Crosstalk between tumor-associated macrophages and tumor cells promotes chemoresistance via CXCL5/PI3K/AKT/mTOR pathway in gastric cancer

Collection of clinical samples

Paraffin-embedded samples of primary lesions from 103 patients with gastric cancer who had undergone 5-FU based neoadjuvant chemotherapy prior to radical resection at Peking Union Medical College Hospital between 2015 and 2017 were used. Patients were divided into two groups according to the evaluation of pathological response based on the guidelines of College of American Pathologists (CAP) [20]. CAP 0, CAP 1 and CAP 2 were defined as pathological response whereas CAP 3 was defined as no pathological response. 67 patients were elected to pathological response group and 36 patients were elected to no pathological response group for further research. Clinical samples were gathered with written informed consent of patients according to a protocol reviewed and approved by the Institutional Review Board of Peking Union Medical College Hospital.

Immunohistochemistry (IHC)

A total of 103 archived clinical samples were fixed in 10% formaldehyde solution, embedded in paraffin and serially severed into 4 μm sections. After deparaffinized in xylene and rehydrated in graded ethanol, microwave heating with sodium citrate retrieval buffer (pH 6.0) was performed for antigen retrieval. The endogenous peroxidase was inactivated by treatment with 3% H2O2 for 10 min. Tissue sections were incubated with blocking buffer followed by incubation with primary antibodies Anti-CD68 (1:100, Cell Signaling Technology, MA, USA), Anti-CD163 (1:100, Cell Signaling Technology, MA, USA), Anti-CD206 (1:100, Cell Signaling Technology, MA, USA) and Anti-CXCL5 (1:200, Abcam, Cambridge, UK) at 4 °C overnight. After washing with PBS, the secondary antibody horseradish peroxidase (HRP)-conjugated Anti-Rabbit IgG (1:100, Cell Signaling Technology, MA, USA) was added for 30 min’ incubation at room temperature. 3, 3ʹ-diaminobenzidine (DAB) regent was applied for visualizing staining subsequent to PBS washing, then all slices were re-dyed with hematoxylin, dehydrated and sealed for microscopic examination. At least three slices were taken from each tumor tissue and five independent fields were randomly selected from each slice for detection. CXCL5 immunoreactivity was scored by multiplying the staining percentage scores (~ 5% scores 0; 5% ~ 25% scores 1; 25% ~ 50% scores 2; 50% ~ 75% scores 3; 75% ~ 100% scores 4) and staining intensity scores (0, no staining; 1, weak; 2, moderate; 3, strong). A final score of 0–3 was defined as low expression, while others were defined as high expression. CD68, CD163 and CD206 immunoreactivity were analyzed by calculating the mean number of positive cells in 5 random 400-fold fields. Two independent pathologists observed and scored the slices without knowledge of the patients’ clinical information.

Cell lines and cell culture

Human gastric cancer cell lines MKN45 and HGC27, and human mononuclear cells (THP-1) were acquired from the Cell Resource Center of Peking Union Medical College (Beijing, China). The cells were maintained in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) incorporating 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) in a humidified 37 °C incubator with 5% CO2. 0.25% trypsin (NCM Biotech, Suzhou, Jiangsu, China) was administered in the logarithmic growth phase for cell digestion and passage.

Gastric cancer cell lines were regarded as 5-FU-sensitive (MKN45-S and HGC27-S) and the IC50 of 5-FU was detected. 5-FU-resistent cell lines (MKN45-R and HGC27-R) were generated by repetitively exposing gastric cancer cells to increasing concentrations of 5-FU over a 10 month period and the acquired 5-FU resistance was confirmed by detecting the IC50 of 5-FU and resistance index. THP-1 monocytes were differentiated into macrophages by 24 h incubation with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St.louis, MO, USA) for 24 h. Adherent cells were washed twice with culture medium followed by 24 h incubation to obtain the resting state of macrophages (M0). Method of detaching PMA-treated THP-1 cells from the culture dish was demonstrated in Additional file 1: Text S1.

Co-culture of cancer cells and macrophages

Transwell chambers (6-well plates, 0.4-μm pore size; Corning, NY, USA) were used for co-culture. MKN45-S, HGC27-S, MKN45-R and HGC27-R cells were seeded onto the upper chambers, and M0 macrophages were placed in the lower chambers. After 48 h of co-culture, TAMs from 5-FU-sensitive TME (MS) and 5-FU-resistant TME (MR) were obtained and harvested for experimental analysis. To investigate the effect of TAMs with different phenotypes on gastric cancer cells, MS and MR were transferred to the upper chambers and gastric cancer cells were placed in the lower chambers for 48 h of co-culture.

Cell counting kit-8 (CCK-8) assay

A cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay was used to evaluate the inhibition of cell growth in response to varying concentrations of 5-FU (0, 5, 10, 20, 40, 80, 120, 160, 200 μg/ml). Briefly, cells were seeded onto 96-well plates at a density of 5 × 103 cells per well in 100 μl of culture medium and incubated at 37 °C with 5% CO2. After incubation for 24 h, varying concentrations of 5-FU diluted with the culture medium were added to each well and co-incubated for another 24 h. Then 10 μl of CCK-8 reagent was administered to each well for 2 h incubation at 37 °C. The optical density (OD) was detected by a microplate reader at 450 nm. Each experiment was repeated three times and each measurement was conducted three times.

