microorganisms was observed during the culture process. No external growth factors or stimulatory cytokines were added during the establishment of the cell lines.

Freshly cultured human cholangiocarcinoma sarcoma cell line CBC2T-2 (p25) and frozen tumor tissues were collected following the manufacturer’s instructions. The genomic DNA of the cells was extracted using an Animal Genome Extraction Kit and then subjected to polymerase chain reaction with fluorescently labeled primers at the 5’ end. The resulting products were sequenced and analyzed for sequence repeats of 21 short tandem repeat (STR) loci, including AMEL, D19S433, D5S818, D21S11, D18S51, D6S1043, D3S1358, D13S317, D7S820, D16S539, CSF1PO, Penta D, D2S441, vWA, D8S1179, TPOX, Penta E, TH01, D12S391, D2 S1338, and FGA. The obtained STR profiles were compared with reference STRs from public cell repositories, including the American Type Culture Collection (ATCC), Deutsche Sammlung Mikroorganismenund Zelkulturen (DSMZ), and the CELLOSAURUS cell database.

Cells in the logarithmic growth phase (p25) were incubated with 10 μg/mL of colchicine for 2 h in a cell culture incubator. Chromosomes were prepared using standard methods and their numbers were mostly distributed across the G-dominant band. Representative images of chromosomes were obtained for karyotype analysis. Karyotype interpretation was based on the International System for Human Cytogenetic Nomenclature (1995)[15].

In total, 1 × 10⁵ cells in the logarithmic growth phase (p25) were digested and inoculated into ultra-low-attachment 96-well plates (Corning). Sphere formation was monitored on days 3, 7, 10, and 14 after inoculation to assess the ability of the cells to form spheres.

CBC2T-2 cells were inoculated into 96-well plates at a density of 5000 cells per 100 μL. Cell counting kit 8 (CCK-8) (APEx BIO) was added at time points of 0, 24, 48, 72, 96, and 120 h at a ratio of 1:10. The cell growth curve was plotted with time on the horizontal axis and absorbance on the vertical axis. The ploidy doubling time (PDT) software was used to calculate the cell doubling time (http://www.doubling-time.com).

CBC2T-2 cells were inoculated into 96-well plates at a density of 4000 cells per 100 μL. The plate was then placed in a Cytation 1 imaging system (Biotek) under a 4 × objective, and the field of view was selected for each well. Images were taken every 2 h for 120 h and processed using the Gen5 Software.

Tumor cells (p25) in the logarithmic growth phase were washed with PBS, digested with ethylenediaminetetraacetic acid-free trypsin, and prepared as a single-cell suspension. The CBC2T-2 cell precipitates were washed twice with PBS and the density was adjusted to 1 × 10⁷/mL. The cells were fixed by adding pre-cooled 75% ice-cold ethanol to each tube for 1-2 h. After incubating away from light for 15-30 min, the cells were stained with 0.5 mL of propidium iodide stain [consisting of 0.5 mL staining buffer, 25 μL propidium iodide staining solution, and 10 μL RNaseA (50 ×)] (Bioscience).

To assess the migratory ability of the cells, a suspension of 200 μL of cells (1 × 10⁵/mL) was evenly distributed in the upper chamber of a transwell without matrix gel (BD). To evaluate the invasive ability of the cells, a suspension of 200 μL of cells (1 × 10⁵/mL) was evenly distributed in the upper chamber of a transwell with matrix gel (Corning). The lower chamber was filled with medium containing 15% FBS and incubated in a cell culture incubator. After 24 and 48 h of incubation, the contents of the upper lumen were removed. After staining the cells with 0.1% crystal violet solution (Beyotime) for 20 min, they were rinsed with PBS; next, they were observed under an inverted microscope and the images were captured. This procedure was repeated using TFK-1 cells from the extrahepatic bile duct as controls for the CBC2T-2 cells.

