Acquisition of specimens
ICC tissues were obtained from surgical specimens resected in the Fourth Department of General Surgery, the First Hospital of Lanzhou University, China. The patients did not receive any preoperative treatment. Fresh ICC tissue specimens were cut under sterile conditions and transported to the laboratory for primary cell isolation and culture in the shortest possible time. The postoperative pathology report revealed a poorly differentiated ICC in the specimens. This study was approved by the Ethics Committee of the First Hospital of Lanzhou University (Approval number: LDYYLL2022-345). Informed consent was obtained from the patient.
BALB/C nude mice
Six-week-old sterile BALB/C female nude mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd (Changzhou, China) and raised in the Animal Experiment Center of Lanzhou University, China. Mice were provided ad libitum access to sterile water and food. Animal experiments were carried out per the requirements of the Laboratory Animal Protection Committee of Lanzhou University, China. When an animal became seriously ill during the experiment, it was euthanized by carbon dioxide exposure followed by cervical dislocation.
Establishment of cell lines
The freshly obtained tissue samples were placed on an ultraclean bench and rinsed three times with phosphate-buffered saline (PBS) at 4 °C; the blood vessels, necrotic tissues, and interstitial tissues on the surface were removed using sterile forceps. The tissue sample was finely chopped and incubated with collagenase type II and neutral protease solution at 37 °C for approximately 2 h in a humidified atmosphere containing 5% CO2. The digested solution was filtered using a 200-mesh sieve and the filtrate was centrifuged at 300×g for 3 min. The supernatant was discarded, the pellet was resuspended in 5 mL PBS, and centrifuged again at 300×g for 3 min. The final pellet was resuspended in the RPMI-1640 medium containing 10% fetal bovine serum (Biological Industries Ltd., Haemek, Israel), 1% penicillin–streptomycin. The suspension was inoculated into culture plates and incubated at 37 °C in a 95% air and 5% CO2 atmosphere. The medium was changed twice a week. When the cells reached 70–80% confluence, cell passage and cryopreservation were performed under optimal conditions.
Control cell line
The human cholangiocarcinoma RBE cell line was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China in 2016. The cell line was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Biological Industries Ltd.), 100 U/mL penicillin, and 100 µg/mL streptavidin-mycin (Biological Industries Ltd.).
Morphology of the ICC-X1 cells
ICC-X1 cells were seeded in the 6-well plates and incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 2 weeks. The cells were harvested every 24 h and analyzed using an inverted microscope to observe their general morphology.
Short tandem repeat (STR) detection
Fifteen passages of logarithmic growth phase ICC-X1 cells were used for these experiments. STR analysis was performed according to the protocols published by the American National Standard Institute (ANSI/ATCC ASN-0002-2011 Authentication of Human Cell Lines: Standardization of STR Profiling).
Chromosomal analysis was performed on the cells at passage. The cells were treated with 0.1 µg/mL colchicine for 1 h and were digested with trypsin/EDTA; slides were prepared according to standard methods. The hypotonic treatment was performed using 0.075 M potassium chloride solution for 20 min at room temperature. The slides with fixed cells were stained using trypsin-Giemsa to identify individual metaphase chromosomes. Fifty chromosomes in the split phase were randomly counted under an oil-immersion objective lens (1000× magnification) of a light microscope and 10 well-dispersed G passage specimens were selected for karyotypic analysis. Abnormal chromosomes were identified according to the International Human Cytogenetic Nomenclature System, 2016 .
Quantification of population doubling time
Forty passages of logarithmic growth phase cells were used for these experiments. Tumor cells in the logarithmic growth phase were digested with trypsin and a single-cell suspension containing 1000 cells/100 μL was prepared. The cells were seeded into 96-well plates at 1000 cells/well and incubated at 37 °C with 5% CO2 and saturated humidity. The cell doubling time was calculated after 24 h. The cells were incubated with a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) for 2.5 h, and absorbance was measured at a wavelength of 450 nm. The cell doubling time was calculated using the formula: Td = t × lg2/lg(N1/N0), where Td is doubling time, T is the time interval, N1 is the endpoint cell number, and N0 is the initial cell number. The growth curve was drawn with the time on the X-axis and the absorbance value on the Y-axis.
