Ethics Statement
Our study complied with all relevant codes of ethics of Southern Medical University. All animal protocols were approved by the Animal Research Ethics Committee of Southern Medical University and were conducted in accordance with the guidelines for the use of laboratory animals. The collection and use of clinical data was approved by the Institutional Research Ethics Committee of Nanfang Hospital of Southern Medical University.
Cell culture, CRC tissue preparation
These methods were performed as described previously51. The MC38 cell line (C57BL/6 J mouse colon adenocarcinoma cells) was obtained from the Chinese Academy of Sciences and cultured in 10% DMEM.
CCK8, colony formation assay, cell wound healing assay, Transwell invasion assay
These assays were performed as described previously51. Co-culture with dHL-60 1×105 dHL-60 cells were added when performing the above experiments. CCK8 and Transwell invasion assays were performed under different conditions (500 ng/ml anti-IL-8, 100 ng/ml recombinant IL-8 cytokine, 25 ng/ml PMA, 0.25 U/ml DNase I, 10 ug/ml ether lipid (PE-O, 18:0–18:1 PE)).
Immunohistochemistry
As previously described51. Primary antibodies used for immunohistochemistry: antibodies against ECI2, CD3, CD19 were from Proteintech; antibodies against CD68, KC (CXCL1) were from Immunoway; antibodies against CD66b, Ly-6G were from Abcam.
RNA extraction, RT-qPCR, and western blot analysis
These assays were performed as described previously51. Primary antibodies used for western blotting: antibodies ECI2, GAPDH, β-actin, PEX19, PEX16, GNPAT were from Proteintech; antibody α-Tubulin was from Abmart; antibodies cit-H3, H3 were from Abcam; antibodies AGPS, PMP70, FAR1 were from ABclonal. RT-qPCR primer sequences are shown in the Supplementary Material.
Lentiviral and plasmid transduction and transfection
Silencing ECI2 lentivirus was constructed from Obio Technology Co. (Y18163, GL413NC, Shanghai, China), and overexpression of ECI2, AGPS-WT, and AGPS-Mu lentivirus was constructed from WZ Biosciences Inc (Shandong, China). Transfection methods were as described previously51.
Animal studies
C57BL / 6J mice were purchased from the Guangdong Animal Center and subjected to a 60h light/dark cycle at 12–18°C and 22–50% humidity. Where quantification was provided, at least three independent experiments were performed for statistical analysis. For the mouse subcutaneous tumorigenic model, each group (NC/ECI2, shNC/shECI2) of MC38 cells (4 × 106) was injected subcutaneously into four-week-old female C57BL/6J mice. Tumor volume (length × width2 × 0.5) was measured every 4 days in mice. The tumor load was less than the maximum diameter (15 mm) approved by the University Animal Research Ethics Committee of Southern Medical University. At the end of the animal study, all mice were euthanized by inhalation of carbon dioxide.
For the mouse colorectal cancer liver metastasis model, 1×106 MC38 shNC or MC38 shECI2 cells were added to 100 µL PBS using a 28 G insulin syringe and injected into the spleen of 8-week-old C57BL/6 J mice via a 3 cm midline open abdomen. For the role of AGPS-Mu, MC38 cells transfected with both shECI2 and AGPS-WT or AGPS-Mu were injected into the spleens of mice. For the effect of DNase I, after injection of MC38 shECI2 cells into the spleens of mice, mice were injected with DNase I (2.5 mg/kg, intraperitoneally, Roche) once a day, and the control group was given PBS. For the effect of anti-KC (CXCL1), after injection of MC38 shECI2 cells into the spleens of mice, mice were injected twice a week intraperitoneally with anti-KC (CXCL1) (1.2ug/kg, PeproTech), and isotype control IgG was given to the control group. for the effect of anti-Ly6G, after injection of MC38 shECI2 cells into the spleens of mice, anti-mouse Ly6G (5 mg/kg, EBioscience) was injected intraperitoneally twice a week, and isotype control IgG was given to the control group. Mice were euthanized 25 days after injection of the cells. Liver metastases (including the size and number of metastatic cancer nodules on the surface of the liver) were observed by dissection. Whole liver tissues were cut and fixed with l0% neutral formalin, and the number of metastatic foci were counted after paraffin embedding, dehydration, serial tissue sections, and HE staining for light microscopic observation. The infiltration of each immune cell and the expression of KC (CXCL1) in the liver tissue were detected by immunohistochemistry; NETs formation (MPO, cit-H3) was detected by immunofluorescence in the liver tissue; and the level of citrullinated histone 3 (cit-H3) in the serums of mice was detected by Elisa.
