Ethics statement
The study protocol was approved by the Animal Ethics Committee of Shanghai Tongji Hospital, Tongji University School of Medicine. Animal use and experiments were carried out in strict accordance with the procedures approved by the National Cancer Institute Animal Care and Use Committee (ACUC).
Establishment of animal model
We utilized a genetically engineered mouse model where a localized infection of colonic tissue with adenovirus expressing Cre recombinase results in a tumor driven by either loss of both copies of APC with wild-type Kras (APC-WT) or a tumor driven by loss of APC with activated Kras harboring a gain in function mutation KrasG12D (APC-KRASG12D) using the methods described previously [13]. APC-KRASG12D mice are produced by hybridization of the homozygous APC allele with a heterozygous LSL-KrasG12D/+ allele. The APC/Krasmut mice were then hybridized to restore the homozygous APC allele and the heterozygous LSL-Kras. At the age of 7 weeks, APC and APC/KRAS mice received an intraperitoneal injection of tamoxifen (20 mg/kg, Sigma, Cat. #T5648) to activate stem cell-specific Cre. The activated Cre could induce APC loss and KRAS G12D allele activation. On average, more than 100 polyps were observed in the distal ileum (ileum) of the genetically engineered mouse 10 weeks after induction of gene mutation. All the APC-WT and APC-KRASG12D mice were euthanized at 8th, 12th, 16th, and 20th weeks (eight mice each time), and the number of polyps was counted.
Neutrophil isolation and sorting
Neutrophils were isolated from peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) of APC-WT and APC-KRASG12D mice. Neutrophils were sorted on a BD-Aria-Plus high-speed sorter after incubation with APC-conjugated anti-mouseLy6G antibody (BD Biosciences, San Jose, California, USA) and APC-Cy7 CD11b (BD Biosciences, San Jose, California, USA).
NETs quantification in vivo
To quantify the NETs in vivo, a capture enzyme-linked immunosorbent assay (ELISA) was performed to identify myeloperoxidase (MPO)-DNA complexes, as MPO was identified as a prominent constituent of NETS. The MPO-DNA complex was detected according to the instructions of the Mouse MPO ELISA kit (HK210–01; Hycult Biotechnology BV, Uden, the Netherlands). Briefly, 100 μL of the sample was added to the culture well, and incubated with 300 μL peroxidase-labeled anti-DNA mAb (Cat. No: 11774424001; Cell Death Detection ELISA PLUS, Roche Diagnostics GmbH, Mannheim, Germany) for 1 h. After that, the absorbance was measured. Values for soluble NET formation are expressed as percentage increase in absorbance above control.
Isolation of exosomes from plasma samples and cell culture supernatants
The plasma samples from APC-WT and APC-KRASG12D mice were centrifuged at 1000 g for 10 min. The harvested supernatant was further diluted in phosphate-buffered saline (PBS) and ultra-centrifuged at 100,000 g for 1 h at 4 °C using a Beckman Coulter Optima LE-80 K ultracentrifuge (Beckman Coulter, Fullerton, CA, USA). The pellet was resuspended in ice cold PBS, filtered with a 0.2 μm pore size filter, and preserved at − 20 °C.
Exosomes were isolated from the conditioned medium of DKs-8 and DKO-1 cells, respectively. The fetal bovine serum (FBS) was ultra-centrifuged at 100,000 g for 8 h in advance to remove the exosomes from the serum. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS until 80% cell confluence. After the cells were cultured in the medium containing 10% exosome-free FBS for 48 h, the cells were collected and centrifuged at 300 g for 10 min to remove cell debris, after which the supernatant was filtered through a 0.22 um polyethersulfone filter (Nalgene, Rochester, NY, USA) and subsequently concentrated using a 100,000 molecular weight cut-off Amicon Ultra centrifugal filter (Millipore, Billerica, MA, USA) and centrifuged at high speed of 150,000 g for 2 h to obtain the exosomes. Protein concentration of the exosomal preparations was determined using a microBCA protein assay kit (Pierce, Rockford, IL, USA), and the number of exosomes per microgram of protein was determined by nanoparticle tracking analysis (NanoSight, Wiltshire, UK).
Exosomes injection in vivo
A total of 5 mg APC-WT and APC-KRASG12D serum exosomes were intravenously injected into the tail vein of mice every 3 days. Exosomes used in these experiments were previously labeled with membrane fluorescent tracer PKH67 (Sigma-Aldrich). The labeled exosomes were washed, collected by ultracentrifugation, and resuspended in RPMI 1640 medium. Exosome-PKH67 and neutrophils (CD11b+ Ly6G+) were evaluated by flow cytometric analysis performed on a FACS LSR Fortessa flow cytometer with FACSDiva software (BD Biosciences, San Jose, CA, USA).
