Patients and tissue samples

Pancreatic cancer tumor samples were collected from patients who underwent surgery at Fujian Medical University Union Hospital in Fuzhou, China, between November 2013 and January 2021. All patients had histologically confirmed diagnoses of PDAC. Exclusion criteria included patients with neoadjuvant treatment, inflammatory diseases, or active infections. A total of 151 patients with PDAC were enrolled in the study. Disease stage was assessed using the American Joint Committee on Cancer V.8. Informed consent was obtained prior to sample collection. Formalin-fixed paraffin-embedded samples were used for IHC analysis.

IHC and IF

IHC experiments were conducted on large tissue sections obtained from paraffin-embedded samples. The IHC protocol, as previously described,46 involved the following steps: Deparaffinization and pretreatment in 1 mmol/L EDTA at 95°C for 20 min for antigen retrieval. Incubation with a 0.3% hydrogen peroxide solution for 15 min to block endogenous peroxidase activity. Primary antibodies were employed to detect ANXA1 (1:500), CD68 (1:500), and c-Casp3 (1:250). For visualization, we used the Elivision super (Mouse/Rabbit) IHC Kit and DAB Kit (20×), following the manufacturer’s instructions. Counterstaining was performed using hematoxylin. IHC analysis of CD68 status was performed as previously described.6 The ANXA1 and c-Casp3 expression levels were categorized based on intensity scores: 0 for negative, 1 for weak, 2 for moderate, or 3 for strong. Additionally, the percentage of cells stained positively for ANXA1 and c-Casp3 were assessed using scores of 1–3, representing<10%, 10–30%, and>30% of cells. Samples were considered to exhibit high expression if the score was≥3. All specimens were evaluated by two pathologists who were blinded to the patients’ clinical information.

IF was performed by our previous protocol.46 The antibodies used were summarized in online supplemental table S1.

Isolation of mouse peritoneal macrophages

Isolation of mouse peritoneal macrophages was referred to previously published articles.47 Intraperitoneal injections of 5 mL phosphate-buffered saline (PBS) were administered to mice. Subsequently, cells were harvested from the mouse peritoneal cavity in PBS. Then, cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin. After 12 hours of incubation, non-adherent cells were discarded, and the adherent macrophages were used for further study.

In vitro and in vivo efferocytosis assay

The thymus derived from mice was subjected to grinding and filtration processes to yield a single-cell suspension. Thymocyte apoptosis was triggered by the application of 2 mM dexamethasone (MedChemExpress, USA) and incubated at a temperature of 37°C for a duration of 4 hours. The apoptotic thymocytes were subsequently labeled with 1 mg/mL of pHrodo red succinimidyl 1 ester (MedChemExpress, USA). These labeled apoptotic cells were introduced to peritoneal macrophages extracted from Anxa1^WT or Anxa1^−/− mice. Following an incubation period of 45 min, apoptotic cells that remained unengulfed were removed through a washing process. The macrophages were then labeled with an anti-CD11b, AF488 antibody (eBioscience, clone M1/70). The process of phagocytosis of the apoptotic cells was monitored and quantified using a fluorescence microscope.

To evaluate in vivo clearance of apoptotic cells in the thymus. Dexamethasone (0.2 mg/25 g) was dosed by intraperitoneal injection to induce thymocyte apoptosis. Then the thymus tissues were collected at 8 hours and 24 hours later, and dissociated into single-cell suspension. Apoptotic cells and dead cells were then detected using the Annexin V-FITC Apoptosis Detection Kit (Solarbio) by flow cytometry.

Cell lines, mice and reagents

The murine PDAC cell line (Panc02) was procured from Shanghai Aolu Biological Technology (Shanghai, China). THP1 were procured from Cellverse (Shanghai, China). The primary mouse PDAC cell line KPC (LSL-Kras^G12D; LSL-Trp53^R172H; Pdx1-Cre) was obtained from professor Xiaolong Liu. Both Panc02 and KPC are syngeneic to C57BL/6 mice. The cell lines underwent genotyping by the Cell Bank of the Chinese Academy of Sciences and were subjected to rigorous testing to eliminate the possibility of mycoplasma contamination.