Ultrastructure of the ICC-X1 cells
Thirty-five passages of logarithmic growth phase ICC-X1 cells were used for these experiments. After washing the cell slides with physiological saline, they were quickly put into 4% glutaraldehyde (SPI-CHEM, USA) in a fixative solution and rinsed three times with phosphate buffer. The cells were dehydrated with 50%, 70%, 80%, 90%, and 100% tert-butanol in a step-by-step gradient (5 min at each concentration). Next, the cells were put into the JEOL JFD-320 cold dryer, and the dried sample was taken out when the temperature equilibrated to room temperature. A conductive paste was used to coat the sample holder with a JEOL JFCC-160 ion sputterer. The specimens were observed and photographed using a scanning electron microscope (HITACHI Regulus 8100).
Transwell chamber migration assay
Thirty passages of logarithmic growth phase cells were used for experiments. The lower chamber was filled with 600 μL of culture medium containing 5% fetal bovine serum and the upper chamber was filled with 1 × 105 cells/well of RBE and ICC-X1; the chambers were then placed in the corresponding lower chambers. After culturing for 8 h, the chamber was taken out, and the cells in the chamber were wiped off and placed in 0.1% crystal violet staining solution for 30 min. The cells passing through the membrane were observed under a light microscope; five fields of view were randomly selected and the cells passing through the membrane were counted.
Plate colony formation assay
ICC-X1 cells of passage 45 and RBE cells in the logarithmic growth phase were digested with 0.25% trypsin. Next, single cells were pipetted into the RPMI-1640 medium supplemented with 10% fetal bovine serum for further use. The cell suspension was diluted in gradient times, and 500 cells/well were inoculated into a 6-well plate. The evenly dispersed cells were then incubated at 37 °C with 5% CO2 and saturated humidity for 2 weeks. The cultures were observed periodically and terminated when macroscopic clones appear in the petri dish. The number of clones with more than 10 cells under the microscope was counted. The colony formation rate (%) was calculated using the formula: (number of clones/number of inoculated cells) × 100.
Spheroid formation assay
Logarithmically growing cells of passage 50 were collected, trypsinized, and cultured in serum-free conditioned stem cell medium (RPMI-1640 medium supplemented with 1 × B27, 20 ng/mL human EGF, 10 ng/mL human FGF, 0.4% bovine serum albumin, and 4 µg/mL insulin). The cultured cells were inoculated (1.5 × 105 cells/well) into a 6-well plate (Ultra-low adherent plate). Spheroid formation was monitored on days 7, 10, and 14 after seeding.
The logarithmically growing cells of passage 40 were collected, trypsinized, and prepared as a single-cell suspension. The tumor cell concentration was adjusted to 1000 cells/100 μL. A volume of 100 μL of the cell suspension was taken, mixed with matrigel 1:1, inoculated into a 12-well plate, and incubated at 37 °C for 30 min to form a gel. To each well, 1 mL of organoid culture medium [RPMI-1640 supplemented with 1% Glutamax (Invitrogen), 1% penicillin/streptomycin, 1× B27 supplement (Gibco), 50 ng/mL EGF (Peprotech), 1.25 mM N-acetylcysteine (Sigma), 10 nM gastrin (Sigma), 10 mM nicotinamide (Sigma), Rspo-1 (Peprotech), 5 mM A83-01 (MCE) and 10 mM Y-27632 (MCE)] was added, and the cell culture plate was placed at 37 °C and humidity in an incubator with 5% CO2. The culture medium was changed every 3–4 days and passaged every 1–2 weeks.
Gemcitabine was purchased from Jiangsu Hansoh Pharmaceutical Group Co., Ltd., China; oxaliplatin was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd., China; 5-fluorouracil (5-FU) was purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., China; and paclitaxel purchased was from Jiangsu Osaikang Pharmaceutical Co., Ltd. Gemcitabine, 5-FU, and paclitaxel were dissolved in normal saline and oxaliplatin was dissolved in 5% glucose solution. The stock solutions were aliquoted as different working solutions to avoid degradation.
