Cell line and culture conditions
The human pancreatic cancer Panc-1 cell line and the human lymphocyte U937 cell line were acquired from American Type Culture Collection. KPCLUC, a mouse pancreatic cancer cell line with luciferase expression was isolated from KrasG12D, Trp53R172H, Pdx-1-Cre (KPC) C57BL/6 mice. All cells were cultured in RPMI 1640 medium (Hyclone, SH30027.02) supplemented with 10% fetal bovine serum (Hyclone), 2 mM L-glutamine, 100 units/mL penicillin, and 100 units/mL streptomycin at 37℃ in a humidified atmosphere of 5% CO2.
The syngeneic orthotopic mouse model
The Cybb-/- mice (B6.129S-Cybbtm1Din/J, No. 002365) purchased from Jackson Laboratory were backcrossed to the C57BL/6 background. After the genetic location confirmation, mice were kept in the Laboratory Animal Center's under pathogen-free conditions at National Cheng Kung University (NCKU). Experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of NCKU (IACUC NO.:103150). Luciferase expressing KPC cells (KPCLUC, 1×107 in 50 mL serum-free medium) were orthotopically injected into the pancreas of aged 8-12 weeks male Cybb knockout (Cybb−/−) mice, or Cybb wild type (WT) C57BL/6 mice.
Isolation and differentiation of murine bone-marrow derived macrophages
Bone marrow cells were isolated from flushing femurs and tibias of 8-12 weeks old WT and Cybb-/- mice. Cell aggregates were dislodged by gentle pipetting, and debris was removed by passing the suspension through a 70 μm nylon web. Cells were washed twice with HBSS, adjusted to the density of 106 cells/mL, seeded into 10 cm low attachment surface dishes containing RPMI medium supplemented with 20 ng/μL rmGM-CSF (PeproTech, 315-03), and cultured at 37 ℃ in a humidified incubator supplied with 5% CO2.
Immunohistochemistry analysis
Paraffin-embedded sections were deparaffinized, and antigens were unmasked by autoclaving at 121℃ for 10 min in sodium citrate buffer (10 mM; pH 6.0). The sections were incubated with primary antibodies at 4℃ overnight followed by incubation with secondary antibodies at room temperature for 30 min. The immunoreactivity was visualized using a DAB chromogen system (DAKO).
Immunofluorescence staining
After the approval of the Institutional Review Board of NCKU Hospital, the pancreatic cancer tissue microarray (TMAs) were constructed from paraffin-embedded blocks of 187 pancreatic cancer patients resected in NCKU Hospital. TMA paraffin blocks were cut to obtain 5-μm sections. TMA sections were stained with primary antibodies at 4℃ overnight followed by incubation with fluorescent-conjugated secondary antibodies at room temperature for 1 hour. The nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI). The panoramic images of IF-stained TMAs were acquired using the FACS-like Tissue Cytometry system (Tissue Gnostics, Vienna, Austria). The percentage of stained cells was quantified using the TissueQuest system software TissueFAX (Tissue Gnostics, Vienna, Austria).