WT C57BL/6J mice, NOD-SCID (NOD/ShiLtJGpt-Prkdc^em26Cd52/Gpt) mice, Anxa1^flox/flox mice (C57BL/6J genetic background) and Lyz2-Cre mice (C57BL/6J) were purchased from GemPharmatech (Nanjing, China, Strain NO. T003822, Strain NO. T016067). Myeloid-specific ANXA1-knockout mice (Anxa1^ΔMɸ, Mɸ: macrophage; Δ: Specific knockout) were generated by crossing Anxa1^flox/flox mice with Lyz2-Cre mice. The mice were maintained under specific-pathogen-free conditions at the laboratory animal room of Fujian Medical University. Male mice of 8–12 weeks were used for all tumor studies. All animal experiments were approved by the Ethics Committee for Animal Research of Fujian Medical University (IACUC FJMU 2023–0087). The protocol for the care and utilization of animals was in strict compliance with the National Regulations for the Administration of Affairs Concerning Experimental Animals.

To establish a PDAC model, both Panc02 and KPC cells (1×10⁶) were subcutaneously implanted into Anxa1^flox/flox and Anxa1^ΔMɸ mice, respectively. When the tumor reached 100 mm³, tumor-bearing mice were randomly divided into groups for further study (4–6 mice, respectively). The groups were treated as follows.

1. Control solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline), BIP-V5 (5 mg/kg, daily intraperitoneally (i.p.));

2. Mouse IgG1 (200 µg/day i.p., Clone No. MOPC-21, Bio X Cell), anti-IFNAR1 (200 µg/2 days i.p., Clone No. MAR1-5A3, Bio X Cell);

3. Control solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline), H151 (750 nmol/2 days, i.p., Clone No. HY-112693, MCE);

4. Control solvent (10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline), RU521 (5 mg/kg, once every 2 days, i.p., Clone No. HY-114180, MCE);

5. Rat IgG2a (200 µg/day i.p., Clone No. 2A3, Bio X Cell), anti-CSF1R (200 µg/2 days i.p., Clone No. AFS98, Bio X Cell);

6. Anti-PD-1 (200 µg/2 days i.p., Clone No. RMP1-14, Bio X Cell), gemcitabine (100 mg/kg, two times a week, Clone No. HY-17026, MCE), shAnxa1AAV (5×10¹⁰ Vector genomes (Vg)/week, injected intratumorally, OBiO), ctrlAAV (5×10¹⁰ Vector genomes (Vg)/week, injected intratumorally, OBiO). shAnxa1AAV and control virus (ctrlAAV) were designed and synthesized by OBiO Technology (Shanghai, China, online supplemental table S2).

After treatment, the mice were sacrificed, and tumors were removed and weighed.

Luciferase-expressing KPC cells (1×10⁶) were injected into the pancreatic capsule of Anxa1^flox/flox and Anxa1^ΔMɸ mice to establish the PDAC tumor in situ model. After 4 weeks, the mice were intraperitoneally injected with D-Luciferin potassium salt (2 mg/mouse, 122799, Revvity), and the tumor growth was monitored according to the IVIS Spectrum (PerkinElmer). Then, the mice were sacrificed and the tumors were recorded.

A humanized mouse tumor model was generated as described previously.25–27 Lentiviruses carrying plasmids overexpressing or knocking down ANXA1 (Ubi-ANXA1-3FLAG-CBh-gcGFP-IRES-puromycin; Ubi-Anxa1-3FLAG-CBh-gcGFP-IRES-puromycin; hU6-ANXA1-CBh-gcGFP-IRES-puromycin) were designed and synthesized by GeneChem (Shanghai, China). The lentiviruses were infected into THP1 to construct a stable cell line for subsequent experiments. To study the role of ANXA1 in macrophages in vivo, we stimulated THP1-Ctrl, THP1-shANXA1, and THP1-ANXA1 cells to M0 polarization, and mixed them with PANC-1, and inoculated them subcutaneously on the backs of immunodeficient mice. In polarization-related experiments, THP1 cells were stimulated with phorbol 12-myristate 13-acetate (200 ng/mL, MCE, HY-18739) for 48 hours and then used in follow-up experiments. 20 NOD-SCID mice were classified into four groups. A control group overexpressing ANXA1 was subcutaneously implanted with 1×10⁶ PANC-1 cells and 1×10⁵ THP1-Ctrl cells, while the THP1-ANXA1 group was subcutaneously implanted with 1×10⁶ PANC-1 cells and 1×10⁵ THP1-ANXA1 cells. A control group with ANXA1 knocked down was subcutaneously implanted with 1×10⁶ PANC-1 cells and 1×10⁵ THP1-Ctrl cells, while a THP1-shANXA1 group was subcutaneously implanted with 1×10⁶ PANC-1 cells and 1×10⁵ THP1-shANXA1 cells.

