Cells

GES-1 cells (BioChannel Biotech) were maintained in DMEM with high glucose. Human GC cell lines including MKN45, AGS, HGC27 (Pricella Biotech) and N87 (Genecarer) cells were cultured in RPMI 1640 medium (Gibco). THP1 cells (Ubigene) were grown in RPMI 1640 supplemented with 0.05 mM 2-mercaptoethanol (Sigma). All cells were incubated at 37 °C with 5% CO2 in medium containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin solution (Beyotime) to ensure optimal growth conditions.

Clinical specimens

Clinical specimens were obtained from patients diagnosed with GC who underwent surgical resection at The Second Hospital of Lanzhou University. Fresh tissue samples for qRT-PCR and western blotting analyses were promptly frozen in liquid nitrogen. Tissue samples designated for immunofluorescence were fixed in 4% formaldehyde (Yuanye Biotech) to ensure optimal preservation for subsequent analysis.

Human mRNA microarray analysis

Gene expression profile analysis of 16 pairs of GC and para-carcinoma tissues was performed using GeneChip™ Human Genome U133 Plus 2.0 microarray (Affymetrix, Santa Clara, USA). Chip scanning and data analysis were conducted by GeneChem Co., Ltd (Shanghai, China).

Bioinformatics analysis

Transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) Stomach Adenocarcinoma (STAD) cohort, available in GDC Data Portal (https://portal.gdc.cancer.gov/), were analyzed for differential gene expression, weighted gene co-expression network analysis (WGCNA) of M2 macrophage-related genes, immune cell infiltration estimation, univariate and multivariate Cox analysis, Kaplan-Meier (KM) survival analysis, correlation analysis of immune checkpoint expression, clinical characteristic evaluation, time-dependent ROC curve analysis, and Gene Set Enrichment Analysis (GSEA) to identify enriched signaling pathways between high and low gene expression groups. Tumor Immune Estimation Resource (TIMER) database (http://timer.cistrome.org/) was used for combined survival analysis of gene expression and M2 macrophage infiltration in GC patients. Single-cell sequencing data from GC tissues (GSE134520, GSE167297) in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) were used for cell clustering and gene expression analysis across different cell types. Transcriptome data from GC tissues (GSE118916, GSE33335 and GSE54129) in the GEO database were used for differential gene expression analysis. Transcription factors (TFs) regulating the miRNAs were predicted using TransmiR v2.0 (http://www.cuilab.cn/transmir), and JASPAR (http://jaspar.genereg.net) was employed to identify TF binding sites within the promoter regions. miRNA target gene prediction was performed using miRDB (http://mirdb.org/), miRWalk (http://mirwalk.umm.uni-heidelberg.de/), and miRTarBase (https://mirtarbase.cuhk.edu.cn/). KEGG pathway enrichment analysis of target genes was conducted utilizing the DAVID database (https://david.ncifcrf.gov/).

TCGA and GEO data were analyzed and visualized using R software (v. 4.2.1), utilizing R packages such as limma, clusterProfiler, pathview, GSEABase, WGCNA, timeROC, survival, survminer, complexheatmap, pheatmap, ggplot2, ggExtra, ggpubr and ggClusterNet. “CIBERSORT” and “Immunedeconv” were used to estimate the relative abundance of tumor-infiltrating immune cells (TIICs) in TCGA-STAD samples from normalized expression data.

Lentiviral transfection

SERPINE1-lentiviral vector and control lentiviral vector were constructed and synthesized by VectorBuilder Biotech (Guangzhou, China) with lentivirus particle concentrations of approximately 6 × 108 TU/ml. The targeting sequences were as follows: shSERPINE1#1, 5′-GTGCCTGGTAGAAACTATTTC-3′; shSERPINE1#2, 5′-AGACCAACAAGTTCAACTATA-3′; shSERPINE1#3, 5′-TCTCTGCCCTCACCAACATTC-3′; and scramble shRNA (shNC) as a non-targeting negative control (NC), 5′-CCTAAGGTTAAGTCGCCCTCG-3′. Lentiviral particles at a multiplicity of infection (MOI) of 18, along with 5 µg/ml polybrene (VectorBuilder Biotech, Guangzhou, China), were introduced into 24-well plates containing cells seeded at a density of 6 × 104 per well. Stable knockdown cell lines were generated by selecting in complete medium supplemented 2 µg/ml puromycin (VectorBuilder Biotech, Guangzhou, China) for two weeks.

