The study was approved by the Ethics Committee of Qilu Hospital of Shandong University, and written informed consent was obtained from each patient.
Cells And Reagents
Human CCA cell lines QBC-939, HUCCT1 and RBE, biliary epithelial cell line HIBEpiC were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The human CCA cell line HCCC-9810 was obtained from the American Type Culture Collection (Manassas, VA, USA). All cell lines were authenticated using short tandem repeat (STR) analysis, and the databases of the Chinese Academy of Sciences or ATCC were used as references. All cells were maintained in Dulbecco's Modified Eagle medium (DMEM, Gibico, New York, USA). In contrast, RBE and HuCCT1 cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Gibico) supplemented with 10% fetal bovine serum (FBS, Gibico) and penicillin/streptomycin (HyClone, Utah, USA) in a humidified atmosphere containing 5% CO2. The information of reagents and antibodies was summarized in Supplementary Table 1. Other reagents without special instruction are purchased from Sigma-Aldrich (Burlington, MA, USA).
Patients And Tissue Microarray (Tma)
A total of 930 CCA patients ( 258 iCCA, 412 pCCA and 260 dCCA)) who underwent surgical resection from 2010 to 2020 in Qilu Hospital of Shandong University were included in the primary cohort. The validation cohort consisted of 58 iCCA patients, 123 pCCA patients, and 101 dCCA patients (Supplementary Table 2) who were selected from the primary cohort according to the criteria as follows: (I) patients who underwent radical resection with clear surgical margin; (II) patients with available formalin-fixed tumor tissues, follow-up and complete medical records, (III) patients with post-operational survival time more than one month, and (IV) patients without a history of other malignancies. The tumors were classified and staged according to the 8th edition of the AJCC/UICC TNM classification system.
The representative paraffin-embedded sections of CCA tissues were used for TMA construction and IHC analysis. Before IHC analysis, hematoxylin and eosin staining was performed to confirm the histological features of all samples. For TMA construction, core biopsies of 1.5 mm in diameter were taken from each sample and arranged into TMA slides.
Ihc And Scoring
IHC was performed according to the standard protocol[3, 34]. For TMA, the slides were submerged in EDTA buffer (pH = 9.0) for optimal antigen retrieval. For the slides of xenografts, sodium citrate buffer (pH 6.0) was used for antigen retrieval. The primary anti-Bmi1 antibody, anti-ki67 antibody, anti-E-cadherin antibody, anti-CD8 antibody, anti-CCL5 antibody, anti-CXCL9 antibody, or ant-CD8A was applied.
Stained slides were screened using a slides scanner (Pannoramic MIDI; 3DHISTECH, Budapest, Hungary). The tumor area was selected by a senior pathologist and evaluated by Quant Center software. The staining intensity was stratified as weak, moderate, or strong, and the area of each staining was calculated. IHC score = (percentage of cells of weak intensity×1) + (percentage of cells of moderate intensity×2) + (percentage of cells of strong intensity×3), according to previous studies. The cohort was divided into different groups according to the cut-offs of IHC scores, which was confirmed as the point with the highest sum of specificity and sensitivity in the receiver operating characteristic (ROC) curves.
Quantitative Real-time Pcr
Total mRNA was extracted from frozen CCA tissues or cells using TRIzol reagent (Invitrogen, USA). Purified RNA was reversely transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Takara, Dalian, China), and qRT-PCR was conducted using SYBR Premix Ex Taq TM (Takara). GAPDH was adopted as an internal control, and target gene relative expression was calculated using the 2−ΔΔCt method. All primers were designed using Primer Express version 5.0 software (Applied Biosystems). The primers used for qPCR are listed in Supplementary Table 3.
Total proteins were extracted from frozen CCA tissues or cells using RIPA lysis buffer (Beyotime, Shanghai, China) with 1% PMSF (Beyotime) on ice for 30 min. Protein concentration was determined using a bicinchoninic acid (BCA) assay kit (Beyotime). Proteins were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and then transferred to polyvinylidene difluoride (PVDF) membranes. Then, proteins were incubated with primary antibodies overnight at 4°C, incubated with secondary antibodies, washed, and subjected to detection using an enhanced chemiluminescence kit (Millipore, Bedford, MA, USA). Quantitative analysis of western blotting was performed using Image J.