Macrophage depletion and reconstitution in mice were conducted as outlined in prior studies.48–50 Bone marrow cells were harvested from the femur and tibia of C57BL/6 mice. The cells were cultured in DMEM supplemented with 10% FBS for 12 hours. Non-adherent monocytes were collected and transfected with a lentivirus overexpressing ANXA1 or a control for 2 days. The transduced cells were then differentiated into BMDMs by treating with 50 ng/mL M-CSF for 7 days. To deplete mice of macrophages, 2 days following subcutaneous tumor formation with KPC cells, PDAC model mice were pretreated with clodronate liposomes (200 µl per mouse; Yeasen, Shanghai, China) via intraperitoneal injection. Three days post-clodronate liposome treatment, lentivirus-transduced BMDMs were intravenously administered to the macrophage-depleted mice to achieve reconstitution. Then, mice were sacrificed 2 weeks later and tumor specimens were dissected for subsequent analysis.

Bone marrow cells were harvested from the femur and tibia of C57BL/6 mice. These cells were cultured in DMEM medium supplemented with 10% FBS for 12 hours. Non-adherent monocytes were collected and transfected with lentiviruses control and overexpressing ANXA1 for 2 days. The transduced cells were then differentiated into BMDMs treated with 50 ng/mL M-CSF for 7 days. For deplete macrophages, 2 days following subcutaneous tumor formation with KPC cells, PDAC model mice were pretreated with clodronate liposomes (200 µl per mouse; Yeasen, Shanghai, China) via intraperitoneal injection. Three days post clodronate liposome treatment, lentivirus-transduced BMDMs were intravenously administered to the macrophage-depleted mice to achieve reconstitution. Then mice were sacrificed 2 weeks later and tumor specimens were dissected for following analysis.

Immunoblotting

Tumor tissues underwent lysis with RIPA Lysis Buffer (Beyotime Biotechnology, P0013B), which was supplemented with a Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, 78440). This was followed by protein quantification using a BCA Protein Assay kit (Beyotime, P0009). The samples were then subjected to equal loading onto a 10% SDS-PAGE and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, FFP33). The membranes were blocked and subsequently incubated with the appropriate primary antibody at a temperature of 4°C overnight. On the following day, the membranes were incubated with HRP-linked secondary antibodies (Cell Signaling Technology, 7074 and 7076). Visualization of the blot was achieved using an enhanced chemiluminescence reagent (Thermo Scientific) on ChemiDoc Imagers (Bio-Rad). The quantification of the WB bands was executed using the ImageJ software. All antibodies used for WB are listed in online supplemental table S1.