Lipidomic analysis
Lipid metabolite analysis of cell samples was performed by the targeted quantitative lipidomic method by Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China). Previously frozen colorectal cancer cells (1 × 107) were homogenized in 1 ml of lipid extract (methyl tert-butyl ether: methanol = 3:1). The samples were stirred for 5 min at 4°C. Then, 200 µl of deionized water was added to the mixture, followed by centrifugation at 12,000 × g for 10 min at 4°C. The extract supernatant was dried and redissolved. Metabolites were quantified using an ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS) system (UPLC, ExionLC™AD, https://sciex.com.cn/; MS/MS, QTRAP® 6500 +, https://sciex.com.cn/). Analysis was accomplished using triple quadrupole mass spectrometry in Multiple Reaction Monitoring (MRM) mode. The software Analyst 1.6.3 was utilized to process the mass spectrometry data. The samples were quality controlled and a total of 834 lipid metabolites were detected.
RNA sequencing and data processing
Total RNA was extracted using Trizol (Life Technologies) according to the manufacturer's instructions, and RNA quality was assayed by both the NanoDrop 2,000 spectrophotometer and the Agilent 2,100/4,200 assay. A total of 3 ug of RNA per sample was used for analysis. Sequencing was sampled from a single replicate. Libraries were constructed using the common library method. After library construction, Qubit 3.0 was used for preliminary quantification, and RT-qPCR was used to accurately quantify the effective concentration of the libraries. RNA-seq was performed by BerryGenomics (Beijing, China) on the Illumina NovaSeq 6000 sequencing platform in PE150 mode. After obtaining the sequencing data, basic data quality control was performed first, and then these high-quality sequences were aligned to the reference genome and analyzed for gene expression level and gene structure. The reference genome for this project can be downloaded from: Genome Download Link (please use IE browser to open this link).
Enzyme-linked immunosorbent assay
Blood samples obtained from mice by venipuncture and cellular serum samples were aliquoted and stored immediately at -80°C according to our institutional specimen storage protocol. Quantification was performed according to the kit manufacturer's instructions, respectively. The kits for human IL-8, human CCL5, human CSF1, and human IL15 were purchased from Proteintech (China). The kit for mouse Cit-H3 was purchased from Ruixin Bio (China).
Isolation of peroxisomes by gradient centrifugation
2×108 cells were collected. Peroxisome extraction buffer (5 mM MOPS, pH 7.65, containing 0.25 M sucrose, 1 mM EDTA, and 0.1% ethanol, protease inhibitor mixture) was added (sigma). The cell suspension was then transferred to a Dounce homogenizer tube and carefully homogenized 25 times on ice using a mortar and pestle B. The tube was centrifuged at 1,000 × g for 10 min at 4°C, and the supernatant was transferred and centrifuged at 2,000 × g for 10 min at 4°C to obtain a pellet (i.e., mitochondrial layer.). The supernatant was transferred to a new centrifuge tube and centrifuged at 25,000 × g for 20 min. The supernatant (peroxisome-free fraction) was transferred to a new tube and the suspension (precipitate) was the crude peroxisome fraction. Peroxisome extraction buffer was added to the precipitate and centrifuged at 100,000 × g for 1.5 h. The substrate was collected for further analysis in western blotting experiments.
In vitro NETs induction method
Autoclave the slides to be used, tweezers, and gun tip were sterilized. Operate on the sterile operating table. Place the slides on the 24-well plate with tweezers, and put one slide per well. Spread 10% polylysine, 500uL per well, and place in 37℃, 5% CO2 incubator for more than 2h. Before spreading the cells, aspirate the poly-lysine in the wells, air dry and set aside. Conditioned medium (CM) was collected or added with anti-IL-8 (500 ng/ml), recombinant IL-8 cytokine (100 ng/ml), PMA (25 ng/ml), DNase I (0.25 U/ml), and ether lipids (18:0–18:1 PE, 10 ug/ml) into 24-well plates with cell crawls for incubation with dHL-60. NET formation was detected after 4 h of incubation.