RNA isolation and quantitative PCR (qPCR)
Total RNA isolation from cultured cells was carried out using Trizol (Invitrogen). The purity and concentration of RNA were determined by the Nanodrop spectrophotometer (Thermo Fisher Scientific; ND-1000). The total RNA from each sample was reverse transcribed into 2mg cDNA using the Deoxyt HiSpec Reverse Transcriptase (Yeastern Biotech). Quantitative assessment of mRNA levels was done by qPCR using KAPA qPCR SYBR Green Master Mix (KAPA) on the StepOne Real-Time PCR System (Applied Biosystems). The reaction conditions were as follows: initial denaturation at 95℃ for 5 min, 40 cycles of denaturation at 95℃ for 10 seconds, and annealing at 60℃ for 10 seconds. The expression of mRNA was determined by the 2–ΔΔCt method (fold difference) and normalized to GAPDH. Human PCR primers used in this study included IL-12p40, forward 5’ -CAGAAGCTAACCATCTCCTGGTTTG-3’ , reverse 5’ -CCGGAGTAATTTGGTGCTCCACAC-3’ ; IL-10, forward 5’ - GATGCCTTCAGCAGAGTGAA-3’ , reverse 5’ - GCAACCCAGGTAACCCTTAAA-3’ ; IFN-γ, forward 5’ -GGCTTTTCAGCTCTGCATCG-3’ , reverse 5’-ATGGGTCCTGGCAGTAACAG-3’ ; CCL2, forward 5’ - AGCCAGATGCAATCAATGCC-3’ , reverse 5’ -GACACTTGCTGCTGGTGATTC-3’ ; IL-17D, forward 5’ -CTTGCGTGGCCACATTACAG-3’ , reverse 5’ - CCGGGTCGTAGGAGATTTCA-3’ ; VEGFA, forward 5’ - ACGAAAGCGCAAGAAATCCC-3’ , reverse 5’ -CTCCAGGGCATTAGACAGCA-3’ ; TGFβ1, forward 5’ -CTGTATTTAAGGACACCCGTGC-3’ , reverse 5’ - TGACACAGAGATCCGCAGTC-3’ ; IL-4, forward 5’ - TGCACCGAGTTGACCGTAA-3’, reverse 5’ -TCAGTTGTGTTCTTGGAGGCA-3’ ; HDAC2, forward 5’ - TGGCCTTTCTGAGCTGATTTT-3’ , reverse 5’ -AGCCACTGAAACAAGACTTCAT-3’; CYBB, forward 5’ -GGAGTTTCAAGATGCGTGGAAACTA-3’ , reverse 5’ -GCCAGACTCAGAGTTGGAGATGCT-3’ ; and GAPDH, forward 5’ -GGAGCGAGATCCCTCCAAAAT-3’ , reverse 5’ - GGCTGTTGTCATACTTCTCATGG-3’ . Mouse PCR primers were: Il-12b, forward 5’ -ATTACTCCGGACGGTTCACG-3’ , reverse 5’ -GACAGAGACGCCATTCCACA-3’ ; Nos2, forward 5’ -AGAGCCACAGTCCTCTTTGC-3’, reverse 5’ - CTGGTCCATGCAGACAACCT-3’ ; Il-10, forward 5’ - GCATGGCCCAGAAATCAAGG-3’ , reverse 5’ -GAGAAATCGATGACAGCGCC-3’ ; Ym-1, forward 5’ -AGGAAGCCCTCCTAAGGACA-3’ , reverse 5’ - CTCCACAGATTCTTCCTCAAAAGC-3’ ; Hdac2, forward 5’ - CCCGTCAGCCCTCTTGTC-3’ , reverse 5’ - TTTCTTCTTGCCGCCTCCTT-3’ ; and Gapdh, forward 5’ -AAGGTCATCCCAGAGCTGAA-3’ , reverse 5’ - CTGCTTCACCACCTTCTTGA-3’ .
ROS detection
To measure ROS levels, 2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (DCFDA) was used. Cells were incubated with 1 μM of DCFDA at room temperature for 30 min followed by washing and resuspension in FACS buffer. Fluorescence was analyzed using a flow cytometer (FACS Canto II).
Analysis of M1 and M2 surface marker expression by flow cytometry
For macrophage phenotype identification, M1 and M2 surface marker expression was detected using flow cytometry. Anti-CD68-BV421 (BD), anti-CD86-APC (BD), and anti-CD204-PE (BD) antibodies were used to identify human macrophage phenotypes, and anti-F4/80-Alex488 (Biolegend), anti-CD206-AF647 (BD), and anti-CD11b-PE (BD) antibodies were used for murine macrophages. Cells (2´105) were stained with antibodies for 15 min. After wash with PBS, cells were analyzed by a flow cytometer (FACS Canto II).
Enzyme-linked immunosorbent assay
Blood samples were collected from the mice and centrifuged at 1,000´g for 10 min to obtain the serum. The secretion levels of Tnf-α, Il12p40, Il10, and Il4 in the serum were measured using ELISA MAXTM Deluxe Sets (No. 430904, 431604, 431414, and 431104; Biolegend) according to the manufacturer’s protocol.
Lentiviral-based stable cell line generation
CYBB, STAT6, HDAC2, and non-target short hairpin RNA (shRNA) vectors were purchased from the National RNAi Core Facility, Academia Sinica, Taipei, Taiwan. Lentiviral transfection with shRNAs in U937 cells was performed in the presence of 8 μg/mL polybrene (Sigma-Aldrich, AL-118). Puromycin (Sigma-Aldrich, P9620) was used to select permanent cell lines.
