2.1 Ethic statement

The study involving animals was conducted following the ARRIVE guidelines and was approved by the ethic Committee of Basic Medical School, Shandong University (ecsbmssdu2021-2–46).

2.2 Mice and cells

Wild-type C57BL/6 J mice were sourced from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. The experimental protocols complied with established ethical standards for animal research. A pIRES human IL-37d expression plasmid was injected into fertilized eggs, which were then implanted into female C57BL/6 mice to generate IL-37dtg mice. To maintain a pure genetic background, the founders underwent backcrossing with wild-type (WT) C57BL/6 mice for more than six generations. Lewis lung cells (LLC) were acquired from Procell Life Technologies Co., Ltd (Wuhan, China). Lewis lung cells are a widely utilized mouse model in cancer research and drug screening, and have been employed in previous studies as a cell line for constructing mouse metastasis models [16, 17, 29].

2.3 Real-time PCR

Total RNA was extracted from 1 × 106 cells using the RNA-fast200 kit (Cat#220,011, Fastagen, shanghai, China). One microgram of RNA was reverse transcribed utilizing the ReverTra Ace qPCR RT Kit(Cat#FSQ-101, Toyobo, Japan). The resulting cDNA was amplified with THUNDERBIRD SYBR qPCR Mix on a StepOnePlus real-time PCR system (Bio-Rad, Hercules, CA, USA). Gene expression levels were normalized against housekeeping genes, specifically 18 s or β-actin, and standardized to a value of 1 in WT-NC cells. The primer sequences are shown in Supplementary Table 1.

2.4 Immunohistochemistry

Lung tissue sections were dewaxed and hydrated in xylene, followed by antigen retrieval. The sections were washed three times with PBS solution and subsequently treated to inhibit endogenous biotin using rabbit serum for 20 min. Following this, the sections were blocked with goat serum for 20 min at 4 °C. The next step involved overnight incubation at 4 °C with rabbit anti-TLR3 antibody (Abcam, Cambridge, MA, USA), rabbit anti-MMP9 antibody (Abcam, Cambridge, MA, USA), and rabbit anti-S100A9 antibody (CST, USA) diluted in PBS. On the following day, the sections underwent a 30-min incubation at 37 °C with MaxVsion Kit containing horseradish peroxidase (HRP)-conjugated anti-rabbit IgG before being stained using the diaminobenzidine tetrahydrochloride (DAB) substrate kit from Fujian MaiXin Company and counterstained with hematoxylin. Finally, the sections were scanned utilizing a Panoramic Digital Section Scanner (VS120) (Olympus, Japan).

2.5 Isolation of pulmonary tissue cells and flow cytometry

To minimize hemorrhaging and facilitate exposure of the heart, the thoracic cavity of the mice was carefully opened using scissors. Subsequently, a continuous infusion of 50 mL of 1xPBS was meticulously administered through the cardiac region. Following this procedure, the lungs were extracted and incubated in a 4 mL digestion solution maintained at 37 °C for one hour. The lung tissue was then homogenized and filtered through a 200-mesh copper mesh into a 10 mL centrifuge tube. During homogenization, phosphate-buffered saline (PBS) was steadily added to ensure cellular cleansing and prevent cell lysis. The tube underwent centrifugation at 500 g for five minutes. The digestion solution consisted of a dissociation mixture containing collagenase IV (100 U/mL) and DNase (1 mg/mL). After removing the supernatant, red blood cells were gently lysed by pipetting and agitation, followed by another centrifugation step at 500 g for five minutes. Approximately 1 × 106 cells were counted and transferred to a flow cytometry tube, which was subsequently centrifuged at 1500 rpm at 4 °C for three minutes before being resuspended in PBS; this washing procedure was repeated twice. Fluorescently labeled antibodies were then added to the cells and allowed to incubate in darkness at 4 °C for sixty minutes. Prior to analysis, cells were carefully filtered through a 200-mesh copper mesh again. The flow cytometry antibodies used in this study included CD11b-PE-cy7, Ly6G-PE, and Ly6C-APC from BioLegend, USA.

2.6 Isolation of murine neutrophils from bone marrow

The musculature surrounding the tibial and femoral bones in mice was carefully dissected. Subsequently, bone marrow cells were gently flushed out using a 25-gauge needle and a 5 mL syringe filled with an ice-cold buffer solution (cell suspension) composed of 1 × PBS (ThermoFisher Scientific, USA), 1% FBS, and 2 mM EDTA (ThermoFisher Scientific, USA). The resulting cell suspension was then meticulously filtered through a 70-μm filter (ThermoFisher Scientific, USA) placed atop a conical 50 mL Falcon tube. To isolate BM Ly6G + cells effectively, the cells were specifically labeled with biotinylated antibodies targeting Ly6G, followed by incubation with streptavidin-coated microbeads (Miltenyi Biotec, Germany). Afterward, the cells were successfully separated using MACS columns (Miltenyi Biotec, Germany) post-incubation.

2.7 Cell migration assay

Following the purification of neutrophils from murine bone marrow, the migratory capacity of unstimulated cells was assessed using a 3 μm pore Transwell system (Corning, USA).A study published in 2018 in Nature Immunology investigated the migration of neutrophils during tumor progression, utilizing Transwell membranes with a pore size of 3 microns to conduct migration and chemotaxis assays [16]. The cellular suspension was subsequently transferred to a sterile 15 mL tube employing serum-free 1640 medium devoid of bovine serum albumin (BSA). Cell enumeration was executed to ensure the adjustment of cell concentration to 5 × 105 cells/mL. In a 24-well plate, 600 μL of complete 1640 medium was meticulously introduced, and the Transwell inserts were gently inserted into the wells, taking care to prevent bubble formation. Subsequently, 200 μL of the single-cell suspension of neutrophils was uniformly dispensed into the Transwell inserts, ensuring the prevention of bubble formation. The plate was then incubated at 37 °C with 5% CO2 for a duration of 1 h. Following this, 10 μL of the medium was extracted from the lower well, and cell enumeration was conducted employing a hemocytometer.

2.8 Mouse metastatic tumor model

To establish a lung cancer metastasis model, 1 × 105 Lewis lung cells were injected into the tail vein of male C57BL/6 and IL-37d mice (6–8 weeks old, n = 7). The mice were euthanized using ether anesthesia 1 to 3 weeks post-injection, and any mice that died unexpectedly were excluded from the analysis. The experiment was repeated to assess survival rates and distant metastasis(n = 7).Animal experiments were submitted to and approved by the School of Medicine at Shandong University(Jinan, Shandong Province, China), ensuring that all experimental procedures and animal welfare measures comply with the Guidelines for the Care and Use of Laboratory Animals established by the National Institutes of Health.

2.9 Statistical analysis

Mean values, standard deviation values, and statistical significance were analyzed by GraphPad Prism 8. Differences between groups were analyzed with the Student’s t test (unpaired), two-way ANOVA followed by the Tukey’s test (for multiple comparisons). P values < 0.05 was considered to be statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns not significant. Results are presented followed by at least three independent experiments of biological replicates. The data presented in this study conform to a normal distribution.