Patients and gastric tissue samples

This research included a total of 250 gastric cancer tissues collected from January 2010 to October 2015; nine samples were excluded due to missing data. The remaining 241 patients were employed for the internal cohort. Gastric tissue specimens were fixed with formalin and embedded in paraffin. The inclusion criteria were as follows: (a) histological identification of gastric cancer; (b) availability of follow-up data and clinicopathological characteristics; and (c) TNM staging of gastric cancer tumours according to the 2010 International Union Against Cancer (UICC) guidelines. The exclusion criteria were as follows: (1) patients with no formalin-fixed paraffin-embedded (FFPE) tumour sample, including the centre of the tumour (CT) and the invasive margin (IM), from initial diagnosis; and (2) patients who received chemotherapy or radiotherapy before surgery. All participating patients with advanced GC routinely received fluorine-based chemotherapy. Comprehensive information of the internal cohort is listed (Table 1). All procedures performed in studies involving human participants were in accordance with the Helsinki declaration. And all patients whose tissue samples were used in this research provided written informed consent. This study was approved by the Ethics Committee of Fujian Medical University Union Hospital (Ethics approval number of scientific research project: 2021KJT031).

Immunohistochemistry and evaluation

The serial sections of the FFPE sample were 4 μm and mounted on a glass slide for IHC analysis. The sections were deparaffinized with xylene and rehydrated with alcohol. We blocked endogenous peroxidase by immersing the slices in a 3% H2O2 aqueous solution for 10 min and microwaved the slices in 0.01 mol/L sodium citrate buffer (pH 6.0) for 10 min for antigen retrieval. The slides were then washed in phosphate-buffered saline (PBS) and incubated with 10% normal goat serum (Zhongshan Biotechnology Co., Ltd., China) to eliminate nonspecific reactions. Subsequently, the sections were incubated overnight with the primary antibody at 4 °C. The negative control was processed with the same methods, but the primary antibody was omitted. After rinsing three times with PBS, the secondary antibody was diluted and incubated on slides for 30 min at room temperature, and the staining was developed with diaminobenzidine (DAB) solution. Finally, the slides were counterstained with haematoxylin, dehydrated, and fixed with a cover glass and neutral resin.

We performed CDK5RAP3 (ab157203, Abcam, 1:100) immunohistochemical staining on tumour tissue of gastric cancer patients. The staining intensity and average percentage of positive cells in 5 randomly selected regions were evaluated to represent the protein expression level. The scoring criteria (Fig. S4A) were as follows: staining intensity was divided into 0 (negative staining), 1 (weak staining, light yellow), 2 (medium staining, yellow-brown), or 3 (strong staining, brown). The proportion of positively stained tumour cells was categorized according to the following thresholds: 0 (≤5% positive cells), 1 (6–25% positive cells), 2 (26–50% positive cells), and 3 (≥51% positive cells). The final expression was calculated by multiplying the staining intensity score by the proportional staining score (total 0–9). Patients with final scores of <4 points were classified as the low expression group, and patients with scores ≥4 were classified as the high expression group.

CD68 (ab213363, Abcam, 1:300) and CD206 (ab64693, Abcam, 1:500) were subjected to immunohistochemical staining. To evaluate the infiltration of immune cells, five representative and independent fields at ×200 magnification were captured at the centre of the tumour (CT) and invasive margin (IM), respectively, on each tissue. Next, we used the ‘measurement’ plug-in in Image-Pro Plus software (6.0, Media Cybernetics Inc.) to assist in marker counting to obtain the number of positive cells in the field. The average number of positive cells in five fields was divided by the area of the field (0.27 mm2) to obtain the infiltration density of immune cells in CT and IM. All the percentages/numbers of positive cells were expressed as the average of five randomly selected microscopic fields.

The IHC results were evaluated by two independent gastroenterology pathologists who were blinded to the clinical prognosis of the patients. Approximately 90% of the scoring results were the same. When the scores of the two independent pathologists diverged, another pathologist checked the results again and selected one of the scores proposed by the first two doctors, or the three pathologists discussed the decision together.