CBC2T-1 and TFK-1 cells were seeded into 6-well plates. When the cells reached 95% confluence, the cell monolayers were scraped into a cross-shape using a pipette tip and gently washed thrice with PBS. Medium containing 10% FBS was added to continue the culture. The images were captured at 0, 24, and 48 h, and the entire scratch area was measured using the ImageJ software.

The cells were digested with 0.25% trypsin, and the cell density was adjusted to 700 cells/well before inoculation into 6-well plates. Colony formation was monitored at 3, 7, 10, and 14 d after inoculation. On day 14, the colonies were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet for 20 min, photographed, and analyzed using ImageJ software.

Patient and mouse tumor tissues and crawling cells were fixed in formaldehyde solution. Subsequently, the specimens were subjected to baking, decolonization, hydration, antigen retrieval, peroxidase blocking, and goat serum closure. The slides were incubated with antibodies against CK19, Vimentin, S100, Desmin, CD56, CD117, and S100. Drops of DAB liquid were added, followed by reaction for 1-5 min before color development was promptly terminated.

Sequencing and data analyses were conducted using the BGI software (Wuhan, China). The exome region DNA was captured using probe capture technology and the target region DNA was sequenced using high-throughput sequencing technology. Genomic DNA was extracted from CBC2T-2 cells and compared with that from normal tissues adjacent to the patient’s resected tumor. Library construction and whole-exome capture of genomic DNA were performed using SureSelect Human All Exon V6 (Agilent Clara, Technologies, Santa CA, United States) and the captured DNA library was sequenced on the DNBSEQ platform. This procedure uses FACETS[16] software to detect somatic copy number variation (CNV) in tumor and normal paired samples, the depth distribution of reads to compare copy number variation with the reference genome, and Ensemble VEP[17] to annotate the CNV. We performed InDel detection using the results of the GATK[18] comparison, followed by the annotation of the detected InDels. We used the GATK MuTect2 tool to identify somatic single-nucleotide variant (SNV) loci and the GATK Funcotator tool to annotate these loci.

We compared somatic mutations with known driver genes from databases and the literature and screened for known driver genes in tumor samples. The reference data sources were Integrative OncoGenomics (IntOGen), the Cancer Gene Census (CGC), three highly cited articles, and pan-cancer data[19-22].

To study the in vivo tumorigenicity of CBC2T-2 cells, cells at a concentration of 1 × 10⁷/mL were injected into the axillae of three 4-wk-old non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The mice were housed in a specific pathogen-free laminar flow animal facility and observed for 8 wk. Tumor diameters were measured every 3 d during this period. After 8 wk, the mice were euthanized, the tumors were excised and photographed, and the samples portions were immersed in a 10 % formalin solution for routine processing.

CBC2T-2 cells (p30) at the logarithmic growth phase were washed with PBS and digested with 0.25% trypsin. The concentration of the cell suspension was adjusted to 10000 cells/100 μL. After culturing the cells for 24 h, different concentrations of chemotherapeutic drugs, including gemcitabine, oxaliplatin, cisplatin, paclitaxel, and 5-fluorouracil were added. Following a drug exposure period of 72 h, the viability of each group was measured by incubating the cells with CCK-8 reagent for 2 h.

The results are presented as the mean ± standard error of at least three independent experiments. Graphs were created using GraphPad Prism 9.0, and a one-way analysis of variance was used to compare the two groups. Statistical significance was set at P < 0.05.

We successfully established the cell line, namely CBC2T-2, from a patient with cholangiocarcinoma sarcoma. The cells were cultured in monolayers for > 13 mo and subjected to > 100 generations. The cell line was established from the original tumor tissue by comparing the 21 STR loci of both the tissue and cell lines using the assay currently recommended by the ATCC. The genomic characteristics of the CBC2T-2 cell line and the originating tumor tissue are presented in Table 1. The assay measurements showed that the cholangiocarcinoma sarcoma cell line was of human origin, and its genetic information was comparable with those from three prestigious culture collections (ATCC, DSMZ, and CELLOSAURUS). No sequences matching other cells were found, indicating that the cells were not contaminated. The cells were maintained at the China Center for Type Culture Collection (CCTCC No. C2022273).

