This study was performed under the approval of the Animal Ethics Committee of Tinghu People’s Hospital. Extensive efforts were made to ensure animal suffering was minimized.
Patient samples and data
This study included patients who were disease-free at the end of the operation, with a complete medical record (i.e., tumor location, hospitalization, lymphatic invasion, vascular invasion, intra-abdominal infection, etc.). Serum samples were collected on postoperative day 1 from patients who underwent curative surgery for colorectal cancer at Tinghu People’s Hospital, between 2016 and 2020. Disease-free survival was defined as time from the completion of surgery to cancer recurrence, or death from any cause.. Patients who were lost to follow-up had their data censored at the last date ofcontact. Written informed consent was obtained by all patients prior to inclusion, and all materials were obtained following approval by the institutional review board of Tinghu People’s Hospital.
The human colorectal cell line HCT116 was purchased from Sigma–Aldrich (St. Louis, MO, USA). The cells were maintained in a subconfluent state using McCoy’s 5a minimum essential media (ThermoFisher, Shanghai, China) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin.
Cells were lysed in NP-40 buffer containing 150 mM sodium chloride, 1.0% NP-40, 50 mM Tris hydrochloride, complete™ Protease Inhibitor Cocktail (Roche Applied Science, Mannheim, Germany), and 1 mM phenylmethyl sulfonyl fluoride for 30 minutes on ice. The supernatant was subjected to protein denaturation with buffer containing 2% sodium dodecyl sulfate (Sigma–Aldrich, St. Louis, MO, USA). Subsequently, it was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a monoclonal antibody against toll-like receptor 9 (TLR9), p38, p-p38, p65, p-p65, c-jun amino-terminal kinase (JNK), p-JNK, Stat, and p-Stat. The any unbound primary antibody was washed away after incubation, and then the membrane incubated with the appropriate horseradish peroxidase-conjugated immunoglobulin secondary antibodies. Afterwards, enhanced chemiluminescence (GE Healthcare) was used to detect the staining.
Mouse neutrophils were isolated from bone marrow extracted from the tibias and femurs. Neutrophils were sorted after incubation with allophycocyanin (APC) -conjugated anti-mouse-Ly6G antibody (BD Pharmingen™, Cat NO. 560599) and APC-Cy7 CD 11b (BD Pharmingen™, Cat NO. 557657).
Experimental metastasis assay
Colorectal metastases were induced in mice as previously described(Nicoud et al. 2007). To understand the mechanism of metastasis, the experiment was conducted. Specifically, 1×104 HCT116 cells or luciferase-labeled HCT116 cells (HCT116-GFP-Luc) were injected via a 1-cm midline laparotomy into the spleens of 8- to 10-week-old nude mice. The tumor cells were allowed to circulate for 15 minutes. In the control group. experimental groups respectively. Prior to the injection of LPS, splenectomy was performed to prevent the formation of splenic tumors. Approval of this experimental protocol was obtained from the Committee on the Ethics of Animal Experiments of Xi’an Jiaotong University (Approval Number: 2011-045).
Detection and quantification of MPO–DNA and citrullinated-histone H3 (Cit-H3)
The detection of serum MPO–DNA complexes was performed using the enzyme-linked immunosorbent assay, as previously described (Nakazawa et al. 2014). In brief, microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA) were initially coated with the monoclonal anti-human MPO antibody (5 µg/ml; AbD Serotec, Kidlington, UK) and incubated overnight at 4°C. After blocking with 1% bovine serum albumin, the sera (final dilution 1:3) and horseradish peroxidase-conjugated anti-DNA antibody in the cell death detection kit (Roche Diagnostics, Tokyo, Japan) were added to the wells, then peroxidase substrate was added according to the instructions provided by the manufacturer. The serum from a patient with MPO-antineutrophil cytoplasmic antibody-associated vasculitis served as a positive control.
Colon sections were prepared and mounted on glass slides to detect Cit-H3 in tissues. Antigen retrieval was performed using citrate buffer and then the retrieved specimens were permeabilized with 0.1% Triton X-100 for 10 min. Subsequently, specimens were blocked with phosphate-buffered saline containing 3% bovine serum albumin and 1% donkey serum. The sections were incubated with primary antibodies, specifically anti-citrullinated-histone H3 (1:100; Abcam, Cambridge, MA, USA). This was followed by incubation with Alexa Fluor 488 donkey anti-rabbit (1:500; Abcam) and Alexa Fluor 647 donkey anti-goat (1:500; Abcam) secondary antibodies overnight at 4℃, respectively. For the detection of DNA, 4’6-diamidino-2-phenylindole was applied.
Treatment with LPS, DNase I, and YW4-03
Initially, injections of LPS (5 µg/ml), DNase I (50 mg/mouse, Roche), or YW4-03 (10 mg/kg) were administered immediately prior to abdominal closure. This was followed by daily administration of intraperitoneal doses of DNAse (thrice weekly for YW4-03).
For the animal studies, the results are expressed either as the mean ± standard error of the mean or the mean ± standard deviation. One-way analysis of variance was used for group comparisons ,whereas Student’s t-test was applied in Comparisons between the experimental and control groups. For the data analysis involving human subjects, human patients, we dichotomized the levels of the MPO–DNA complex at the median and compared the baseline characteristics for each group. Besides categorical variables were examined using the chi-squared test or Fisher exact tests, while continuous variables were analyzed using Student’s t-test or the Wilcoxon rank-sum test. The analysis of disease-free survival was conducted using the Kaplan–Meier method. For all analyses, a two-tailed p < 0.05 denoted statistical significance.