Transient transfection

SERPINE1 overexpression plasmid and negative control plasmid were constructed and synthesized by VectorBuilder Biotech (Guangzhou, China). pcDNA3.1-EGFP-STAT3 plasmid and negative control plasmid were also obtained from VectorBuilder Biotech (Guangzhou, China). pCMV3-C-Myc-SOCS7 plasmid was constructed by Sino Biological Inc. (Beijing). Transfection complex was prepared by diluting 10 µl Lipo2000 and 4.0 µg plasmid in 250 µl Opti-MEM (Gibco) each, followed by gentle mixing. 3 × 105 cells were seeded in 6-well plates, incubated with 1.5 ml Opti-MEM and 500 µl transfection complex for 6 h, and then cultured in complete medium for an additional 48 h.

AntagomiR negative control (NC, 5’-CAGUACUUUUGUGUAGUACAA-3’) and antagomiR-let-7 g-5p (5’-AACUGUACAAACUACUACCUCA-3’) were synthesized by GenePharma (Shanghai) and dissolved in 125 µl DEPC-treated water to prepare a 20 µM stock solution. THP1 cells (4 × 104/well) were seeded in 24-well plates, incubated with a transfection complex (20 pmol antagomiR, 1 µl Lipo2000 and 100 µl pti-MEM) and 400 µl Opti-MEM for 6 h, followed by replacement with PMA-containing medium and cultured for 24 h.

Immunofluorescence (IF)

Tissue sections were heated at 58 °C for 2 h, then deparaffinized in xylene and rehydrated through a graded ethanol series. Cells were washed, fixed with 4% paraformaldehyde, and permeabilized using 0.2% Triton X-100 (Solarbio). Antigen retrieval was performed using sodium citrate buffer (pH 6.0, 98 °C), followed by goat serum blocking for 1 h. Sections were incubated with PAI-1 (rabbit, 1:200, Immunoway), CD163 (mouse, 1:200, Immunoway), CD206 (mouse, 1:200, Proteintech), F4/80 (rabbit, 1:200, Bioss), iNOS (rabbit, 1:200, Bioss), Arg1 (rabbit, 1:200, Proteintech), STAT3 (rabbit, 1:200, Bioss) antibodies overnight at 4 °C, reactivated, stained with Cy3-conjugated goat anti-rabbit IgG (Abcam) and Alexa Fluor 488-conjugated goat anti-mouse IgG (Abcam) for 30 min, counterstained with DAPI, and imaged using fluorescence microscopy (IX51, Olympus) for ImageJ analysis.

Flow cytometry assay

THP-1 cells were differentiated into macrophages using 150 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) for 24 h and subsequently co-cultured with cancer-derived exosomes or GC cells in 6-well plates with 0.4-µm membranes for 72 h. Harvested macrophages were converted into single-cell suspensions, stained with Elab Fluor 488 anti-human CD68 (Mouse, 1:20, ElabScience) and APC anti-human CD206 (Mouse, 1:20, ElabScience) antibodies, and analyzed for CD68+CD206+ populations by flow cytometry (Accuri C6, BD).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using the Hifair III 1st Strand cDNA Synthesis Kit (Yeasen). Quantitative PCR (qPCR) was performed on a Real-Time PCR System (7500, Applied Biosystems) using Hieff UNICON Universal Blue QPCR SYBR Green Master Mix (Yeasen). Total miRNA was isolated with the miRNeasy Mini Kit (Qiagen) and reverse transcribed into first-strand cDNA using the Mir-X miRNA First Strand Synthesis Kit (Takara). qPCR was conducted using the Mir-X miRNA qRT-PCR TB Green Kit (Takara). Relative quantification (2−ΔΔCT) was normalized to GAPDH and U6 snRNA. Primer sequences are provided in the Supplementary Materials.

