Background
Gallbladder carcinoma (GBC) is an invasive adenocarcinoma that originates from the epithelial linking of the digestive system [
1]. GBC is a common aggressive malignant neoplasm and the fifth most deadly cancer, initiating from the gallbladder or cystic duct. Chronic cholecystitis (CC) with gallstones, dietary factors, chronic gallbladder infections, and environmental exposure to specific chemicals are considered as main risk factors for the development of GBC [
2], which has a wide incidence worldwide [
3‐
5].
Despite the progress in therapeutic strategies, the overall survival rate has remained poor, mainly due to late diagnosis, early metastasis, ineffective surgical resection, and insensitivity to chemoradiation [
1,
6‐
8]. Therefore, it is essential to further investigate its biological behaviours, mechanisms, and potential treatments. In recent years, cancer cell lines originating from patients have proven to be a powerful tool that can be used for drug screening, drug resistance research, analysis of the tumour microenvironment, gallbladder cancer pathogenesis and the mechanism of metastasis [
9,
10]. Previously, only a few GBC cell lines derived from primary tumours have been established but insufficiently elaborated upon [
11‐
27]. This situation necessitates the establishment of more novel GBC cell lines for studying it in detail.
In this study, a novel gallbladder cell line derived from a primary GBC, referred to as ZJU-0430, was successfully established. All our data together confirmed that it as a potentially useful model for the further study of this disease.
Methods
Patient history
This study was performed in accordance with the Declaration of Helsinki of 1975, and the official recommendations of Chinese Community Guidelines, and was approved by the Ethics Committee and Institutional Review Board of the Sir Run Run Hospital. Written informed consent was obtained from the patient.
A 74-year-old male patient with pain in the upper abdomen was admitted in our centre. Gastroscopy showed the presence of a gastric antral ulcer (stage S2), and irregular deep concave ulcer at the gastric angle, and cancer priority. Abdominal computed tomography (CT) also found a 1.5-cm thick wall at the bottom of the gallbladder. A radioimmunoassay showed that the patient’s serum levels of a variety of biomarkers were normal (CA19-9, CA-125, AFP, CEA, and PSA) except for ferritin, which was high (409.3 ng/ml, 30–400 ng/ml). A radical resection of stomach and gallbladder were performed and a pathological examination showed that gastric carcinoma was a poorly differentiated adenocarcinoma derived from signet-ring cell carcinoma, whereas the primary gallbladder cancer was a well differentiated adenocarcinoma.
Cell lines as control
GBC-SD cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China), and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Carlsbad CA, USA) supplemented by 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), and 1% penicillin/streptomycin and amphotericin B (Invitrogen).
Cell culture
The approach of primary culture was as previously described [
28]. Single-cell suspensions were obtained according to the manufacturer’s protocol (Miltenyi Biotec GmbH, Germany). Specifically, the surgically resected gallbladder specimens were immediately transferred to the lab, rinsed several times in cold Dulbecco’s Phosphate Buffered Saline (DPBS) containing 1% penicillin/streptomycin and amphotericin B, cut into small pieces (1 mm
3), and transferred into a gentleMACS™ C tube containing the buffer from the Tumor Dissociation Kit (human), and subjected to a gentleMACS™ Tissue Dissociator. Cells were cultured in complete growth RPMI 1640 medium (containing additives) at 37 °C in a humidified incubator containing an atmosphere of 5% CO
2. Fibroblasts were removed by differential trypsinisation as described previously [
29,
30]. Serial passages were carried out every 3–4 days, routinely at a ratio of 1:2, and the medium was replaced when colour changed. The ZJU-0430 cell line was cultured for > 100 passages and showed no changes in its morphology.
Single cell RNA sequencing
ZJU-0430 cells were grown in mycoplasma-free complete growth RPMI 1640 medium without antibiotics. Cells were grown to 60% confluence, and washed with 4 ml of DPBS, and then treated with StemPro™ Accutase™ Cell Dissociation Reagent (Invitrogen). Cells were then centrifuged at 300 rcf for 30 s at room temperature and the supernatant removed without disturbing the cell pellet. Next, 0.04% bovine serum albumin fraction V (BSA) solution (Sigma-Aldrich, St. Louis, MO, USA) was added and then centrifugation at 300 rcf for 5 min. The resuspended cells were then filtered through 40 µm cell strainer (Corning Incorporated, Corning, NY, USA) and following Trypan Blue staining, the cell density and viability were determined using haemocytometer.