CBC2T-2 cells (P1, P10, P20, and P50) were observed microscopically as they actively proliferate and grow in a monolayer against the wall. Two types of cell morphology were observed: Typical polygonal cholangiocarcinoma cell morphology and long spindle-shaped chondrosarcoma cell morphology (Figure 2A). Transmission electron microscopy revealed the presence of numerous mitochondria, rough endoplasmic reticulum, ribosomes, and irregular nuclei with deep indentations in the nuclear membrane. Scanning electron microscopy demonstrated microvilli-like protrusions on the cell surface as well as tight junctions and intercellular bridges (Figure 2B).

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Figure 2 Morphological features, doubling time and cell cycle of CBC2T-1 cells. A: Bright field morphology of CBC2T-2 cells in the primary,1^st, 10^th, 20^th and 50^th passages (scale bars, 100 μm); B: SEM and TEM electron microscopy were used to observe the cellular ultrastructure (scale bars, 100, 30, 10 μm); C: Flow cytometry of CBC2T-2 cells; D and E: Cumulative growth curve of CBC2T-2 cells.

Flow cytometry was used to detect cell cycle progression in CBC2T-2 cells. Diploid cells were observed in G0/G1 phase, tetraploid cells in G2/M phase, and hyperdiploid cells in S phase (Figure 2C). The PDT of CBC2T-2 cells was calculated to be approximately 47.11 h using both live cell imaging and CCK-8 with the PDT software (PDT) (Figures 2D and E). A short PDT indicated active cell proliferation.

The novel cell line CBC2T-2 was inoculated into an ultra-low-attachment 96-well plate and observed for 14 d. The cells demonstrated their ability to form spheroids (Figure 3A). The results of the wound healing assay showed that CBC2T-2 cells showed higher levels of wound repair at both 24 and 48 h (Figures 3B and C). Furthermore, compared with TFK-1 cells, CBC2T-2 cells exhibited greater migratory and invasive capacities (Figure 3D). Moreover, CBC2T-2 cells exhibited significantly stronger clonogenic ability than TFK-1 cells (Figures 3E and F). These findings indicate that the cell line CBC2T-2 has a high capacity for healing, invasion, migration, and sphere formation, making it a valuable tool for studying the pathogenesis of cholangiocarcinoma sarcoma.

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Figure 3 Characterization of CBC2T-2 cell behavior. A: Representative images of the ability of CBC2T-2 cells to form spheres at days 7 and 14; B and C: Wound healing assay of CBC2T-2 and TFK-1 cells after 24 and 48 h; D: The migratory and invasive abilities of CBC2T-1 and TFK-1 cells were detected by transwell assay; E and F: Representative images showing the colony formation of CBC2T-1 and TFK-1 cells. Scale bars, 100 μm. ^aP < 0.01; ^bP < 0.001.

Karyotype analysis of representative single cells from the CBC2T-2 cell line revealed abnormalities in both chromosome number and structure, with polyploidy chromosomes ranging from 69 to 79 and structural aberrations, including gain, deletion, and translocation (Figure 4A). When the value of the black line exceeds 2, it indicates an increase in the copy number, whereas a value below 2 suggests a decrease. Copy number alterations occurred throughout almost the entire chromosomal section of the cells and tumor tissues (Figure 4B). Single-nucleotide polymorphism analysis showed that the cell line CBC2T-2 had extensive insertions and deletions in almost the entire chromosomal fraction (Figure 4C). Somatic SNVs are single nucleotide variants of somatic mutations. On average, 521 SNVs were found in all samples, with an average of 91 missense mutations, eight nonsense mutations, and zero SNV invalidating the termination codon (Figure 4D).

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Figure 4 Cytogenetic analysis and genomic characteristics. A: Chromosome karyotype analysis shows that the cells are triploid and there is variation in the number of structures; B: Cells and tumor tissues have copy number increases and decreases in almost the entire chromosome section; C: Histogram of the distribution of somatic cell InDel annotation results; D: Somatic somatic single-nucleotide variant annotation result distribution map.

