Materials and Reagents
Palmitic Acid (CH3(CH2)14COOH, molecular weight: 256.42), Phorbol 12-myristate 13-acetate (PMA), and Resatorvid (TAK-424) were purchased from Merck & Co (Darmstadt, Germany), IL-4, IL-13, γ-interferon (γ-IFN), Macrophage Colony Stimulating Factor (M-CSF), and lipopolysaccharide (LPS) were purchased from PeproTech (NJ, USA).
Cell line and cell culture
Human gastric cancer cell lines HGC-27 and AGS-1, Murine gastric cancer cell line mouse forestomach carcinoma (MFC) and the human monocyte cell line THP-1 were obtained from the Cell Bank of the China Science Academy (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) and RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) in a 5% CO2 incubator at 37 °C.
Macrophages polarization
THP-1 was inoculated in six-well plates and stimulated for 18-24 h using 200nM/ml PMA, then replaced with medium containing 20% FBS, 1μg/ml LPS, 20ng/ml γ-IFN polarized for 48 h and induced as M1 macrophages. Then Stimulation with 20ng/ml IL-4 and IL-13 for 48 h induced M2 macrophages.
Bone marrow-derived macrophage isolation. Six-week-old C57BL/6 male mice were sacrificed and the bones were removed using scissors. The bone marrow was flushed out using a 3 ml syringe needle, resuspended in 20% FBS DMEM medium, centrifuged at 1500 rpm for 5 min, and 1 ml of erythrocyte lysate was added, centrifuged again, and resuspended in 20% FBS DMEM. The cells were counted and spread out in 6-well plates, 20 ng/ml of M-CSF was added, the medium was changed on the third day, and the polarization stimulating factor was added for seven days.
RNA isolation and RT-qPCR
RNA-Quick purification Kit (ES science) was performed to extract total RNA from BMDMs, and Prime Script RT reagent Kit (TaKaRa) was conducted to synthesize cDNA following the manufacturer’s introductions. Table 1 shows the forward and reverses primer sequences used for RT-PCR. Quantitative real-time PCR was performed with the SYBR Green (Thermo) by Applied Biosystems 7500 plus according to the manufacturer’s protocol. Relative quantitation was conducted with the 2(-ΔΔCT) method normalize for GAPDH expression.
Cell survival assay
Cell Counting Kit-8 (Solarbio, Beijing, China) was used to evaluate cell viability, cells were seeded to 96-well plates (2 × 103 cells/ml) and treated with serial concentrations of PA (0-0.6μM) or M1-CMs, then incubated for 24 h at 37°C. CCK-8 solution (10μl/well) was added and incubated for 2 h at 37°C, then measured at 450 nm absorbance after 1-4 h of incubation using a plate reader (Cytation5; Bio-Tek) to calculate the cell viability.
Flow cytometry
Macrophages were harvested, washed with PBS, incubated with a blocking reagent, and then stained with antibodies according to the manufacturer's instructions. The following antibodies were purchased from Biolegend (CA, USA): anti-mouse CD206 APC, anti-mouse F4/80 PE, anti-mouse CD68 APC and anti-mouse CD86 FITC. Finally, the stained cells were incubated with a secondary antibody for 1 hour, then analyzed for surface marker using the CytoFLEX S (Beckman Coulter, CA, USA) and FlowJo V10 software.
Conditioned medium preparation
Macrophage cells were stimulated with or without different concentrations of PA + γ-IFN for 72 h, then the medium was replaced with an FBS-free medium for 24 h, and the supernatant was centrifuged at 10,000 rpm for 5 min, after which the supernatant was collected as a conditioned medium and stored at -80°C.
Cell migration, invasion
Cell invasive and migration ability were determined using transwell chambers (Corning Life Science, Acton, MA, USA) coated with or without an extracellular matrix gel (BD Biosciences, San Jose, CA, USA) and incubated at 37°C with 5% CO2. HGC-27 or AGS cells were inoculated at a density of 1 x 104 per well (in 100μL medium) in the upper Chamber, 600ul CMs were added to the lower Chamber and incubated for 24-48 h. The upper Chamber of cells was removed with a cotton swab and washed 3 times with PBS, then fixed in paraformaldehyde for 30 min and incubated for 20 min with 10 ug/ml DAPI solution. The stained cells were photographed using an inverted fluorescent microscope, and five random fields per well were taken (40x magnification) and quantified using Image J software.
Wound healing assay
Gastric cancer cells were cultured in 24-well plates (4 x 105 cells/well), scratches were made using a disposable pipette tip when cells adhered to the wall, and detached cells were washed three times with PBS. Cells were stimulated with or without PA + γ-IFN-CMs. After 48 h of incubation, cell invasion was obtained using a phase contrast inverted microscope. ImageJ software selected and analyzed three random fields along the scratched line.
Western blot analysis
BMDMs were prepared in RIPA lysis buffer (Solarbio, Beijing, China) containing protease and phosphatase inhibitors and protein concentration was detected by BCA Protein Assay Kit (Beyotime, Shanghai, China). About 25μg of protein was separated by a 6% SDS-PAGE and then electro-transferred to the PVDF membrane (Beyotime, Shanghai, China). 5% non-fat milk was used to block the membrane at room temperature for 2 h and incubated with the appropriate dilution of primary antibodies at 4°C overnight. The following primary antibodies were used: Anti-Akt (1:1000, #4691, Cell Signaling Technology), anti-p-Akt (Ser473) (1:1200, #4060, Cell Signaling Technology), anti-PI3K p85(1:1000, #17366, Cell Signaling Technology), and anti-p-PI3K p85 (1:1000, #4228, Cell Signaling Technology). Anti-NF-κB/p65(1:1000, 66535‐1‐Ig, Proteintech), anti-p/p651:1000, #3033, Cell Signaling Technology, and anti-TLR4 (1:1500, 66350-1-Ig, Proteintech). After incubation with an HRP-conjugated secondary antibody for 2 h at room temperature, Membranes were visualized with Thermo Pierce enhanced chemiluminescence (ECL).
Animal model
5-7 week(s) male C57/BL6 mice were obtained from Shanghai SLAC Laboratory Animal, Co., Ltd. (Shanghai, China). 2.0×106 MFC cells were injected into the right side of each mouse and were randomly divided into four groups (n=6) five day(s) later. The control group was gavaged with PBS per day. The two control treatment groups received γ-IFN (3μg/tumor) by intra-tumoral injection and the PA (10.26mg/kg) by gavage per day respectively as described elsewhere. (Lin et al. 2017; Sun et al. 2021) The combination group was treated with PA + γ-IFN simultaneously. The tumor volumes were measured every 3 day(s). All mice were killed on the 26th day, and tumors were removed for Flow Cytometry (FITC) and Immunohistochemistry (IHC).
Immunohistochemistry (IHC)
Immunohistochemistry was performed to evaluate the macrophage expression level in tumor tissues. The protocol of IHC was conducted as described elsewhere.(Boulter et al. 2012) Primary antibodies: iNOS (Absin, 1:3600); CD206 (Abcam, 1:24000); FoxP3 (Cell Signaling Technology, 1:600); CD8 (eBioscience, 1:400); Ly-6G (Biolegend, 1:60000); ImageJ software was used for morphometric evaluation of the positive cells, then five image fields per tumor section were randomly selected by a NanoZoomer S210 (Hamamatsu, Japan).
Statistical Analysis
All results are presented as mean ± SD from a minimum of three replicates. Differences between the groups were analyzed using the one-way analysis of variance and performed with Prism 8 (GraphPad, San Diego, CA, USA). Statistical significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001. A p-value less than 0.05 was regarded as statistically significant.