Single-cell RNA-seq libraries were prepared with Chromium Single cell 3′ Reagent v2 (or v3) Kits according to the manufacturer’s protocol. Single-cell suspensions were loaded on the Chromium Single Cell Controller Instrument (10 × Genomics) to generate single cell gel beads in emulsions (GEMs). In order to do this, ZJU-0430 single cells were suspended in DPBS containing 0.04% weight/volume BSA. About cells were added to each channel with a targeted cell recovery estimate of cells. After generation of GEMs, reverse transcription reactions were performed to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads Myone Silane Beads (Thermo Fisher Scientific, MA, USA). The cDNA was then amplified by PCR with appropriate an appropriate number cycles which depending on the recovery cells. Subsequently, the amplified cDNA was fragmented, end-repaired, A-tailed, index adaptor ligated and library amplification. These libraries were sequenced on the Illumina sequencing platform (HiSeq X Ten) and 150 bp paired-end reads were generated.
The Cell Ranger software pipeline (version 2.2.0) provided by 10 × Genomics was used to demultiplex cellular barcodes, map reads to the genome and transcriptome using the STAR aligner, and down-sample reads as required to generate normalized aggregate data across samples, producing a matrix of gene counts versus cells. We processed the unique molecular identifier (UMI) count matrix using the R package Seurat (version 2.3.4). To remove low quality cells, we filtered those cells which with UMI larger than 8000 and gene lower than 2000. And also we discarded low-quality cells where > 20% of the counts belonged to mitochondrial genes. After applying these QC criteria, 11,083 single cells and 20,118 genes in total remained and were included in downstream analyses. Library size normalization was performed in Seurat on the filtered matrix to obtain the normalized count. Top variable genes across single cells were identified using the method described in Macosko et al. [
31]. Principal component analysis (PCA) was performed to reduce the dimensionality on the log transformed gene-barcode matrices of top variable genes. Cells were clustered based on a graph-based clustering approach, and were visualized in 2-dimension using tSNE and UMAP. Likelihood ratio test that simultaneously test for changes in mean expression and in the percentage of expressed cells was used to identify significantly differentially expressed genes between clusters. Enriched biological function were analysis with DAVID website and fgsea packages in R.
Tumorigenicity
All BALB/c nude mice experiments were carried out in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, and were approved by the Zhejiang University Animal Care and Use Committee (ZUACUC) (Project License 11627). To determine the oncogenicity of the ZJU-0430 cell line, cells were subcutaneously injected into the hind flanks of four nude mice (BALB/c nu; 4–6 weeks old) (SLAC Laboratory Animals Company, Shanghai, China), Mice were maintained in laminar flow cabinets under specific pathogen-free conditions. A total of 1 × 106 cells were injected using a 27-gauge needle and the tumours monitored at intervals using digital callipers. After 3 weeks, mice was killed by cervical dislocation under sodium pentobarbital (45 mg/kg) anaesthesia. Tumours were removed, fixed in 10% formalin and subjected to pathological examination and IHC evaluation.
Light and electron microscopic analysis
Both the morphology and ultrastructure of the ZJU-0430 cells were examined following seeding into 25-mm
3 tissue-culture flasks. The general morphology of the cells was observed daily under a phase-contrast microscope and histopathologically compared to that of the original tumours. Electron microscopy was conducted as previously described [
32] using the GBC-SD cell line as the control. Cells were harvested, pelleted, fixed (2.5% glutaraldehyde), post-fixing (2% osmium tetraoxide) and then embedded in EPON resin. Ultrathin sections were stained and examined under a HT7700 microscope (Hitachi, Tokyo, Japan).
In vitro growth kinetics and cell cycle analysis
The cell growth curve analysis was obtained, as described previously [
33]. The ZJU-0430 cells and GBC-SD cells were plated at 5000 cells per well, cultured at 12 h intervals for 3 consecutive days, and cell growth determined using an MTT assay, respectively. The growth curve was plotted, and doubling time was calculated as described (
http://www.doubling-time.com/index.php).
ZJU-0430 and GBC-SD cells were plated, harvested, washed twice with cold DPBS, and fixed overnight in 70% ethanol at 4 °C, treated for 30 min with RNase A (2 μg/ml), and then with propidium iodide (PI) at room temperature at the dark. The cell cycle distribution was determined using a FACSCanto™ II flow cytometer (Becton–Dickinson, Mount View, VA, USA) and analysed using appropriate software.
Cytogenetic analysis and STR genotyping
Cells at the primary and 100th passage were used for analysis. Karyotyping was performed as described previously [
34]. Briefly, cells were treated with 0.02 μg/ml Colcemid for 90 min at 37 °C until exponential proliferation was reached. The cell pellet was resuspended with potassium chloride (0.075 mol/l), incubated for 20 min at 37 °C, fixed in Carnoy’s solution (methanol: acetic acid = 3:1 by volume), and analyzed using trypsin G banding.
