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  • br Quantification of apoptosis and


    Quantification of apoptosis and the cell cycle was performed on a FACSCalibur Flow Cytometer (Becton Dickinson, USA), as described [31]. NETs-treated and untreated cancer cells were harvested, treated with 20 μg/mL of RNase A and 50 μg/mL of PI (R4642-10MG, 81845-25MG, Sigma-Aldrich) after fixation with 70% ethanol, or stained with FITC-Annexin V and PI (556547, BD Pharmingen), then analyzed im-mediately.
    For the scratch wound healing assay, cancer cells (5 × 104 cell/mL) were incubated overnight. Migration was calculated according to the measurements at time 0 and 8 h after making a scratch and adding 0.5 × NETs. In addition, 24-well Transwell chambers (cat# 3422, Corning) with 8 μm-pore insets were used. Cells (2 × 105 cell) in serum-free media were planted on the inset for 6 h incubation in the migration assay, and plated on the inset coated with Matrigel (354234, BD Biosciences) for 48 h incubation in the invasion assay. RPMI-1640 containing 0.5 × NETs was added on to the inset and media containing 10% FCS was in the lower compartment as a chemoattractant. Cells invading the lower surface of the filter were stained with 0.1% crystal violet, and then counted in 5 random fields. The stained crystal violet was extracted using 33% acetic FG 4592 to obtain the absorbance at 570 nm.
    2.9. CCK-8 and FCM analysis of NETs-induced PBMC
    Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from a Ficoll-Paque density gradient (17-5442-02, GE Healthcare). PBMCs (1 × 105) were stimulated with cell-free NETs (0.5, 1, and 2 ×), in 96-well plates. The media served as a negative control. After 24 h, 48 h and 72 h of culture, supernatants were used to measure cytokine levels and cells were analyzed by CCK-8 for survival.
    The cells were stained using FITC-labeled anti-CD4 (130-092-358, Miltenyi Biotech) and PerCP-Cy5.5- labeled anti-CD8 antibodies (565310, BD Biosciences), then analyzed on FCM and processed using FlowJo 7.6.4. The isotype IgG served as the control.
    2.10. Analysis of cytokine levels with ELISA
    Cytokine levels of IL-8 (D8000C, R&D Systems), TNF-α (BMS223HS, eBioscience) and HMGB1 (ST51011, IBL International GmbH) in su-pernatants of BCG-treated cancer cells and IFN-γ (cat# 430107, BioLegend), TNF-α, IL-12 and IL-2 (BMS223HS, BMS238TEN, BMS221HS, eBioscience) in supernatants of NETs-treated PBMCs, were measured using the corresponding human ELISA kits, according to the manufacturers' instructions. 
    2.11. Mouse tumor models treated by BCG-induced NETs
    C57BL/6 mice (6–7-weekold) were purchased from HFK Bioscience Co., Ltd. (Beijing, China). All animal experiments were approved by the institute Ethics Committee. Murine bladder tumor cells (MB49) were obtained from M.A. O'Donnell (University of Iowa, IA). The mice were inoculated subcutaneously with 106 syngeneic MB49 cells to evaluate the role of BCG-induced NETs in vivo.
    Mouse marrow was taken for isolating neutrophils by centrifugation on a Ficoll-Hypaque density gradient. Neutrophils were purified using a mouse Neutrophil Isolation Kit (130-097-658, Miltenyi Biotec) for preparation of cell-free BCG-induced NETs. On day 10 following tumor implantation, the mice with tumors of similar size were treated in-tratumorally every 3 day with NETs, DNase- or heat-treated NETs, or medium as a control, for 4 times with 8 mice in each group. The mice were accessed for tumor weight, survival, and terminal morbidity. Tumor volume was calculated according to the following formula: major axis × minor axis2 × 0.52.
    NETs-treated mice were sacrificed on day 24 following tumor im-plantation, and the tumors were resected for pathologic observation after hematoxylin and eosin (H&E) staining. For immunochemistry (IHC), sections of paraffin-embedded samples were incubated with primary antibodies against mouse CD3 (555,273, BD Biosciences, Germany) or CD14 (ab182032, Abcam), then with peroxidase-con-jugated secondary antibodies to quantify T cells or mononuclear cells. The number of infiltrating immune cells, expressed as the average number/high-power field (HPF), was determined based on 10 randomly fields at ×400 magnification.
    TUNEL assays were conducted with the in Situ Cell Death Detection Kit (cat# 11684817910, Roche Applied Science), according to the manufacturer's protocol. Slides were counterstained with hematoxylin to identify the nuclei.
    2.12. Statistical analysis
    SPSS.20.0 software was used for statistical analysis and graphing. Unless otherwise stated, all data are presented as the mean ± SD of at least three independent experiments. A two-tailed Student's t-test or one-way ANOVA followed by Tukey's multiple comparison test was used, as indicated. A P < .05 was considered statistically significant.
    3. Results
    3.1. BCG induces NETs formation, which mainly consist of DNA and proteins