Cell culture
DKs-8 (WT allele), DKO-1 (KRAS mutant) cells were cultured in serum-containing DMEM (Mediatech, Manassas, VA, USA). Human peripheral blood neutrophils (PMN) were prepared as described elsewhere [14].
Three-dimensional cell culture
Three layers of collagen were used in a 48-well cell culture dish for cell culture in a three-dimensional collagen gel matrix. Neutrophils were trypsinized, syringed, and resuspended in serum-free DMEM at a concentration of 5 × 105 cells/mL before being plated in collagen-coated dish. The upper and lower layers contained 150 μL/well of PureCol collagen (Advanced Biomatrix, San Diego, CA) which were diluted to 2 mg/ml in serum-free DMEM. The intermediate layer consisted of 2 mg/mL collagen in serum-free DMEM (150 μL/well) and 5 × 103 neutrophils. Serum-free medium or serum-free medium supplemented with 50 g of DKs-8- or DKO-1-derived exosomes. The medium was renewed twice a week, and the neutrophils was cultured for 2 weeks. The experiment was repeated independently three times.
Detection of peptides via LC-multiple reaction monitoring (LC-MRM)
DKs-8, DKO-1 cells and PMN were grown to 80% confluence. After serum starvation overnight, one million cells were cocultured with 100 g of the indicated exosomes or mock-treated for 1 h under constant rotation at 37 °C. Cells were then pelleted and rinsed three times with ice-cold PBS, and the detection of peptides was conducted as described previously [15]. The mobile phase consisted of 0.1% formic acid solution (A) or 90% acetonitrile (B). An 80 min gradient was performed with a 15 min washing period (100% A). Following that, the gradient was increased to 60% B for 43 min treatment, followed by an increase to 95% B for 49 min treatment. At last, the cells were allowed to stand for 11 min before the gradient returning to 97% A. Transitions for each peptide (WT KAS LVVVGAGGVGK, KRAS G13D LVVVGAGDVGK) were selected using the Skyline software package.
In vitro NET formation
Neutrophils were incubated with tumor exosomes, plated on coated plates for 1 h, and then treated with Phorbol-12-myristate-13-acetate (PMA, 100 nM, Sigma-Aldrich) at 37 °C 5% CO2 for 4 h. Equivalent cells were also lysed with 0.1% Triton X-100 (Union Carbide Chemicals, Cleveland, OH, USA) or frozen as a control for the total DNA. Cells were incubated at 37 °C for 2–4 h. The membrane-impermeable DNA-binding dye, Sytox Green (Invitrogen) was added (0.1 μM) to bind extracellular DNA, and fluorescence was quantified using a fluorescence spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Interaction of exosomes with NETs under static conditions
Neutrophils (1 × 105) were seeded onto 13 mm coverslips (Glasscyto) and treated with 50 nM Phorbol-12-myristate-13-acetate (PMA, 100 nM, Sigma-Aldrich), a potent inducer of NETosis at 37 °C, 5% CO2 for 4 h. The neutrophils were then cocultured with 1 μg of exosomes which were labeled with 3 mM DiD in advance. The neutrophils were fixed with 500 uL of 4% paraformaldehyde for 10 min, then washed 3 times with PBS and incubated for 10 min with blocking solution (PBS, 10% FBS, 5 mg/ml BSA). The samples were subsequently stained with Hoechst 33342 (1:1000) for 2 h. Images were captured with a confocal microscope (LEICA DMI 4000 with laser system TCS SPE) and analyzed using Fiji Image J software.
Adhesion, migration and invasion assays
For the adhesion assays, 5 × 105 neutrophils were encapsulated in 24-well plates for 1 h at 37 °C in 5% CO2. DKs-8 cells were labeled with CFSE (Molecular Probes) for 10 min, and then 1 × 105 DKs-8 cells were added to the wells containing 100 nM PMA neutrophils, DKs-8 exosomes (DKs-8 exos); DKO-1 exosomes (DKO-1 exos); DKs-8 exos and 1000 U DNAse, DKO-1 exos and IL-8 or DKO-1 exos and anti-IL-8). Wells without PMA were used as controls. After incubation for 4 h at 37 °C in 5% CO2, wells were fixed with 4% paraformaldehyde. Cell adhesion was evaluated as the number of CFSE-labeled DKs-8 cells in 4 random high power field at × 20 using Olympus Floview 500 microscope.