Colony formation assay

Cells were seeded onto 6-well plates at a density of 500 cells per well for adhesion-dependent colony formation. 5-FU was added to the culture medium at a final concentration of 15 μg/ml and the culture medium that contained 5-FU was changed every 3–4 days. After 2 weeks, visible colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet staining solution for 10 min. Then, the formed colony units were photographed and counted for analysis.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of CXCL5 and CCL18 in culture supernatants from M0, MS, MR, MKN45-S, HGC27-S, MKN45-R and HGC27-R cells (1 × 106 cells) were quantified by ELISA kits (Cell Signaling Technology, MA, USA) according to the manufacturer’s instructions. Absorbance was measured using a microplate reader. The concentration of the sample were estimated from the standard curve and the levels below the detection limit of the assay were perceived as zero.

Recombinant protein

Recombinant human CXCL5 (rhCXCL5) were purchased from R&D Systems (Minneapolis, MN, USA). 100 μg/ml stock solution of rhCXCL5 was achieved by dissolving 25 μg powder in 250 μl PBS, followed by adding 0.1% BSA in the final solution, and cells were treated with 10 ng/ml rhCXCL5 for 48 h.

Chemotaxis assay

THP-1 cells were seeded onto the upper chamber (6-well plates, 0.8 μm pore size; Corning, NY, USA) at a density of 2 × 105 in 200 μl serum-free medium. M0, MS and MR cells were cultured with serum-free medium for 24 h, then the supernatants from above cells were collected and added to the corresponding lower chamber with or without CXCL5 neutralizing antibody (0.5 μg/ml; Abcam, Cambridge, UK). After incubation for 24 h at 37 °C with 5% CO2, THP-1 cells that migrated to the lower chamber were measured by fixing and staining the inserts with 0.1% crystal violet staining solution and counting under a microscope (100-fold fields). Non-migratory cells were removed before the membrane was observed.

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized from 1 μg of total RNA using 5 × PrimeScript RT reagent Kit (Takara, Dalian, China), and RT-qPCR was performed using TB Green Premix Ex Taq II (Takara, Dalian, China). Reactions were performed in triplicate and the relative mRNA expression was analyzed by the 2−ΔΔCt method using GAPDH as an internal control. The forward and reverse primer sequences for the targeted genes are listed in Additional file 2: Table S1.

Protein extraction and western blot analysis

Total protein was extracted out of cells using ice-cold RIPA buffer (Thermo Scientific, Rockford, IL, USA) with Halt Protease and Phosphatase inhibitor Cocktail (Thermo Scientific, Rockford, IL, USA) for 15 min. Protein samples were sonicated followed by centrifugation at 12000 g for 15 min at 4 °C and the concentrations were detected by BCA Protein Assay Kit (Beyotime, Shanghai, China). Approximately 30 μg of denatured protein was fractionated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked by TBST solution containing 5% skimmed milk for 1 h at room temperature and then incubated overnight at 4˚C with the primary antibodies against P-gp, Bcl-2, Bax, PTEN, PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR (1:1000, Cell Signaling Technology, MA, USA) and GAPDH (1:500, Cell Signaling Technology, MA, USA). Next, the membranes were incubated with corresponding secondary antibody HRP-conjugated Anti-Rabbit IgG (1:10000, Cell Signaling Technology, MA, USA) at room temperature for 1 h. SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA) was used for visualizing the blots in a Kodak Image station (Tanon, China) (Additional file 3: Table S2).

Flow cytometry analysis

M0, MS and MR cells were washed twice by PBS and filtered through a 100 μm mesh for flow cytometry. Then cells were counted, diluted to 1 × 106 cells per 100 μl and subsequently stained with FITC-CD11b, PE-CD86, APC-CD163 and APC-CD206 antibodies (BioLegend, San Diego, CA, USA) followed by incubating in darkness at 4 °C for 15 min. Finally, the labeled cells were analyzed by BD Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA). The process was conducted in triplicate and data were analyzed by FlowJo software (Tree Star, Oregon, OR, USA) (Additional file 4: Figure S1).

Apoptosis assay

Cell apoptosis was measured using Annexin-V-FITC Apoptosis Detection Kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. In brief, cells were washed twice with PBS, after centrifugation, cells were suspended in 100 μl of 1 × binding buffer. Then 5 μl Annexin V-FITC and 5 μl propidium iodide (PI) were added to stain cells for 15 min in the dark. The stained cells were maintained on ice until apoptosis was measured using BD Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA). The process was conducted in triplicate and data were analyzed by FlowJo software (Tree Star, Oregon, OR, USA).

Statistical analysis

Data were expressed as mean ± standard deviation (SD) of at least three separate experiments. Student’s t-test or one-way analysis of variance (ANOVA) was used for difference analysis. Correlation of the expression level of CD163 or CD206 with CXCL5 was determined using Spearman rank-order correlation. Survival curves were analyzed using Kaplan–Meier and log-rank methods. All statistical analyses were calculated by SPSS 22.0 (SPSS Inc., Chicago, IL, USA) in conjunction with GraphPad Prism 8 (GraphPad Prism Software, Inc., San Diego, CA, USA), and P < 0.05 was considered statistically significant.

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