Multiplexed immunofluorescence staining

We performed multiplexed immunofluorescence staining to identify the expression of CDK5RAP3 (ab157203, Abcam, 1:50), CD68 (M0876, DAKO, 1:500), CD206 (CL488-60143, Proteintech, 1:300) and INOS (ER1706-89, HuaAn, 1:100) in 28 gastric cancer tissues. Tumour cytokeratin was stained with CKpan. All nuclei were stained with DAPI. Briefly, formalin-fixed paraffin-embedded tissue sections were cut into 4-mm thick sections, thawed at 70 °C for 45 min, deparaffinized and fixed with formaldehyde: methanol (1:10). Then, in a pH 8.0 EDTA buffer and heat-induced antigen recovery was performed at 100% power in an 800 W standard microwave until the boiling point, and then 30% power was used for 15 min. The tissue sections were then cooled and washed in 0.02% Tris-buffered saline-Tween 20 (TBST) with gentle stirring. Then, the sections were blocked with blocking buffer (Dako, X0909) for 10 min at room temperature and then incubated with the primary antibody at 4 °C overnight. Then, the horseradish peroxidase (HRP)-conjugated secondary antibody (PerkinElmer) was incubated at room temperature for 1 h, and then the tyramide-based HRP was activated at 37 °C for 20 min. The stained signal was further amplified using Opal 540 Acetamide Signal Amplification (TSA) reagent (PerkinElmer) and incubated with TSA dilution at room temperature. Using TSA, HRP-conjugated secondary antibodies mediate the covalent binding between the Pax-5 protein and different fluorophores. After this covalent reaction, additional antigen recovery (pH 6.0 citrate buffer) was performed for 20 min to remove the bound antibody. Note: Repeat all steps in sequence for each primary antibody. Then, after counterstaining with 4′,6-diamidino-2-phenylindole (Life Technologies) at room temperature, all sections were washed five times in 0.02% TBST for 5 min each for 2 min and stored in a 4 °C lightproof box C until imaging.

Establishment of cell lines

The cell lines were established as previously described [19].

Cell culture and reagents

The human gastric cancer cell lines BGC-823 and AGS were obtained from the Cell Line Bank, Chinese Academy of Sciences. All the cell lines were confirmed to be free of mycoplasma contamination by PCR and culture. The species origin was confirmed with PCR. The identity of the cell line was authenticated with short tandem repeat (STR) profiling. These cell lines were cultured in 1640 (Gibco, Grand Island, NY) supplemented with 10% foetal bovine serum (FBS) (Gibco, Grand Island, NY) and incubated at 37 °C in a humidified atmosphere containing 5% CO2. The human monocyte line THP-1 was purchased from Guangzhou Cellcook Biotechnology Co., Ltd. To obtain macrophages, 3 × 105 THP-1 cells were seeded in 0.4 μm pore inserts treated with 200 nM PMA (Sigma-Aldrich, CN) for 24 h and polarized into macrophages. To obtain TAMs, THP-1 macrophages were cultured by the addition of conditioned media from a gastric cancer cell line (BGC-823) for 24 h. Macrophage and gastric cancer cell cocultivation was conducted using a noncontact coculture Transwell system (Corning, CN). Inserts containing TAMs or THP-1 macrophages were transferred to 6-well plates seeded with gastric cancer cells (1 × 105 cells per well) in advance and cocultured. After 48 h of coculture, TAMs or gastric cancer cells were harvested for further analyses.

PDTC (ab141406, ammonium pyrrolidinedithiocarbamate, Abcam), a selective NF-κB inhibitor, was purchased from Abcam Co., Ltd., Shanghai, China. RS 102895 hydrochloride (ab120812, Abcam), a selective CCR2 receptor antagonist, was purchased from Abcam Co., Ltd., Shanghai, China. MMP-2 inhibitor (ab145190, Abcam) was purchased from Abcam Co., Ltd., Shanghai, China. PMA (P1585, Sigma-Aldrich) was dissolved in PBS containing 0.1% BSA and used at a final concentration of 100 ng/ml.

Cell coculture system

BGC-823 cells were seeded into the lower chamber, and THP-1 cells or primary monocytes (1:1 ratio) were added into the upper chamber of a 6-well Transwell apparatus (Corning Costar). A membrane with a pore size of 0.4 μm separated the cell lines, preventing physical interaction between monocytes and BGC-823 cells while allowing signalling and crosstalk to take place. Coculture times are indicated in the text.