Quantification of cfDNA

Panc02 tumor cells were inoculated into Anxa1^ΔMɸ and Anxa1^WT mice, and allowed tumors to establish. Four weeks post inoculation, whole blood was collected by cardiac puncture into cfDNA tubes. Plasma was obtained by a double spin procedure (1,600 g for 10 min, separation, followed by 16,000 g for 10 min). Commence by taking a 1.5 mL centrifuge tube and adding 1 mL of plasma, followed by the addition of 100 µL of 10%SDS and 30 µL of Proteinase K. Invert the mixture for thorough mixing, then place it in an incubator set at 65°C for a duration of 20 min. Next, take a 5 mL centrifuge tube and add 15 µL of MagMAX Cell-Free DNA Magnetic Beads and 1.25 mL of MagMAX Cell-Free DNA Lysis/Binding Solution. Ensure complete mixing of the contents. Place the centrifuge tube in a QT-1 vortex mixer and vortex thoroughly for 10 min. Subsequently, place the centrifuge tube on a DynaMag Magnet magnetic separation rack and let it stand for 5 min or until the solution becomes clear and the beads concentrate on the wall of the centrifuge tube. Carefully remove the supernatant using a pipettor. Resuspend the magnetic beads in 1 mL of MagMAX Cell-Free DNA Wash Solution and transfer the suspension to a new 1.5 mL centrifuge tube. Place the 1.5 mL centrifuge tube on a DynaMag-2 Magnet magnetic separation rack and let it stand for 20 s. Repeat the standing process for an additional 2 min or until the solution becomes clear and the beads concentrate on the wall of the centrifuge tube. Remove the supernatant using a 1 mL pipettor and remove the residual supernatant with a 200 µL pipettor. Remove the centrifuge tube from the magnetic separation rack, add 1 mL of MagMAX Cell-Free DNA Wash Solution, and vortex for 30 s. Remove the centrifuge tube from the magnetic separation rack again, add freshly prepared 80% alcohol, and vortex for 30 s. Place the centrifuge tube on a DynaMag-2 Magnet and let it stand for 2 min, then remove the supernatant. Remove the residual alcohol, and allow it to dry at room temperature for 5 min. Remove a centrifuge tube from the magnetic separation rack, add 22 µL of MagMAX Cell-Free DNA Elution Solution, and vortex for 5 min. Return the centrifuge tube to the magnetic separation rack and let it stand for 2 min, or until all magnetic beads are adsorbed on the wall of the centrifuge tube to make the solution clear. Finally, transfer 20 µL of the supernatant (cfDNA lysate) into a centrifuge tube and store at −20°C. Sample analysis was performed using an Agilent 2100, and cfDNA (ng/mL of plasma) was calculated as the quantitative outcome.

Cell proliferation assay

Per hole of 96-well plate was seeded with 3×10³ viable Panc02 cells and grew for 24 hours. Next, the cells were allowed to grow in complete media for 5 days with treatment of BIP-V5 or control solvent (DMSO). The viability of Panc02 cells were determined using the Cell Counting Kit-8 (Dojindo, Japan) and measured at 450 nM with the BioTek Gen5 system (BioTek, Winooski, Vermont, USA).

ELISA analysis

Tumor tissues were subjected to a homogenization process in a solution of PBS, which was supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). This process was conducted in tubes using a three-dimensional cryogenic grinder (Servicebio, KZ-5F-3D), adhering strictly to the manufacturer’s protocol. For each 100 mg of tumor tissue, a volume of 3 mL of buffer was employed. Tumor homogenates were subsequently clarified through a centrifugation process at a force of 12,000 g for a duration of 20 min at a temperature of 4°C. ELISA was performed for the measurement of IFN-β (Multi Sciences, Zhejiang, China) in tumor tissue homogenates according to the manufacturer’s instructions.

The BMDMs obtained from Anxa1^ΔMɸ and Anxa1^WT mice were polarized into M1 and M2 macrophages, respectively. The supernatants were clarified by centrifugation at 12,000 g for 20 min at 4°C. For each sample, an equal volume was employed. IL-1β, IL-6, TNF-α, IL-10 and CCL2 (Multi Sciences, Zhejiang, China) were assayed in supernatants according to the manufacturer’s instructions.

qPCR for general

For the execution of qPCR assays on samples, the total RNA was initially extracted using TRIzol (Invitrogen, 15596026). This was followed by a RT-PCR process conducted with the PrimeScript RT Reagent Kit (Takara, RR047A). The resulting complementary DNA (cDNA) was then subjected to a qPCR assay using the SYBR Green qPCR Master Mix (HY-K0501A, MCE), according to the manufacturer’s recommended protocol. All the qPCR assays were performed at least three times independently on the Applied Biosystems 7500 Real-Time PCR System. Sequences for qPCR primers are provided in online supplemental table S3.

Bulk RNA sequencing

Tumor tissues from the mouse were subjected to a grinding process and subsequently lysed using TRIzol (Invitrogen). The total RNA was then extracted using an RNA extraction kit (procured from Vazyme), strictly adhering to the manufacturer’s protocol. The sequencing and analysis process was performed by OE Biotech, Shanghai.