Cell migration and invasion were tested using 6.5-mm Costar Transwell chambers with 8-μm pores (Corning, NY, USA). The plate coated with 50 μL Matrigel (YB356234, Shanghai Yubo Biological Technology, Shanghai, China) diluted by serum-free medium at 4 °C was then placed into the upper chamber of the Transwell apparatus. Then, 200 μL of cell suspension in medium containing 20% FBS was added to each well of the upper chamber and 800 μL of conditional medium containing 20% FBS was added to the lower chamber. Tumor cell suspension in the upper chamber were treated with neutrophil media containing DKs-8 exos + PMA-stimulated neutrophils, DKO-1 exos + PMA-stimulated neutrophils, DKO-1 exos + PMA-stimulated neutrophil media and 1000 U of DNAse, DKO-1 exos + PMA-stimulated neutrophil and 10 μg/ml IL-8 neutralizing antibodies, DKO-1 exos + PMA-stimulated neutrophil and 10 μg/ml anti-IL-8, or culture media alone (control). After incubation for 24 h at 37 °C, transwell plate was immersed in formaldehyde for 10 min, and then stained with 0.1% crystal violet for observation and cells were counted under an inverted microscope.
Cell counting kit-8 (CCK-8)
Cell viability was detected by CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan). DKs-8 cells (1 × 103 cells/well) were seeded onto 96-well plates and incubated for 48 h before CCK-8 detection. Ten μL of CCK-8 test solution was added into each well for incubation at 37 °C. At each time point (12, 24, 48 h), the optical density (OD) at 450 nm wavelength was read with a microplate reader.
Elisa
The IL-8 concentration in the cell culture supernatant (1 × 106 cells/mL) and the concentrations of TNF-α, IL-6, IL-8, and IL-17 in the serum were measured according to the ELISA kit instructions (R&D Systems, Minneapolis, MN, USA). The concentration of G-CSF in mouse serum was measured using a Mouse G-CSF ELISA Kit (ab197743, Abcam Inc., Cambridge, UK).
RNA extraction and quantification
Total RNA was extracted using miRNeasy Mini Kit (217,004, QIAGEN, Germany). The total RNA concentration and purity were determined on a nanodrop2000 micro-ultraviolet spectrophotometer (1011 U, nanodrop, USA). The RNA was reversely transcribed into cDNA using TaqMan MicroRNA Assays Reverse Transcription Primer kit (4,427,975, Applied Biosystems, USA). IL-8 primers and probes were purchased from Applied Biosystems (Test ID: Hs00174103_m1). The following primers and probe sequences and concentrations were used for human β-actin control detection: 5’Primer: GCCACCCCACTTCTCTCTAAGG at 50 nM, 3’Primer: GGGCACGAAGGCTCATCATTC at 300 nM, and TaqMan Probe: VIC-CCCAGTCCTCTCCCAAGTCCACACAGG-TAMRA at 50 nM. Real-time quantitative polymerase chain reaction (qPCR) was performed using ABI 7500 quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA). The final data were analyzed using the 2-ΔΔCt method and experiments were repeated independently three times.
Western blot analysis
Total protein was extracted and quantified using 2D Quant kit (GE Healthcare, Amersham Biosciences, Uppsala, Sweden). Then, 20 μg protein samples was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto membrane. After blocking, the membrane was incubated with primary antibodies overnight at 4 °C. KRAS (ab180772, 1:500), CD63 (ab68418, 1:1000), CD9 (ab223052, 1:1000), CD81 (ab155760, 1:1000), and Calnexin (ab22595, 1:1000) were purchased from Abcam (Cambridge, UK). After intensive washing, the membrane was further incubated with IgG goat anti-rabbit secondary antibody (1:1000, A21020, Abbkine, Scientific Co., Ltd., Wuhan, China) for 1 h at 37 °C, and was then developed by enhanced chemiluminescence (ECL) reagent. GAPDH worked as an internal control. The experiment was repeated independently three times.
Statistical analysis
SPSS 21.0 (IBM Corporation, San Jose, CA, USA) was used for statistical analysis. The measurement data with normal distribution was expressed as mean ± standard deviation. The unpaired t-test was used to compare the two independent groups that data conformed to the normal distribution and equal variance. Data comparisons between groups were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Data analysis at different time points was performed by repeated measurement ANOVA with Bonferroni post hoc test. The difference was regarded as statistically significant when p < 0.05.