Drug sensitivity test
Logarithmically growing RBE and ICC-X1 cells of passage 50 were collected separately and prepared as single-cell suspensions after trypsinization. The cells were inoculated into 96-well plates at 10,000 cells/well, and each group was inoculated into 6 wells. After cell adherence, different concentrations of antitumor drugs were added to the experimental group and equal volumes of solvent of each drug group were added to the vehicle control group. After 72 h of incubation, the complete medium was replaced with 100 μL of serum-free medium containing 10% CCK-8 reagent. The optical density was measured at 450 nm after 2 h.
Tumorigenicity in BALB/C nude mice
The cells were prepared at passage 25 to determine their tumorigenicity in BALB/C nude mice. The cultured cells (1 × 107 cells/mL) were collected, washed, resuspended in 0.1 mL of complete RPMI-1640 medium, and injected subcutaneously into the middle and posterior left axilla of three 6-week-old female BALB/C nude mice. Tumor diameters and body weight were measured every week after cell injection and tumor volume was calculated as ½ × length × (width2) . Tumor-bearing mice were sacrificed after 4 weeks. Tumor tissue was excised and fixed with 10% formalin, and routine histopathological and immunohistochemical examinations were performed.
Cells at passage 36 were digested and grown on sterile glass slides. At 48 h, slides were washed with PBS, fixed with 4% paraformaldehyde for 15 min, air-dried, and treated with 0.5% Triton X-100 for 20 min. For primary tumor tissues, transplanted tumors, and organoids, the specimens were immersed in a formaldehyde solution for fixation, dehydration, embedding, and sectioning. The slides were then covered with the following antibodies: mouse mAb against human cytokeratin (CK)7 (1:300; Servicebio), mouse mAb against human CK19 (1:1000; Servicebio), rabbit mAb against Ki-67 (1:200; Servicebio), mouse mAb against human p53 clonal antibodies (1:200; Servicebio), mouse mAb against human E-cadherin (1:600; Servicebio), and mouse mAb against human vimentin (1:1000; Servicebio). The slides were incubated with the antibodies for 60 min and washed thoroughly with PBS. Biotinylated rabbit anti-mouse IgG (1:200; Servicebio) was then added for 15–20 min and the slides were washed. Then, a 3,3′-diaminobenzidine solution was added to the slides for 1–5 min at room temperature. Finally, slides were rinsed with distilled water, stained with hematoxylin and eosin (H&E), and examined under a light microscope.
The mRNA profiles of ICC-X1 cells were compared to that of RBE cells to better understand their molecular features and altered pathways. First, the purity, concentration, and integrity of the extracted RNA samples were determined to ensure that qualified samples are used for transcriptome sequencing. The library was constructed using qualified samples by (1) enriching eukaryotic mRNA with magnetic beads with oligo(dT); (2) adding fragmentation buffer to randomly interrupt mRNA; (3) using mRNA as a template and synthesizing, the first cDNA chain with random hexamers and the second cDNA chain by adding buffer, dNTPs, RNase H, and DNA polymerase I, and purifying the cDNA using AMPure XP beads; (4) subjecting the purified double-stranded cDNA to end repair, adding a tail, and connecting a sequencing adapter, and then using AMPure XP beads to select fragment sizes; and (5) obtaining a cDNA library by PCR enrichment. After the library was constructed, quantitative PCR was used to accurately quantify the effective concentration of the library (library effective concentration > 2 nM) to ensure the quality of the library. After passing the library check, sequencing was performed on the Illumina platform (Illumina, Inc., USA). This project used the reference genome: Homo_sapiens.GRCh38_release95.genome.fa as a reference for sequence alignment and subsequent analysis. The reads on the StringTie alignment were used for assembly and quantification after the alignment analysis was completed.
The statistical significance between the experimental and control group was determined using the Student’s t-test. The statistical significance was set at p ≤ 0.05.