Lentivirus Transduction And Stable Cell Lines
To enhance the expression levels of Bmi1, the human Bmi1 gene was PCR-amplified from cDNA and cloned into a pLenti-EF1a lentiviral vector (GeneChem, China). To silence Bmi1, HEK293T cells were transiently transfected with two short hairpin RNAs (shRNAs) and packaging plasmids GV, pHelper1.0, and pHelper2.0. QBC-939 cells were plated at 2×106 cells per 6-well plate and transfected with 10 µg of the indicated plasmids. After 48h, lentiviral supernatants were collected and used to infect QBC-939 cells in the presence of 8µg/ml of polybrene (Sigma-Aldrich). Stable cell lines were selected with media containing 2µg/ml of puromycin for 14 days. The knockdown and overexpression efficiency levels were validated by western blotting and quantitative real-time PCR. The sequences of shRNAs wereThe related sequences are listed in Supplementary Table 4.
Cck8 Proliferation Assay
Cells transfected with the indicated shRNAs or vector were plated into 96-well plates (5×103 cells/well) and treated with PTC-209 (10 µM) if indicated. After incubation for the indicated times, CCK8 reagent (Dojindo, Japan) was added, and cells were incubated at 37°C for 1 h. The optical density at 450 nm was detected using a spectrophotometer (Molecular Devices Company, San Jose, CA, USA).
Colony Formation Assay
Cells were seeded into 6-well plates (500 cells/well) and cultured for 2 weeks at 37°C. After fixation with methanol for 15 min, cells were stained with 0.1% crystal violet (Sigma-Aldrich) for 20 min. The number of colonies (more than 50 cells) was counted under a microscope at 10× magnification.
Wound Healing Assay
Cells were seeded in 6-well plates (2×105 cells/well) and cultured at 37°C. After attachment, a wound was created in the cell monolayer using a sterile pipette tip. Cells were washed twice with cold phosphate-buffered saline (PBS), and the initial wound size was measured using a microscope. Then, cells were cultured with serum-free medium for 24 h at 37°C, and the wound size was measured again. The wound closure percentage = (1–[final wound size / initial wound size])×100.
The 8.0 µm pore transwell chamber was purchased from BD Biosciences (Franklin Lakes, NJ, USA). The upper chamber was pre-coated with Matrigel (BD Biosciences) for the invasion experiments. Cells were starved of serum-free medium for 6 h before the experiment. 2×105 cells were suspended plated into each upper chamber. The lower chambers contained media with 10% FBS to facilitate the migration or invasion of cells. After incubation for 12 h, cells adhering to the lower surface were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich) for 20 min. Migrated or invaded cell number was counted from three randomly selected visual fields with a microscope.
Conditioned Medium (Cm) Preparation
CCA cells at 80% confluence were maintained in DMEM supplemented with 10% exosome-depleted FBS for 48 h, the medium of cells was collected and concentrated for 1000×g to discard the pellets. The supernatant was filtered with a low-protein-binding filter (0.2µm) (Millipore) and concentrated ten times with Amicon Ultra 15 ml filters at 4000 g.
Exosome Isolation And Identification
Exosomes were isolated from CM using the ultracentrifugation method. Briefly, CM was sequentially centrifuged at 300 g for 30 min, 3 000 g for 30 min, 20 000 g for 30 min, and 100 000 g for 80 min at 4℃. The pellets were washed with cold PBS and purified by another centrifugation at 100,000 g for 80 min at 4℃. Subsequently, the purified exosomes were resuspended in PBS for protein detection and functional assays. Transmission electron microscopy (JEM-1-11 microscope, Japan) was used to image exosomes at 100 keV. The quality of exosomes was determined by the Nanosight NS300 instrument equipped with ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The protein concentration of exosomes was determined using a BCA Protein Assay Kit (Beyotime).
