Cell culture and human sample collection
Human tumor cell lines GBC-SD were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), SGC-996 was obtained from Tongji university (Shanghai, China). The other human GBC cell lines EH-GB1 and NOZ were purchased from the Health Science Research Resources Bank(Osaka, Japan). GBC-SD, EH-GB1, and SGC-996 were cultured in DMEM,and NOZ cell line was cultured in William’s medium. All cells were cultured in their corresponding media supplemented with 10% fetal bovine serum (FBS)at 37°C in a humidified atmosphere of 95% air and 5% CO2.All 49 GBC tissues were collected from patients who underwent radical surgery at the Xinhua Hospital Affiliated to Shanghai Jiao Tong University
School of Medicine, Shanghai, China. We retrospectively obtained clinicopathological data from these patients, including age, sex, T stage regional lymph node status, TNM stage, and differentiation. The usage of these specimens and the patient data were approved by the Ethics Committee of the Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.
Human peripheral blood neutrophil isolation
Whole blood was collected from patients with GBC and layered over PolymorphprepTM(Axis-Shield, Norway). After centrifugation at 500 ×g for 30 min at 18–22°C, the top band at the sample-medium interface comprised of mononuclear cells and the lower band comprised of polymorphonuclear cells were separated while pelleted erythrocytes were discarded. The polymorphonuclear fraction was then diluted with one volume of 0.45% NaCl solution or culture medium at 0.5 N to restore normal osmolality. The cell suspension was transferred to a 15ml tube. After centrifugation at 400 ×g for 10 min, the cells were washed with the medium followed by spinning down again and finally resuspended in culture medium.
NETs generation
For the in vitro NET generation assay, 1×105neutrophil were cocultured with 5×105tumor cells for 3.5 h in RPMI1640 complete medium at 37°C and 5% CO2.
IHC and immunofluorescence
IHC was performed to assess SCEL and Ki-67 expression in GBC tissues. In brief, paraffin-embedded sections of GBC and cholecystitis tissue were deparaffinized and then heated in a pressure pot for 2.5 min to retrieve antigens. Thereafter, the sections were incubated with primary anti-SCEL andanti-Ki-67 antibodies (1:100, Abcam, Burlingame, CA, USA) overnight at 4°C.Antibody binding was detected using a peroxidase–conjugated secondary antibody at37°C for 40 min. A DAB Substrate Kit was used to perform the chromogenic reaction. The intensity of the staining was evaluated in accordance with the following criteria: 0, negative; 1, low; 2, medium; 3, high. The extent of staining was scored as follows: 0, 0% stained; 1, 1–25% stained; 2, 26–50%stained; 3, 51–100% stained. Five random fields (20× magnification) were evaluated using a light microscope. The final scores were determined by multiplying the scores of the intensity with those of the extent and dividing the samples into four grades: 0, negative (-); 1–2, low staining (+); 3–5, medium staining (++); 6–9, high staining (+++)(15) (16).Immunofluorescence staining was performed to detect the neutrophils. In brief, NETs were added on 13-mm glass coverslips (Fisher Scientific) and allowed to adhere overnight at 4℃on coverslips followed by culture in the presence of anti-citH3(1:100,NOVUS, USA) and NE (1:100, NOVUS) antibody at 4°C over night. Thereafter, cells were incubated with fluorescent dye-labelled secondary antibodies at room temperature for 1 h. The cells were again incubated with anti-fade DAPI solution (1:1000), and images were captured with a confocal fluorescence microscope.
Establishment of an in vivo tumor model
All animal studies were approved by Xinhua Hospital Ethics Committee Affiliated to Shanghai Jiaotong University School of Medicine, and 4-week-oldfemale BALB/c nude mic mice were maintained in a barrier facility on high-efficiency particulate air-filtered racks. Tumor cells were harvested via trypsinization, washed in PBS, and resuspended at 1×107 cells/ml in Matrigel. A total of 1×106 cells were subcutaneously injected into each mouse to develop
tumors as previously described (2). Tumor size was measured weekly.
RT-PCR
The total RNA of the cells was extracted with TRIzol (Invitrogen) in accordance with the manufacturer's instructions. Thereafter, the mRNA was reverse-transcribed to single-stranded cDNAs using a reverse-transcription PCR (RT-PCR) system (TaKaRa). The primers were listed in Supplementary Table S1. Thereafter, real-time fluorescent quantitative PCR or semi-quantitative PCR was performed to analyze the gene expression levels.
Western blotting
Whole-cell extracts were prepared by lysing the cells with RIPA lysis buffer supplemented with a proteinase inhibitor cocktail (Sigma). In total, 30 mg protein lysate was separated via SDS–PAGE and then the target proteins were detected via western blot analysis with the following antibodies: anti-SCEL,anti-PI3Kα110, anti-EGFR, anti-P-AKT(ser473),anti-pan-AKT, anti-FLAG, andanti-β-actin (Supplementary Table 1).
CCK8 assay
CCK8 assays were performed in accordance with the manufacturer’s instructions. In brief, 1,000 cells in 100ul culture were added into each well of a 96-well plate. The plate was incubated for a period (0, 1, 2,3, 4, and 5 d) in an incubator and then 10 ul of CCK-8 solution was added to each well of the plate. The plate was again incubated for 2 h in the incubator and the absorbance was measured at 450 nm using a microplate reader. To per form colony formation assay, GBC cells were seeded on six-well plates (500cells/well). After culturing for 2 weeks, colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% Crystal Violet (Beyotime)for 20 min. Thereafter, stained colonies were photographed and the numbers of colonies (>50 cells/colony) were counted after rinsing twice with PBS. Three independent experiments were performed.
Plasmid construction and stable cell line establishment
The complete coding region of human SCEL (NM_003843.4) was cloned into pCMV Puro vector. Lentiviruses were produced in 293T cells for the stable transfection of the cell lines, in accordance with the manufacturer’s instructions, and an empty vector was transfected into cells as a control. In total, 1×105 tumor cells in 2 ml medium with 8 μg/ml polybrenewere infected with 1 ml lentivirus supernatant. After 48 h, cultures were supplemented with puromycin (4 μg/ml) for 3 d for drug-resistance selection.
Co-immunoprecipitation assays (Co-IP)
EH-CB1 cells were transfected with different expression plasmids and lysed. Cell lysates were incubated with anti-FLAG antibody conjugated beads at 4°C overnight. The beads were washed with lysis buffer three times followed by western blotting.
ELISA
GBC cell lines (2 × 105 cells) were implanted in 6-well plates and cultured for 72 h, and the conditioned medium was collected after centrifugation at 700g for 5 min at 4°C.IL8protein was quantified using IL8 kit(BOSTER Systems) according to the manufacturer’s instructions. The same culture medium was used as a control.
Statistical analysis
Statistical analyses were performed using the IBM SPSS Statistics Program. Each experiment was performed in triplicate, and the values are presented as the mean ± SD, unless otherwise stated. The variance between the groups was statistically compared. Student’s t-test was used to compare the mean values. Kaplan–Meier curves were analyzed for relevant variables. The log-rank test was used to analyze the differences in survival times among the patient subgroups. All probability values had a statistical power of 90%, and a 2-sidedlevel of 5%. A P-value < 0.05 was considered statistically significant.