To verify that ZJU-0430 cell line was indeed derived from humans and had no-contamination, genomic DNA from ZJU-0430 cell lines was extracted using the Gentra Puregene Cell Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s protocol. STR profiling was performed by amplifying eighteen loci simultaneously in a single tube and analyzing by capillary electrophoresis on an ABI Prism® 3500 × 1 Genetic Analyzer. Data were matched with the American Type Culture Collection (ATCC) STR database (ATCC sales order number: SO0146233).
Test for mycoplasma contamination
The cell-culture supernatant was collected and evaluated using the PCR Mycoplasma Test Kit (HuaAn Biotechnology, Hangzhou, Zhejiang, China), following the manufacturer’s instructions. PCR products were separated by electrophoresis in 1% agarose and documented by photography.
Immunophenotypic characterization of ZJU-0430 cells by flow cytometry
The primary and 100-passage cells were collected for flow cytometry analysis using a FACSCanto™ II flow cytometer (Becton–Dickinson, Mount View, VA, USA), respectively. The panel of monoclonal antibodies used included those specific for CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184, CD326, and CD338. All antibodies and reagents were purchased from BioLegend (San Diego, CA). Data were obtained using FCS Express Software (De Novo Software, Los Angeles, CA). Antigen expression was scored as positive based on a significant shift in staining in comparison to corresponding isotype control.
Wound-healing assays
This procedure was conducted by the Cell Comb™ Scratch Assay according to the manufacturer’s instructions (Merck, Darmstadt, Germany). Briefly, a wound was created in the cell monolayer using cell comb, when the cells reached semi-confluence, and they were cultured thereafter for an additional 72 h at 37 °C and 5% CO2. Eight scratched fields were randomly chosen for cell counting.
Migration and invasion assays
The in vitro migration and invasion assays were performed as previously described [
35,
36]. Briefly, 1 × 10
4 cells were seeded into cell-culture insert wells (Corning Incorporated, Corning, NY, USA) with or without Matrigel-coating (BD Biosciences, Bedford, MA, USA) in RPMI 1640 medium. The extent of migration and invasion was determined after cultured for 48 h. The cells on the lower surface were fixed and stained with Giemsa solution.
A colony formation assay was used to assess the proliferative ability of the ZJU-0430 cells. A single-cell suspension was prepared and plated on 6-well plates (200 cells/well) in triplicate and cultured for 14 days. The colonies were stained with 1% crystal violet for 30 s after fixation with 4% paraformaldehyde for 5 min.
Immunostaining
Tissue section slides from original patient (GBC and gastric cancer), ZJU-0430 tumours grown in mice, and cytospins of ZJU-0430 cells were used in this experiment as described previously [
37,
38]. For cytospins, ZJU-0430 cells were plated onto glass coverslips, fixed with 4% paraformaldehyde (PFA), blocked with 10% goat serum, incubated with primary antibodies (vimentin, CK-pan, CK7, and CK19 all from cell signaling technology (CST)) at 4 °C overnight, followed by the addition of immunofluorescent labelled antibodies [FITC/PE goat anti-rabbit IgG (H + L)]. For tissue slides, 5 μm sections were fixed onto the slides, dewaxed, rehydrated, heated for antigen retrieval, incubated in 3% hydrogen peroxide to inactivate endogenous peroxidase, blocked with 5% BSA, and then incubated with primary antibodies specific for the following proteins, MUC1, MUC2, MUC4, MUC5AC, MUC6, AFP, Hepatocyte, Glypican-3, GS, CK20, CAD17, CDX2, beta-catenin, STAB 2, vimentin, MMP-2, MMP-9, CK7, and CK19. A 3,3′ diaminobenzidine tetrahydrochloride (DAB) horseradish peroxidase colour development kit (Boster) was used for visualizing target protein and Mayer’s haematoxylin (Boster) was used for counterstaining.
Discussions
Human GBC is an extremely malignant disease owning to its relative rarity, histological heterogeneity, and proximity to vital structures. Surgical resection, and chemoradiotherapy are ineffective [
1,
8,
40], and the median survival is less than 10 months [
41] while the 5-year survival rate is less than 5% [
42]. In recent years, a series of human carcinoma GBC cell lines have been established from peritoneum effusion, surgical and autopsy tissues, biopsy specimens, and metastatic lesions [
19,
26,
27,
37,
43,
44]. Novel GBC cell lines provide an ideal experiment model to investigate the biological behavior of GBC cells and may aid in the development of novel adjuvant therapies, potential anticancer agents, or new diagnostic strategies.