Western blotting analysis
The harvested cells were washed twice with PBS and lysed in ice for 30 min with RIPA lysis buffer (Millipore). Lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C, and protein concentration was measured by the Bradford assay (Bio-Rad Laboratories, 500-0006). For Western blot analysis, cell lysates were boiled for 5 min with sample buffer before being resolved in SDS-polyacrylamide gels. After separation, the proteins were transferred to the PVDF membrane (Millipore, IPVH00010). The membrane was blocked with 5% nonfat dried milk in TBST buffer (20 mM Tris-HCL, pH7.4, 150mM NaCl, 0.1% Tween 20) for 1h and then incubated overnight at 4°C with specific primary antibodies. Subsequently, the membrane was washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibodies (LEADGENE) for 1h at room temperature. The blot signals were developed using an enhanced chemiluminescence kit (GeneTex, GTX14698) and captured by the iBright 1000 Series Image System (Thermo Fisher).
Chromatin immunoprecipitation (ChIP)
ChIP was performed using the EZ-ChIP Chromatin Immunoprecipitation Kit (Millipore) according to the manufacturer’s protocol. Briefly, cells were fixed with 1% formaldehyde for 15 min, and unreacted formaldehyde was quenched by adding 125mM glycine for 15 min. The chromatin was sonicated to the average length of 200-1000bp. Samples were reacted with anti-STAT6 (1:50, Cell signaling, #5397S) and anti-HDAC2 (1:50, Cell signaling, #57156S). The DNA sample precipitated with the target antibody was detected by PCR using the primers covering -896/-719 region of the CYBB promoter: forward 5’– TGACACAATCTCGGCTCACTGCAA-3’ and reverse 5’-TCACGCCTGTAATCCCAGCACTTT-3’.
Luciferase reporter assay
The CYBB-WT and CYBB-deletion reporter constructs were generated by standard gene synthesis and cloning of the promoter sequences in a Luciferase reporter system (pGL4.17 vector, Promega). U937 cells were transfected with the above reporter plasmids by electroporation (NEPA21 electroporator). Cells (1x106) were suspended in OPTI-MEM (100μl) and mixed with 10μg of plasmid DNA in an electrode chamber. Twenty-four hours after transfection, cells were stimulated with IL-4 or without IL-4 for 8h, and then were harvested and lysed for luciferase and Renilla measurements using the Dual-Luciferase Assay System (Promega). To assess transfection efficiency, co-transfection of the pRL-TK vector as an internal control allowed normalization of transfection by Renilla luciferase activity.
STAT6-CYBB decoys Oligodeoxynucleotide (ODN)
STAT6-CYBB decoy ODNs and scrambled ODNs were synthesized with selected sequence targets. The STAT6-CYBB decoy ODN is a double-stranded phosphonothioate 28mer DNA that exhibits a similar antagonistic inhibitor of the transcription factor STAT6. Sequences utilized were as follows: STAT6-CYBB decoy ODN, 5’ -CTG ACT CCC AGG TTC AAG TGA TTC TCC T-3’ and 3’ -GAC TGA GGG TCC AAG TTC ACT AAG AGG A-5’; mutated decoy ODN, 5’ -CTG ACT CCC GAG TTC AAG TGA TTC CCC T-3’ and 3’ - GAC TGA GGG CTC AAG TTC ACT AAG GGG A -5’; scrambled decoy ODN, 5’ -CGA AAA TTC GTT AAA TCA CTA GCT TAC C-3’ and 3’-GCT TTT AAG CAA TTT AGT GAT CGA ATG G-5’. Synthetic ODNs were dissolved in sterile annealing buffer (1M Tris pH 7.5, 0.5M EDTA, pH 8.0, 5M NaCl and ddH2), and were annealed for 3 hours during which the temperature was reduced from 90°C to 25°C. After annealing, the reaction mixtures were held at 4 °C.
Statistical analysis
Each experiment was performed at least three time independently under identical conditions, and data are expressed as the mean ± standard error of the mean (SEM). Student’s t-test and one-way ANOVA were used to analyze the differences. Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA) and SigmaPlot 12.0 (Systat Software Inc., CA, USA). Statistical probability (p) is expressed as *** p < 0.001, ** p < 0.01, and * p < 0.05.