Proteome analysis in Liquid Chromatography-Mass Spectrometry/Mass Spectrometry analysis

Samples were added to the SDT buffer and transferred to 2 mL tubes containing a specific amount of quartz sand. The lysate was homogenized using an MP Fastprep-24 Automated Homogenizer at 6.0M/S for 30 s, repeated twice. The homogenate was then sonicated and boiled for 10 min. Following centrifugation at 14,000 g for 15 min, the supernatant was filtered using 0.22 µm filters. The filtrate was quantified using the BCA Protein Assay Kit (P0012, Beyotime). For each sample, 20 µg of proteins were mixed with 6× loading buffer and boiled for 5 min. The proteins were then separated on a 12% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) gel and protein bands were visualized by Coomassie Blue R-250 staining. For each sample, 50–200 µg of proteins were reduced with 100 mM Dithiothreitol (DTT) for 5 min at 100°C. Subsequently, the detergent, DTT, and other low-molecular-weight components were removed using UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.5) through repeated ultrafiltration (Sartorius, 30 kD). A solution of 100 µl iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues, and the samples were incubated for 30 min in darkness. The filters were washed three times with 100 µl UA buffer and twice with 100 µl 50 mM NH4HCO3 buffer. Finally, the protein suspensions were digested with 4 µg trypsin (Promega) in 40 µl 50 mM NH4HCO3 buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptide segment was desalted using a C18 column. The peptide content was estimated by UV light spectral density at 280 nm, using an extinction coefficient of 1.1 of 0.1% (g/l) solution that was calculated based on the frequency of tryptophan and tyrosine in vertebrate proteins. LC-MS/MS analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) coupled to EASY-nLC (Thermo Fisher Scientific). The MS data were analyzed using MaxQuant software V.1.6.17.0. The cut-off of the global false discovery rate for peptide and protein identification was set to 0.01. Protein abundance was calculated based on the normalized spectral protein intensity (LFQ intensity). Proteins with a fold change greater than 2 or less than 0.5 and a p value (Student’s t-test) less than 0.05 were considered to be DEPs. All protein sequences were aligned to the database using NCBI BLAST on a Linux server, retaining only the sequences in the top 10 and with an E-value less than or equal to 1e-3. The GO term of the sequence with the top bit-score was selected by Blast2GO. Annotation from GO terms to proteins was completed by Blast2GO Command Line. After the initial annotation, InterProScan was used to search the European Bioinformatics Institute (EBI) database by motif and then add the functional information of the motif to proteins to improve annotation. Further improvement of annotation and connection between GO terms were carried out by ANNEX. Fisher’s Exact Test was used to enrich GO terms by comparing the number of DEPs and total proteins correlated to GO terms. Pathway analysis was also performed using the KEGG database. Fisher’s exact test was used to identify the significantly enriched pathways by comparing the number of DEPs and total proteins correlated to pathways.

Tissue digestion

Single cells were isolated from tumor tissues by using gentle enzymatic dissociation with a Tumor Dissociation Kit (Miltenyi Biotec, 130-096-730), as previously described.51 Then, single-cell suspension was resuspended in PBS with 0.04% BSA at a density of 1×10⁶ cells/mL in preparation for further analysis.

Flow cytometry

The discrimination between live and dead cells was conducted using the Fixable Viability Stain 700. Single-cell suspensions underwent incubation with anti-mouse CD16/CD32 to inhibit the non-specific binding of the Fc receptor, followed by staining with antibodies in accordance with established protocols.52 For the labeling of intracellular cytokines, cells were subjected to stimulation with a Leukocyte Activation Cocktail supplemented with GolgiPlug (550583, BD Pharmingen) for a duration of 4 hours at a temperature of 37°C and 5% CO₂ concentration in RPMI medium. The Transcription Factor Buffer Set (562574, BD Pharmingen) was employed for intracellular staining, adhering to the guidelines provided by the manufacturer. Online supplemental figure S3A illustrates the representative gating for flow cytometry. Flow cytometric assays were executed using BD FACSCelesta, and the resultant data were analyzed employing FlowJo software (Treestar). A comprehensive list of all antibodies used for flow cytometry is provided in the online supplemental table S1.

scRNA-seq of tumor tissues from mouse models

DEGs analysis

The DEGs analysis method was performed as described before.53 To identify DEGs, we used the scanpy.tl.rank_genes_groups() function based on the Wilcoxon rank-sum test with default parameters, and selected the genes expressed in more than 10% of the cells in either of the compared groups of cells and with an average log (fold change) value greater than 1 as DEGs. Adjusted p value was calculated by Benjamini–Hochberg correction and the value 0.05 was used as the criterion to evaluate the statistical significance.