A comparison of the detected mutated genes with the CGC database was used to screen for possible cancer susceptibility genes (Table 2). The major tumor susceptibility genes in CBC2T-2 cells included BALK, BARD1, KDR, FAT1, WRN, HNF1A, BRCA1, AXIN2, and SETBP1. We compared somatic mutations in CBC2T-2 with known driver genes from databases and the literature and identified known driver genes (Table 3). TP53 and ARID1A are the most frequently reported genetic alterations in extrahepatic cholangiocarcinoma[23].

Xenograft tumor formation assays were performed and tumor growth was monitored weekly to confirm the tumorigenic ability of CBC2T-2 cells in NOD/SCID mice. Within 3 wk, tumors developed in all the three mice, indicating that the established cell lines exhibited good tumorigenicity and could be used to establish an in vivo model (Figures 5A-C).

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Figure 5 Verification the tumorigenic ability of CBC2T-2 cells in non-obese diabetic/severe combined immunodeficient mice. A: Tumor formation in non-obese diabetic/severe combined immunodeficient mice; B: Body weight gain curves in xenograft mice; C: Tumor size over time in three xenograft mice; D: Hematoxylin and eosin staining and immunohistochemical (CK19, CD68, CD117, Vmention, Desmin) results of primary tumors, CBC2T-2 xenografts and CBC2T-2. Scale bars, 50 and 100 μm. HE: Hematoxylin and eosin.

After 8 wk, the mice were euthanized, and their tumors were removed for measurement and photography. HE and immunohistochemical staining were performed on the tumor xenografts. We observed tumors in mouse tumor grafts, CBC2T-2 (P30) cells, and the patient’s primary tumor tissue using HE staining. The histological consistency was confirmed by the staining procedure (Figure 5D, first row). In addition, CK19, S-100, Vimentin, Desmin, CD117, and CD68 were positively expressed. These findings demonstrated that the primary tumor tissue had both an epithelial component (carcinoma) and a mesenchymal component (chondrosarcoma). Furthermore, in vivo and in vitro experiments were conducted to demonstrate histological concordance through immunohistochemistry (Figure 5D).

The sensitivity of CBC2T-2 cells to the first-line anticancer drugs used for the treatment of cholangiocarcinoma was assessed. Among the chemotherapeutic agents tested, oxaliplatin (IC₅₀ = 77.51 μM), paclitaxel (IC₅₀ = 0.002 μM), 5-fluorouracil (IC₅₀ = 7.516 μM), cisplatin (IC₅₀ = 19.24 μM), and gemcitabine (IC₅₀ = 0.009 μM) were evaluated. Our results demonstrated that among the five anticancer drugs, paclitaxel was the most sensitive, followed by gemcitabine. These findings provide clinical guidelines for the treatment of patients with cholangiocarcinoma sarcoma, suggesting a preference for combined paclitaxel and gemcitabine as the most effective treatment option (Figure 6).

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Figure 6 Sensitivity of CBC2T-2 cell line to anticancer drugs. A-E: Sensitivity of CBC2T-2 cells to paclitaxel, gemcitabine, 5-fluorouracll, cisplatin, and oxaliplatin.

Cholangiocarcinoma sarcoma is a rare tumor composed of epithelial and mesenchymal cells, and the sarcomatous component in carcinosarcoma is differentiated from spindle cells or pleomorphic cells.

There is no extrahepatic cholangiocarcinoma sarcoma cell line.

To establish and characterize a new extrahepatic cholangiocarcinoma sarcoma cell line.

Establish cell lines through primary culture and subculture, and identify their biological characteristics.

In this study, we successfully established and characterized of a extrahepatic cholangiocarcinoma sarcoma cell line CBC2T-2, from the primary tumor of a patient with cholangiocarcinoma sarcoma.

To date, first report of the extrahepatic cholangiocarcinoma sarcoma cell line CBC2T-2.

The establishment of extrahepatic cholangiocarcinoma sarcoma cell line provides suitable experimental models for further study of cholangiocarcinoma sarcoma.