Cell lines derived from primary tumours are more likely to reflect the characteristics of the original primary tumour more accurately. Based on this idea, here we report the establishment of a GBC cell line designated as ZJU-0430, which was derived from a 74-year-old male patient who had some serum ferritin abnormalities, and GBC that was a well-differentiated adenocarcinoma. The cell grew in adherent monolayer pattern with at least two morphologies that co-existing. scRNA-Seq results revealed that ZJU-0430 cell line was no too much heterogeneity although the morphological difference. Optical microscopy, ultrastructural examination, immunostaining of epithelial markers for positive (CK-pan, CK7, and CK19) and negative mesenchymal expression (vimentin), and H&E staining of cells displayed features that were typical of malignant epithelial cells. The STR genotyping analysis showed the ZJU-0430 cell line was of human origin and that it differed from other established cell lines. A mycoplasma examination confirmed ZJU-0430 to be free of mycoplasma. The PDT was approximately 19.81 h, which was shorter than GBC-SD cells. Taken together, these results suggest that ZJU-0430 cell line is a representative of the disease and may be used for pre-clinical investigation of the pathogenesis and treatment of GBC.
Recently, Devendra Chaudhary, et al. reported that 46.7% of GBCs have an abnormal karyotype [
45]. In the present study, the karyotypes of ZJU-0430 cell line were found to be near-triploid both at primary and at P100, with the structural aberrations including gains, losses, and additional material of unknown origin on chromosomes 6 and 17 (one derivative chromosome 6 and two derivative chromosome 17) resulting from an unbalanced translocation with others chromosomes (chromosome 6 to an unknown chromosome, and chromosome 17 to the short arm of chromosome 5). An isochromosome was found for the short arm of chromosome 21; and two unknown marker chromosomes were also detected. None of these structural aberrations matched those in the previous cytogenetic studies of GBC [
26,
37]. Cytogenetic abnormalities with mutations are usually related to oncogenic activation; therefore, further analysis may yield potential molecular targets in the context of this disease.
Mycoplasma can impede a whole range of cellular properties and functions and hinders the use of cells and their derived products in pharmacological area [
46]. The culture supernatant derived from the ZJU-0430 cells was subjected to PCR analysis, and was found to be mycoplasma free. We have therefore identified the ZJU-0430 cell line as a useful tool to study GBC.
Although numerous GBC cell lines have been documented, including G-415, NOZ, KMG-A, GBK-1, FU-GBC-1, FU-GBC-2, PTHrP-GBK, GB-d1, TGBC1TKB, TGBC2TKB, OCUG-1, TUGBK-1, HAG-1, GBC-SD, SGC-996, EH-GB1, EH-GB2 and TJ-GBC2 [
11‐
20,
22,
23,
25,
27,
37,
47,
48], the surface markers present in GBC have yet to characterized. Meanwhile, cancer stem cells have highly clinically relevant in the development, progression, aggressiveness, recurrence, and metastasis of tumours. Our study is the first to characterize progenitor cells in a newly established GBC cell line. Our results are consistent with other reports [
49‐
51] and since we found these cells to be positive for CD24, CD44, CD29, and CD133, partially positive for CD184, and CD326, and negative for CD34, CD90, CD117, and CD338. Taken together, these data showed that ZJU-0430 cell had stemness properties.
GBC is highly aggressive in patients [
52]; therefore, the malignant phenotype of ZJU-0430 cell line was explored both in vitro and in vivo. Our data showed that the migration, invasion, and proliferation abilities of ZJU-0430 are much greater than of GBC-SD cells, and in addition there were higher levels of expression of metastatic-related marker MMP-2. The cells were capable of forming solid tumours that were histologically identical to the original surgical specimen, indicating that the GBC cell line could be used as a reliable model for in vivo preclinical research.
An immunoblotting assay showed ZJU-0430 cells to be positive for biliary tract cancer epithelial marker (CK-pan, CK7, and CK19), which are found to be positive at high rates in real GBC specimens. The mesenchymal marker vimentin was also tested, but it is not found to be expressed. Since the patient (source of ZJU-0430) had been diagnosed with gastric carcinoma before GBC, gastric carcinoma markers were also explored on the xenograft tumour. The gastric carcinoma markers MUC2, MUC5AC, and MUC6 were expressed weakly or not at all. Since the colon and liver are contiguous with each other, the origin of ZJU-0430 cell was a little unclear. In this study, liver markers (AFP, Hepatocyte, Glypican-3, and GS) and colon markers (CK20, CAD17, CDX2, β-catenin, and SATB2) were used for staining of xenograft-derived tumours, with the result that all markers were either expressed only weakly, or were not express at all. In addition, the colon cancer marker CDX2 protein were also not found to be expressed on original GBC. Therefore, ZJU-0430 cell line was verified to be originated from the gallbladder primary site.
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