Cell culture
Esophageal epithelial cell line (HEEC) and ESCA cells (TE-1 and OE19) were purchased from TongPai Biotechnology (Shanghai, China). All these cells were maintained in the culture medium containing 90% Roswell Park Memorial Institute (RPMI)-1640 (Gibco, Grand Island, NY, USA), 10% fetal bovine serum (Gibco), and 1% penicillin-streptomycin (Beyotime, Shanghai, China) in an incubator at 37°C with 5% CO2.
To downregulate PD-L1 expression, 15 µg/mL anti-PD-L1 antibody (αPD-L1) was added into the culture medium for incubation of 48 h.
Cell viability determination
HEEC, TE-1, and OE19 cells were seeded into 96-well plates and incubated with 0, 100, 200, 400, 800, and 1000 µM of I3C (Fig. 1A; MCE, Monmouth Junction, NJ, USA) for 24 h. After that, 10 µL of cell counting kit8 (CCK8) solution from the CCK8 kit (Beyotime) was added to incubate with the cells for 3 h. The absorbance was measured at 450 nm using a microplate reader.
Flow cytometry
Peripheral blood mononuclear cells (PBMCs) were purchased from Procell (Wuhan, China) and were cultured in RPMI-1640 medium. To activate T cells, PBMCs were incubated with Dynabeads Human T-Activator CD3/CD28 (Gibco) for 3 days. The cells after activation were co-cultured with TE-1 or OE19 cells treated with I3C at 37°C for 2 days. The harvested PBMCs were fixed and permeated using the BD Cytofix/Cytoperm fixation/permeabilization solution kit (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The cells were stained with FITC conjugated anti-mouse CD3 and APC conjugated anti‐mouse CD8 at 4°C for 30 min in the dark. The samples were analyzed using a flow cytometer.
Cytotoxicity analysis
PBMCs were incubated with TE-1 or OE19 cells at 37°C for 6 h. Cytotoxicity was evaluated using a lactate dehydrogenase (LDH) cytotoxicity assay kit (Beyotime). Briefly, the cell culture medium was centrifuged at 400 g for 5 min. The supernatant was incubated with 60 µl LDH working solution at room temperature for 0.5 h in dark place. The absorbance was measured at 490 nm.
Enzyme-linked immunosorbent assay (ELISA)
The supernatant obtained after centrifugation of the cell culture medium was used for ELISA. The levels of interferon (IFN)-γ and interleukin (IL)-2 were measured using the human IFN-γ ELISA kit and human IL-2 ELISA kit, respectively, according to the manufacturer’s instructions.
Bioinformatic analysis
The proteins that can be bound with I3C were predicted using the SwissTargetPrediction tool. The expression of CES1 in ESCA was predicted using the GEPIA and UALCAN online databases. The prognosis analysis with high or low CES1 was performed using the Kaplan-Meier Plotter database.
Cell transfection
TE-1 and OE19 were placed in six-well plates and cultured until over 70% cell confluence. CES1 shRNA (shCES1) and its negative control (shNC) purchased from GenePharma (Shanghai, China) were transfected into cells using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in line with the manufacturer’s protocols. After 48 h, the cells were harvested.
Quantitative real-time polymerase chain reaction (qPCR)
TRIzol reagent (Invitrogen) was used to extract total RNA from ESCA cells. RNA concentration was detected by a NanoDrop 2000 system (Thermo Fisher Scientific, Waltham, MA, USA). About 1 µg RNA was reversed to cDNA using the AdvanceFast 1st strand cDNA synthesis kit (Yeasen, Shanghai, China). mRNA expression was measured using the Hieff qPCR SYBR green master mix (Yeasen) and calculated using the 2−ΔΔCt method.
RNA binding protein immunoprecipitation (RIP)
GesSeq RIP kit (CloudSeq, Shanghai, China) was used. ESCA cells were lysed using the lysis buffer and the lysate was collected after centrifuging at 14000 g for 10 min. In addition, PGM beads were mixed with 5 µg antibodies against IgG and anti-CES1 at room temperature for 30 min. The lysate was incubated with bead-antibody at 4°C for a night. After washing, RNA was purified and PD-L1 expression was measured using qPCR.
Dual-luciferase reporter assay
PD-L1 sequences were inserted into the pGL3 basic vector (Promega, Madison, WI, USA) to synthesize wild-type (WT)-PD-L1. Similarly, mutant PD-L1 sequences were inserted into the same vector to synthesize mutant (MUT)-PD-L1. TE-1 and OE19 cells were transfected with WT-PD-L1 or MUT-PD-L1, vector/shNC or CES1 overexpression plasmids/shCES1, and pRL-TK vector (Promega) using lipofectamine 2000 for 48 h. The firefly and Renilla luciferase activities were measured using the dual luciferase reporter gene assay kit (GenePharma), and relative luciferase activity was calculated by firefly/Renilla luciferase activity ratio.
Data analysis
All data were analyzed using the GraphPad Prism 8.0 software. Results are represented as mean ± standard deviation. The difference between two or more groups was analyzed using the unpaired student’s t-test or one-way ANOVA. P value < 0.05 was set as statistical significance.