Western blotting

Tissues, cells and exosomes were lysed using RIPA buffer (Solarbio) supplemented with 1% PMSF (Sigma), and the lysates were centrifuged at 12,000 × g for 15 min at 4 °C. Protein concentrations were quantified via a BCA assay (Solarbio). Proteins were then separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking with 5% fat-free milk, membranes were incubated overnight with primary antibodies (Supplementary Materials). Chemiluminescent substrates (Affinity) were used for detection, and blots were visualized using SH-Compact523 Chemiluminescence Gel Imaging System (Shenhua).

Cell viability

Cell viability was assessed using a CCK8 kit (PUMOKE), and absorbance at 450 nm was measured with a microplate reader (iMark, Bio-Rad).

EdU incorporation assay

Cell proliferation was evaluated using EdU staining, following the manufacturer’s instructions (Solarbio).

Colony formation

A colony formation assay was conducted using 0.3% soft agar. In each well of a 6-well plate, 400–800 cells were seeded and cultured for two weeks. Colonies were then stained with 0.5% crystal violet.

Exosome isolation and identification

2 × 10⁵ GC cells silencing or overexpressing SERPINE1 were seeded into T75 culture flasks and cultured to approximately 80% confluence. The medium was then replaced with serum-free media (Umibio) and incubated for an additional 48 h. Subsequently, equal volume of conditioned medium was collected for exosome isolation by ultracentrifugation (XPN-100, 32Ti rotor, Beckman) and re-suspended in 200 µl of cold 1 × PBS. Exosome concentration and size were analyzed via BCA protein assay (Solarbao), nanoparticle tracking analysis (ZetaView PMX110, Germany), while their morphology and size were confirmed by TEM (Hitachi-7500, Japan). Protein markers were detected using western blotting. Dil-labeled exosomes (Yeasen) internalized by macrophages were visualized using confocal laser scanning microscopy (CLSM; SP8, Leica).

Migration and invasion assays

Transwell inserts (8 μm, Corning) were employed to assess cell migration and invasion. 500 µl complete medium was added to the lower chamber of a 24-well plate, and 100 µl of cell suspension (approximately 2 × 104 cells) was placed in the upper chamber for 48 h. Invasion assays were conducted by coating the upper chamber membranes with 100 µl Matrigel Matrix (Corning), followed by the same steps as the migration assay. Invading cells were methanol-fixed, stained with crystal violet, and imaged using an IX51 microscope (Olympus).

Transmission electron microscopy (TEM)

Exosome solution was diluted 1:10 with 1 × PBS, and 10 µl was applied onto a piece of parafilm, followed by placement of a 200-mesh Formvar-carbon copper grid for 20 min. The grid was fixed in 2.5% glutaraldehyde for 5 min, washed in deionized water, stained with 4% uranyl acetate for 10 min, stained with methylcellulose-UA on ice for 10 min and observed by Hitachi-7500 TEM.

Small RNA sequencing (sRNA seq) of exosomes

sRNA sequencing was performed by RiboBio Biotech (Guangzhou). Total RNA was isolated from six exosome samples (three from the SERPINE1 knockdown group and three from the control group) using the miRNeasy Micro Kit (QIAGEN). RNA quality and integrity were verified with an ND-1000 Spectrophotometer (NanoDrop Technologies) and a Bioanalyzer 2100 (Agilent). A sequencing library was constructed from 1 µg of total RNA per sample using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB), and sequencing was conducted on the Illumina HiSeq2500 platform. Differential expression of exosomal miRNAs was evaluated using reads per million (RPM) and DESeq2 (V1.26.0).

Protein microarray analysis

Protein microarray analysis was conducted using the CSP100 Plus Microarray (HWayen), which immobilizes highly specific antibodies involving 16 signaling pathways. Each antibody had six technical replicates, scanned using an Agilent SureScan Dx Microarray Scanner, and image intensities were analyzed with GenePix Pro v6.0 software (Axon).

Xenograft experiments

Specific pathogen-free (SPF)-grade BALB/c-nu mice (male, 5 weeks old) were obtained from Chengdu Yaokang Biotech Co. Ltd and kept under SPF conditions. Twelve mice were randomly divided into two groups: MKN45/AGS cells transfected with scrambled shRNA or shSERPINE1#3 (n = 3/group). Each mouse was subcutaneously injected with 5 × 10⁶ cells mixed with 50% Matrigel (Corning). Tumor volumes were measured weekly. After 42 days, mice were sacrificed, and tumors were excised, weighed, and divided for western blotting and immunofluorescence analyses.

