Cell lines and cell culture
The doxycycline-inducible T-Rex/K-RasG12V cells (source: fetus) were constructed as previously described  and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% tetracycline-free fetal bovine serum (FBS). Panc-1 (source: male), SW1990 (source: male), HCT116 (source: male), and the hTERT (human telomerase reverse transcriptase) immortalized HPNE cell lines (source: male) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were cultured in DMEM with 10% FBS, except for HCT116 which was cultured in McCoy’s 5A medium with 10% FBS. HPNE cells stably transfected with mutant K-RasG12V were provided by Prof. Paul Chiao (MD Anderson Cancer Center, Houston, TX, USA) and cultured in DMEM with 10% FBS as previously described . All cell lines were confirmed to be mycoplasma-negative (LookOut mycoplasma polymerase chain reaction [PCR] detection kit, Sigma, St. Louis, MO, USA), and authentication of cell lines was performed by STR (short tandem repeats) genotyping (Microread Genetics, Beijing, China). Doxycycline, glucose oxidase, and catalase were purchased from Sigma (St. Louis, MO, USA). Human recombinant IL-1α was from Thermo Fisher Scientific (Rockford, IL, USA). Neutralizing IL-1α antibody was from R&D systems (Minneapolis, MN, USA; #MAB200).
Human pancreatic cancer tissues were provided by Shanghai Outdo Biotech (Shanghai, China) as a sample array containing 15 de-identified tumor samples. De-identified serum samples from healthy donors and pancreatic cancer patients were obtained from the tissue bank of Sun Yat-sen University Cancer Center (Guangzhou, Guangdong, China). Studies using de-identified human samples were reviewed and approved by Committee for Ethical Review of Research involving Human Subjects of Sun Yat-sen University.
Quantitative reverse transcription-polymerase chain reaction
Total RNA was isolated using Trizol (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. RNA was reversely transcribed using the Primer Script RT reagent Kit with gDNA Eraser (Takara Bio Inc, Kusatsu, Shiga, Japan). The primer sequences for human CD137 were 5′-TCCGCAGATCATCTCCTTCT-3′ (forward) and 5′-AGTTTCTTTCTGCCCCGTTT-3′ (reverse). The primer sequences for human IL1A were 5′-TGTGACTGCCCAAGATGAAG-3′ (forward) and 5′-CCCAGAAGAAGAGGAGGTTG-3′ (reverse). The elongation factor 1 (EF1) was used as the reference gene; primer sequences for EF1 were 5′-GCTTCACTGCTCAGGTGAT-3′ (forward) and 5′-GCCGTGTGGCAATCCAAT-3′ (reverse). Real-time PCR was performed using the SYBR Premix Ex Taq RNase H+ kit (Takara) and analyzed using the Bio-Rad detection system (Bio-Rad, Hercules, CA, USA). The samples were first incubated for 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. The results were calculated using the formula 2−(Ct target−Ct EF1) and matched to the control samples.
The level of soluble CD137 (sCD137) in serum samples from pancreatic cancer patients and healthy donors was measured using enzyme-linked immunosorbent assay (ELISA) kit (Ray Biotech, Norcross, USA; #ELH-TNFRSF9) according to the protocol provided by the manufacturer.
The small interfering RNAs (siRNAs) against MAPK1 and p65 (RelA) were designed and synthesized by RiboBio (Guangzhou, Guangdong, China). The sequences of siRNAs against MAPK1 are 5′-CGAGCAAATGAAAGATGTA-3′ and 5′-CAAGAAGACCTGAATTGTA-3′. The sequences of siRNAs against p65 are 5′-CTTCCAAGTTCCTATAGAA-3′ and 5′-GGACATATGAGACCTTCAA-3′. Cells were incubated with doxycycline to induce K-Ras expression for 48 h before siRNA transfection, using lipofectamine RNAi Max reagents (Invitrogen) according to the manufacturer’s instructions. Transfection was performed for 24 h with a 100 nmol/L siRNA solution in the presence of doxycycline. Assays for the expression of target molecules were performed 48 h after the transfection.
For the detection of membrane CD137, cells were fixed with 4% formaldehyde in phosphate buffer saline (PBS) and stained with primary antibodies with a dilution of 1:100 for 2 h at room temperature. Rabbit anti-human CD137 antibody (#62634; Cell Signaling, Danvers, MA, USA) was used. Cells were then washed and incubated for 30 min at room temperature with PBS containing anti-rabbit immunoglobulin G (IgG) (#11-4839; eBioscience, San Diego, CA, USA) antibody coupled with FITC. Cells were then collected and washed twice with PBS before flow cytometry analysis (Gallios; Beckman Coulter, Brea, CA, USA). For each experiment, at least 10,000 cells per sample were analyzed using FlowJo software (https://www.flowjo.com).
Pancreatic cancer tissue microarrays (Shanghai Outdo Biotech, Shanghai, China) were first dried at 58 °C for 1 h, dewaxed and rehydrated before epitope retrieval by heating at 100 °C in 10 mmol/L sodium-citrate (pH6.0) for 4 min. The sections were cooled down to room temperature for 30 min. To eliminate the endogenous peroxidase and alkaline phosphatase activity, the tissue sections were treated with 3% hydrogen peroxide for 20 min. The sections were then incubated with individual primary antibodies overnight, followed by incubation with secondary antibodies for 1 h. 3,3′-diaminobenzidine (DAB) was then applied as a substrate to reveal the antigen. Hematoxylin was used for counterstaining. The primary antibody used in this study was rabbit anti-CD137 (#62634; Cell Signaling). All other reagents were from ZSGB-Bio (Beijing, China).
Illumina HiSeq_RNASeqV2 RSEM normalized gene expression profiles for human pancreatic adenocarcinoma were retrieved from The Cancer Genome Atlas (TCGA) Pan-Cancer atlas (paad_tcga_pan_can_atlas_2018) using the Cancer Genomics Data Server (CGDS)-R (R functions for querying the CGDS) package. A total of 166 samples with expression data for CD137 and IL1A were included for analysis.
All experiments were repeated at least three times. Q–Q plots were used to compare and determine data distribution. Data are expressed as mean ± SEM (standard error of the mean) unless otherwise specified. Student t-tests were used to evaluate the statistical significance of the difference between two groups of samples with normal distributions. Despite a large sample size, the relationship between CD137 and IL1A expression in human pancreatic carcinoma tissues was assessed using a Spearman’s rank correlation because of the nature of data (integer scores). Statistical analyses were performed using the GraphPad Prism software (San Diego, CA, USA). No statistical method was used to calculate sample sizes, which were determined empirically. All tests were two-tailed, and a P value of 0.05 or less was considered statistically significant.