Immunofluorescence detection of NETs formation
For paraffin-embedded tissue samples, sections were made at a thickness of 4 mm. After deparaffinization, antigen repair was performed in a citrate solution in a microwave oven (95°C, 30 min). For cell samples, cells (1 × 105) were inoculated on coverslips and fixed using 4% paraformaldehyde for 15 min. permeabilized in 0.1% Triton X-100 for 5 min. The slides or coverslips were then closed in PBS containing 2% bovine serum albumin for 1 h at room temperature. The slides or coverslips were incubated overnight at 4°C in a mixture of the two primary antibodies. The following primary antibodies cit-H3 (anti-rabbit antibody, Abcam), MPO (anti-mouse antibody, Immunoway) were used. The next day slides or coverslips were washed using cold PBS and incubated for 1 hour in the dark at room temperature with a mixture of two secondary antibodies produced in different species. The following secondary antibodies were used: anti-rabbit labeled with Alexa Fluor 488 and anti-mouse labeled with Alexa Fluor 594. Slides and coverslips were restained using 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) to visualize nuclei. The slides were sealed with a sealer containing an anti-fluorescence quencher. Each sample was examined under a fluorescence microscope and photographed. NETs formation was determined to be the percentage of field-of-view positivity for citrullinated histone 3 (cit-H3).
Chemotaxis assay
The dHL-60 cells (1 × 105 cells) were added to the upper chamber added to the Transwell device (Corning, 3402). Conditioned medium of differently treated CRC cells was added to the lower chamber as a chemoattractant. By adding different treatment conditions (anti-IL-8 (500 ng/ml), recombinant IL-8 cytokine (100 ng/ml), PMA (25 ng/ml), DNase I (0.25 U/ml), and ether lipids (18:0–18:1 PE, 10 ug/ml)) reagents were added to the lower chamber, and after 3.5 hours of incubation optical microscopy was observed to count the number of cells located in the lower chamber of the number of dHL-60.
Induction of dHL-60 cell differentiation
HL-60 (ATCC: CCL-240), a human myeloid neutrophil-like cell, was cultured in RPMI-1641 (Sigma, USA) with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin and maintained in a 5% CO2 incubator at 37°C. The cells were incubated with 1.25% DMSO in 5% CO2 at 37°C for 5 days to allow them to differentiate into neutrophils, which were recorded as dHL-60. Differentiation was verified by flow cytometry and Giemsa staining.
Flow cytometry
Pre-differentiated and post-differentiated HL-60 cells (5×106 cells) were collected. Goat serum was blocked on ice for 30 min, and the cells were incubated with goat anti-rat CD11b antibody coupled with Alexa fluor647 dye (BD Biosciences) and goat anti-rabbit CD16 antibody coupled with Alexa fluor488 dye (BD Biosciences) for 30 min at 4°C protected from light. Finally, the cells were resuspended in 100 µl PBS and the cell suspension was transferred to flow cytometry tubes for flow cytometry analysis (CytoFLEX S, Beckman). Analysis was performed using Flow-jo (V10.8.1) software (BD, USA).
Actinomycin D assay
To determine whether IL-8 accumulation occurs at the transcriptional level, CRC cells were pretreated with actinomycin D (2 ug/ml; FDbio, China) for 30 min, followed by treatment with 18:0–18:1 PE (10 ug/ml) for 3 h, and RNA was extracted for RT-qPCR. To determine whether IL-8 post-transcriptional regulation differed in the experimental and control groups, CRC cells were treated with 18:0–18:1 PE (10 ug/ml) for 3 h prior to actinomycin D (2ug/mL) treatment. RNA was extracted at different time points for RT-qPCR detection of IL-8 mRNA. The cDNA generation and RT-PCR methods were as described previously51. IL-8 mRNA abundance was analyzed with GAPDH as an endogenous control.
Luciferase assay
IL-8 activation was measured using the IL-8-dependent luciferase reporter gene (pGL3-IL8-Luc (-1481 + 44)). All experiments were performed according to the kit instructions (Promega). Cells were inoculated in 96-well plates and cell lysis buffer was transferred to black microtiter plates. Firefly luciferase reaction solution was then added and firefly luciferase activity was measured. The activity was measured after incubation with Renilla luciferase reaction solution.
Statistics and Reproducibility
Error lines indicate mean ± SD. Statistical analyses were performed using Prism9 (GraphPad software), ImageJ software, or SPSS software Statistics 22 (IBM Corp.). To compare two groups, P values were calculated using a two-sided Student's t test. For comparison of more than two groups, P values were calculated using ANOVA. A two-sided χ2 test was used. Survival curves were plotted using the Kaplan-Meier method. Correlation analysis was performed using Correlation of Ozone correlations. The relationship between ECI2 expression and clinicopathological characteristics was analyzed using the chi-square test and Pearson correlation. P < 0.05 was considered significant (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns denotes no statistical difference). Each experiment was repeated independently at least three times.