Co-immunoprecipitation (Co-IP)

Macrophages induced by PMA were exposed to exosomes isolated from GC cells and transfected with pCMV3-C-Myc-SOCS7 plasmids as before, with or without antagomir-92b-5p. Co-IP was conducted using the Pierce Co-IP Kit (Thermo Scientific). Cell lysates were pre-cleared with Protein A/G beads to remove non-specific proteins, with 20% reserved as input control. The remaining lysates were incubated overnight at 4 °C with anti-STAT3 (Rabbit, 1:50, Cell Signaling), anti-Myc (Rabbit, 1:200, Beyotime) or IgG (Rabbit, 1:50, Abcam), followed by Protein A/G bead capture. After washing to remove unbound proteins, complexes were eluted, denatured, and analyzed via western blotting.

Fluorescence in situ hybridization (FISH)

FISH analysis was performed using the miRNA FISH Kit (GenePharma). A Cy3-labeled hsa-let-7 g-5p probe (GenePharma) was synthesized as follows: 5′ Cy3-AACTGTACAAACTACTACCTCA-3′ and provided by GenePharma. Briefly, paraffin Sect. (5 μm) were incubated at 60 °C for 30 min, deparaffinized with xylene, and rehydrated through graded ethanol. Proteinase K digestion (20 min at 37 °C) and denaturation (8 min at 78 °C) followed. Cells were fixed with 4% formaldehyde for 15 min at room temperature, washed with PBS, and hybridized with denatured probes at 37 °C for 12 h. Nuclei were counterstained with DAPI (GenePharma), and images were captured using an Olympus BX51 fluorescence microscope.

Chromatin immunoprecipitation (ChIP)

ChIP assays were performed using a ChIP Kit (Beyotime) in accordance with established protocols. MKN45 cells were transfected with pcDNA3.1-EGFP-STAT3 plasmids as before. Cells at 24 h post-transfection were crosslinked with 1% formaldehyde at 37 °C for 10 min, followed by quenching with 125 mM glycine at room temperature for 5 min. Cells were subsequently collected, lysed, and subjected to sonication to fragment the DNA into 200–750 bp segments, confirmed by agarose gel electrophoresis. A 10% aliquot of each chromatin complex was reserved as an input control. IP was performed using anti-pSTAT3 (Rabbit, 1:50, Cell Signaling Technology) or Rabbit IgG (1:50, Abcam) antibodies, with Protein A/G Agarose/Salmon Sperm DNA to capture immune complexes associated with the let-7 g-5p promoter (site position: -1666 to -1483). After sequential washes, qPCR was employed to quantify the immunoprecipitated DNA, with ΔΔCt values calculated according to previous literature [9]. Specific primers used are provided in the Supplementary Materials.

Dual-luciferase reporter gene assay

The potential STAT3-binding sites on the let-7 g-5p promoter were identified using JASPAR (http://jaspar.genereg.net/). The promoter region (-1692 to -1420) of let-7 g-5p was synthesized and inserted into the pRP[Pro]-hRluc/Puro-Luciferase reporter plasmids including negative control, wild-type and mutant-type (VectorBuilder), which were transfected into MKN45 cells. Luciferase activity was evaluated using the Dual-Luciferase Reporter Assay System (Promega). Relative luciferase activity was calculated following the method described in the previous literature [9].

Statistical analysis

Data analysis and visualization were conducted using SPSS 26.0 (IBM, Chicago, IL, USA) and GraphPad Prism v9 (GraphPad Software, San Diego, CA, USA). Categorical variables were analyzed with a chi-squared test, and correlations were determined using Spearman’s correlation test. Nonparametric data were analyzed using the Mann-Whitney U test for two-variable comparisons and the Kruskal-Wallis test for multiple variables. Normally distributed continuous data were analyzed using Student’s t-tests or paired t-tests for two-group comparisons and one-way ANOVA with Tukey’s post hoc test for multiple comparisons. All experiments were independently replicated at least three times. Statistical significance was set at p < 0.05 and shown as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns (not significant) p > 0.05.