Mouse xenograft models

All male BALB/c nude mice (4–5 weeks old) used in our study were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. For tumour growth experiments, BGC-823 cells (5 × 105), TAMs (5 × 105), BGC-823 cells (5 × 105) with TAMs (5 × 105) in 200 μl were subcutaneously injected into the right axillary fossa of nude mice. Tumour volume was measured every 3 days and calculated with the following formula: V = (L × W2)/2 cm2 (V, tumour volume; L, length; W, width). The mice were sacrificed at 3–4 weeks after injection, and the tumours were weighed. For the lung or liver metastasis model, BGC-823 cells (5 × 105), TAMs (5 × 105), BGC-823 cells (5 × 105) with TAMs (5 × 105) in 200 μl were injected into the tail veins or spleen of nude mice. Forty-five days later, the mice were sacrificed, and the lungs or livers were dissected to examine the histopathological metastatic loci. All animal experiments were performed according to the Animal Protection Committee of Fujian Medical University (Fuzhou, China) and approved by the Ethics Committee of Fujian Medical University/Laboratory Animal Center (Fuzhou, China). The tumour tissues and liver or lung tissues of mice were further examined by H&E, IHC staining, or RT-PCR assay.

All animal experiments were performed according to the Animal Protection Committee of Fujian Medical University (Fuzhou, China) and approved by the Ethics Committee of Fujian Medical University/Laboratory Animal Center (Fuzhou, China).

Flow cytometry

For flow cytometry, cells were washed twice with PBS and blocked with FcR Blocking Reagent (human/mouse) (Miltenyi Biotec GmbH). Cells were then stained with CD206-APC (mouse), CD16/32-PE. Cy7 (mouse), CD11b-FITC (mouse), F4/80-APC (mouse), CD206-APC (human), CD86-PE (human), CD11b-PE. Cy7 (human) antibodies (Invitrogen eBioscience, Thermo Fisher Scientific Co., Ltd., China). Separated mouse macrophages were permeated and fixed using a Cytofix/CytoPerm Plus™ kit (BD Biosciences) following the manufacturer’s instructions and then stained with CD206 and CD16/32 antibodies. Cells were resuspended in FACS buffer (PBS with 10% FBS) and run on a BD Flow Cytometer LSRFortessaX-20 (BD Biosciences). Data were analysed using FlowJo Software v10.0 (Ashland, OR). All tests were controlled by homologous isotype control antibodies.

RNA isolation and RT-PCR

Total RNA of cells was extracted with TRIzol reagent (Invitrogen, USA), and cDNA was synthesized using a Reverse Transcription Reagent Kit (Takara, Dalian, China) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was conducted using a SYBR® Premix DimerEraser kit (Takara) with a Stratagene MX3005P cycler according to the manufacturer’s instructions. The relative quantification of the expression of each gene was normalized to the relative quantification of the expression of β-actin mRNA. The sequences of specific primers are listed in Table S4.

Isolation of macrophages and tumour cells from mouse tumour tissue

Single-cell suspensions were prepared from fresh tumours using a Tumour Dissociation Kit (Miltenyi Biotec GmbH). Cells were then immediately separated using a negative magnetic bead-assisted sorting assay (Mouse Cell Depletion Kit). TAMs were separated from the positive cell suspensions using CD11b + magnetic beads. All operations were performed according to the manufacturer’s protocol.

Western blot assay

Cells were plated into 60-mm dishes and cultured to 80% confluence. The cells were then scraped and lysed in RIPA buffer and the lysates were centrifuged at 10,000 × g (4 °C for 10 min). Protein concentrations were determined using a BCA Protein Assay Kit (Thermo) according to the manufacturer’s instructions. A total of 40 µg protein from each sample was denatured, loaded into a well in a polyacrylamide gel, separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membranes were blocked with 5% nonfat milk at room temperature for one hour and incubated overnight with primary antibodies in PBST (1:1000). After the membranes were washed with PBST, they were incubated for 1 h at room temperature with the corresponding horseradish peroxidase-conjugated secondary antibody at the appropriate dilution and then washed three times with PBST. The protein bands on the membranes were detected using enhanced chemiluminescence (Amersham Corporation, Arlington Heights, IL, USA). Western blot analysis was performed using the following antibodies: CDK5RAP3 (ab157203, Abcam, 1:1000), CCL2 (MCP1) (ab214819, Abcam, 1:1000), CCR2 (ab203128, Abcam, 1:1000), NF-κB p65 (ab16502, Abcam, 0.5 µg/ml), NF-kB p65 (phospho S536) (ab16502, Abcam, 1:1000), MMP2 (ab92536, Abcam, 1:2000), E-cadherin (ab40772, Abcam, 1:2000), N-cadherin (ab76011, Abcam, 1:5000), vimentin (ab92547, Abcam, 1:1000), Snail (ab216347, Abcam, 1:1000), β-ACTIN (ab8226, Abcam, 1:2000), and GAPDH (ab8245, Abcam, 1:1000).