Exosome Labeling And Tracing
According to the manufacturer's instructions, isolated exosomes were labeled with a PKH26Red or PKH67 Green Fluorescent Cell Linker Kit (Sigma-Aldrich). QBC-939 or RBE cells were exposed to labeled exosomes at 37℃ for 6 h. Cells were washed with cold PBS, then fixed with 4% paraformaldehyde for 30 min, and stained with DAPI for 20 min at room temperature. Finally, cells were observed under a confocal microscope (Leica Microsystems AG, Germany).
Human CCL5 (RANTES) ELISA Kit (Cat. No. RAB0076) and CXCL9 (MIG) ELISA Kit (Cat. No. RAB0138) were obtained from Sigma-Aldrich. Concentrations CCL5 or CXCL9 in CCA cells medium were measured according to the manufacturer's instructions. To detect the protein levels of CCL5 or CXCL9 of supernatant, QBC-939 or RBE cells were treated with PTC-209 or shBmi1 for 48 h. The collected medium was concentrated to 1ml. For ELISA detection, 100 µl of standard or sample was added to the appropriate wells and incubated for 2 h at room temperature. After washing, 200 µl of CCL5 or CXCL9 conjugate was added and incubated for 2 h at room temperature. Substrate solution was added for 30 minutes at room temperature. After adding 50 µl of stop solution, the ELISA absorbance values could be read on the BIOTEK spectrophotometer (Vermont, USA) at 450 nm.
The enzymatic ChIP kit was obtained from Cell Signaling Technology (Cat. No. 9003), and ChIP-qPCR assays were performed according to the manufacturer's instructions. Briefly, QBC-939 cells were sequentially treated with DTBP solution (Sigma-Aldrich Cat. No. D2338) and formaldehyde and harvested with a cell scraper. Then, the cell pellet was lysed with ChIP lysis buffer and sonicated to generate fragmented chromatin samples. Chromatin samples were incubated with anti-Bmi1 or anti-Ubiquityl-Histone H2A antibodies overnight at 4℃. The rabbit IgG (Cell Signaling Technology, Cat. No. 2729) was used as the negative control, and a non-immunoprecipitated sample (2%) was used as the input control. The purified DNA levels were quantified by qPCR. The primers used for ChIP-qPCR are listed in Supplementary Table 5.
Tumor Xenograft Models
Female BALB/c nude mice (5-6 weeks old, 16-18 g) were purchased from GemPharmatech Co., Ltd. (Nanjing, China). Mice were randomly divided into three groups (n=6). QBC-939 cells were transfected with EGFP-shBmi1 by the lentivirus (GeneChem, China). After screening with puromycin, stable high EGFP-expressing QBC-939 cells were observed by fluorescence microscopy and selected by flow cytometry. Stable clones of QBC-939 cells (5×106 cells), transfected with Bmi1 knockdown, were subcutaneously injected into the right flanks of nude mice. The tumor was monitored by a live imaging system (IVIS Spectrum). Radiant efficiency was measured to quantify the tumor burden of mice. Tumor diameters were measured with an external caliper every 2 days. Tumor volume was calculated according to the formula as follows: V = (L×W2 )/2, where V is the volume (mm3), L is the length (mm), and W is the width (mm). The tumors were excised, photographed, and weighed after 28 days.
For the in vivo metastasis assay, stable clones of QBC-939 cells (2×106 cells) with Bmi1 knockdown were injected into the tail vein of BALB/c nude mice (n=6). Mice weights were measured every week. After 8 weeks, the mice were sacrificed, and the weights of livers and lungs were calculated to assess the actual tumor burden. The number of nodules on the livers and lungs was confirmed by histological staining and counted. If necessary, isolated exosomes (100µl per mice i.v.) were injected weekly via intravenous injection. All animal experiments were approved by the Medical Ethics Committee of Shandong University.
All values were represented as mean±standard deviation (SD). Statistical analyses were carried out using SPSS 26.0 and GraphPad prism 8.1 software. The Chi-square test assessed the correlation between Bmi1 and clinicopathologic characteristics. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The independent prognostic significance of clinicopathological characteristics was analyzed using multivariate analysis with the Cox proportional hazards regression model. Linear correlations were analyzed with Pearson correlation analysis. In vitro and in vivo experiments, one-way or two-way ANOVA was used to compare the statistical difference between groups. P values less than 0.05 were considered statistically significant.