Pathway enrichment analysis

In order to explore the potential functions of the DEGs, analyses were conducted using GO and the Kyoto Encyclopedia of Genes and Genomes (KEGG) in conjunction with the “clusterProfiler” R package V.3.16.1.55 Pathways with an adjusted p value (p_adj) of less than 0.05 were deemed to be significantly enriched.

Pseudotime trajectory analysis

The trajectory of cell differentiation for monocyte subtypes was reconstructed using Monocle 2 V.2.10.0.56 The construction of this trajectory involved the selection of the top 2,000 highly variable genes using the FindVariableFeatures() function from Seurat V.3.1.2. Dimension reduction was subsequently performed using the DDRTree() function. The resulting trajectory was visualized using the plot_cell_trajectory() function in Monocle 2.

Cell type recognition with cell-ID

Cell-ID is a multivariate approach that extracts gene signatures for each individual cell and performs cell identity recognition using hypergeometric tests (HGT).55 Dimensionality reduction was performed on a normalized gene expression matrix through multiple correspondence analysis, where both cells and genes were projected in the same low-dimensional space. Then a gene ranking was calculated for each cell to obtain the most featured gene sets of that cell. HGT were performed on these gene sets against reference from SynEcoSys database, which contains all cell type’s featured genes in the specific organ/tissue. Identity of each cell was determined as the cell type has the minimal HGT p value. For cluster annotation, frequency of each cell type was calculated in each cluster, and cell type with the highest frequency was chosen as the cluster’s identity.53 The cell type identification of each cluster was determined according to the expression of canonical markers from the reference database SynEcoSys (Singleron Biotechnology). SynEcoSys contains collections of canonical cell type markers for single-cell seq data, from CellMakerDB, PanglaoDB and recently published literatures.

Cell–cell interaction analysis (CellPhoneDB)

The cell–cell interaction analysis was performed by CellPhoneDB V.2.1.057 based on known receptor–ligand interactions between two cell types/subtypes, as described previously.

Immune Repertoire analysis

The pipeline of TCR clonotypes assignment was conducted using Cell Ranger (V.4.0.0) as a reference. A TCR/BCR diversity metric, encompassing clonotype frequency and barcode information, was procured. For the TCR, only cells possessing one productive TCR α-chain (TRA) and one productive TCR β-chain (TRB) were retained for subsequent analysis. Each unique pair of TRA(s)-TRB(s) was designated as a clonotype. If a clonotype was present in a minimum of two cells, the cells containing this clonotype were deemed to be clonal. The number of cells with such pairs served as an indicator of the degree of clonality of the clonotype.

Isolation and stimulation of mouse BMDMs

Bone marrow cells were isolated from the femur and tibia of mice and cultured with culture medium (high-glucose DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 mg/mL streptomycin, 10 ng/mL M-CSF (MedChemExpress, USA)) for 5 days, allowed to obtain the adherent BMDMs. Then, these macrophages were induced to different phenotypes. The obtained BMDMs were stimulated with 100 ng/mL LPS and 20 ng/mL IFN-γ for 12 hours for inducing BMDMs into M1 macrophages, while stimulated with 20 ng/mL IL-4 for 24 hours for inducing BMDMs polarized into M2 macrophages.

Statistics and bioinformatics

All data were represented as the mean±SD deviation (SD). Each experiment was conducted a minimum of three times for consistency. The specific statistical details pertaining to the experiments are delineated in the associated legend or methods section. The two-tailed Student’s t-test was employed to ascertain the statistical significance between two distinct groups. For the purpose of correlation analysis, the Spearman Rank correlation test was used. The comparison of categorical variables was conducted using either the χ² test or the Fisher’s exact test. The measurement of OS was taken from the date of diagnosis until the date of death from any cause, or until the last censored follow-up. The methodologies used for survival analysis have been delineated in our preceding study. The data were analyzed using either the Statistical Package for Social Science V.26.0 or GraphPad Prism (V.8.3.0.). p<0.05 was considered statistically significant, with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANXA1 expression data were obtained from the Cancer Genome Atlas (https://tcga-data.nci.nih.gov/tcga/) and TIMER databases (http://timer.comp-genomics.org/timer/).