Immunofluorescence assay

Cells grown on coverslips were rinsed with PBS and fixed with ice-cold 4% paraformaldehyde for 5 min at RT. Subsequently, the cells were blocked with 0.2% Triton X-100 for 30 minutes followed by 5% BSA for 1 h, washed for 30 min, and incubated with primary monoclonal antibodies against CDK5RAP3 and NF-κB p65 overnight at 4 °C. The next day, the coverslips were incubated for 1 h in a dark room with fluorescently conjugated secondary antibody (1:200). Furthermore, the coverslips were stained with DAPI (Vector Laboratories, Burlingame, CA, USA) for 5 min at 4 °C. Finally, a laser scanning confocal microscope (Leica, Germany) was used to observe the expression in cells.

ELISA

ELISA was conducted according to the instructions. Concentrations of CCL2 (human), IL4 (human), IL10 (human), and MMP2 (human) in the culture supernatant of treated cells were measured with the use of a commercially available kit (CUSABIO).

MTT assay

BGC-823 cells were seeded into 96-well plates (5 × 103 cells per well) and cultured overnight. Subsequently, 10 μL of MTT solution (5 mg/ml; Solarbio, China) was added to each well at 24, 48, 72 and 96 h. Following incubation for 2 h, the absorbance was measured at 450 nm using a spectrophotometer (FLX800, Bio - TEK).

Colony formation assay

BGC-823 cells were seeded into well plates (500 cells per well) and cultured for 14 days. The cells in every well were fixed with 4% paraformaldehyde for 30 min and then stained with 0.2% crystal violet at room temperature for 30 min. The number of cell colonies was counted.

Transwell migration and invasion assay

The cell migration test was performed in 24-well Transwells (pore size 8 μm; Corning, USA) without Matrigel coating. Twenty-four-well Transwells precoated with Matrigel (Falcon 354,480; BD Biosciences, USA) were used for the cell invasion test. A total of 1 × 105 cells in 500 μl RPMI 1640 containing 1% foetal bovine serum were added to the upper chamber, and 750 μl RPMI 1640 containing 10% foetal bovine serum was added to the lower chamber. After 48 h of incubation, Matrigel and the cells remaining in the upper chamber were removed with a cotton swab. The cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. The cells were counted and photographed in five microscopic fields (200×). All experiments were performed in triplicate.

Processing of genomic data and gene set enrichment analysis

We used publicly available data from TCGA, which was downloaded from the Genomic Data Commons (https://portal.gdc.cancer.gov) on 15 June 2020, and this download included clinical information and mRNA expression data. The mRNA expression data were presented as counts and were normalized with R software (version 4.0.0) and the “limma” package. Gene set enrichment analysis (GSEA) performed by the Molecular Signature Database (MSigDB) was used to identify the pathways that were significantly enriched in CDK5RAP3low tumour samples. If a gene set had a positive enrichment score, the majority of its members had higher expression accompanied by a higher risk score, and the set was considered ‘enriched’. Specific information on the method used for the analysis can be found in Table S5.

Statistical analysis

All data were processed using SPSS 25.0 (SPSS Inc. Chicago, IL) and R software (version 4.0.0). The cut-off value for CDK5RAP3 expression was the median value. Data are presented as the mean ± SD and were analysed using Student’s t-test or one-way ANOVA. We used the χ² test or Fisher’s exact test to compare categorical variables of clinical characteristics. The Kaplan–Meier method was used for overall survival analysis, and a log-rank test was used to compare differences. We defined the survival time of patients who were lost to follow-up as the time from surgery to the last follow-up time, and the survival time of patients who were still alive at the end of the study was defined as the time from surgery to the database deadline. Two-tailed P values < 0.05 indicated significant differences.