Cell lines and mice. The murine melanoma cell line, B16F10, and Lewis Lung carcinoma (LLC) cells were purchased from the Chinese Academy of Sciences Cell Bank. C57BL/6 mice (males; age, 6-8 weeks, number. 40) were obtained from SPF (Beijing) Biotechnology Co., Ltd. All animal experiments were approved by the Ethics Committee of the Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Science.
Chemicals and regents. Paclitaxel (cat. no. S1150) and ticagrelor (cat. no. S4079) were purchased from Selleck Chemicals, and prepared as 5 or 20 mM stock solution, and stored at -80℃. FBS (cat. no. 10270-106), penicillin/streptomycin solution (cat. no. 2029632) and high glucose DMEM (cat. no. 10313021) were obtained from Gibco; Thermo Fisher Scientific, Inc. The Annexin V-FITC kit (cat. no. LHK601-100) was obtained from Jiamay Biotech Co., Ltd., while the Cell Counting Kit-8 (CCK-8; cat. no. GB707) from Dojindo Molecular Technologies, Inc. The MatrigelTM Basement Membrane Matrix (cat. no. 354234; BD Biosciences) was obtained from Corning, Inc., and the antibodies against Bad (ca. no. sc-8044) and Bax (cat. no. sc-493) from Santa Cruz Biotechnology, Inc. Finally, the anti-caspase-3 (cat. no. A0214), anti-GAPDH (cat. no. AC035), HRP Goat Anti-mouse IgG (cat. no. AS003) and HRP Goat Anti-Rabbit IgG (cat. no. AS014) antibodies were purchased from ABclonal Biotech Co., Ltd.
Cell viability assay. Cells were grown in culture flasks, harvested in trypsin/EDTA solution, and resuspended in DMEM supplemented with 2% FBS. Subsequently, a total of 5,000 cells were seeded into 96-well plates. Following incubation for 6 h, cells were treated with paclitaxel (5 nM), ticagrelor (20 µM) or paclitaxel (5 nM) combined with ticagrelor (20 µM). Each experiment was repeated for four times per group independently. The cell viability assay was performed according to the manufacturer's instructions (CCK-8; cat. no. GB707; Dojindo Molecular Technologies, Inc). Briefly, 10 µl CCK-8 reagent was added into each well and cells were cultured for 48 h. Following incubation for 2 h at 37℃, the optical density (OD) at 450 nm was measured in each well.
Platelet isolation. C57/BL6 wild-type mice (ag, 6-8 weeks) were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (130 mg/kg,) and fixed by pin. Following anesthesia for 5 min, fresh blood was collected (1 ml/mouse) from the inferior vena cava using a 1/7 volume of acid-citrate-dextrose (2.5% trisodium citrate, 2.0% d-glucose, 1.5% citric acid) as anticoagulant. When needed, mice were sacrificed by cervical dislocation. Subsequently, the blood was centrifuged at 200 x g for 10 min and the supernatant was transferred into new tubes and centrifuged at 800 x g for 3 min. The precipitate was then washed with CGS buffer (0.9% NaCl, 0.03 M d-glucose, 0.01 M trisodium citrate, pH 7.0) and resuspended in modified Tyrode's buffer (MTB; 12 mM NaHCO3, 0.9% NaCl, 5.6 mM d-glucose, 2.6 mM KCl, 2.4 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, and 0.1% BSA, pH 7.4). The platelets were counted using the Sysmex XP-100 hematology analyzer (Sysmex Co.) and incubated at room temperature for 1 h.
Co-culture of B16F10 or LLC cells with platelets. Cells were harvested from culture flasks after trypsinization (with EDTA) and resuspended in DMEM containing 2% FBS. Subsequently, 2x105 cells/well were seeded into 6-well culture plates. Following incubation for 4 h, the prepared platelets (2x106/well) were added into each well and cultured for an additional 48 h. Subsequently, cells/platelets were treated with paclitaxel (5 nM), ticagrelor 20 µM or paclitaxel (5 nM) + ticagrelor (20 µM).
Colony formation assay. Cells were harvested from culture flasks by trypsinization and resuspended in DMEM supplemented with 10% FBS. Subsequently, 500 µl (300 cells) of the cell suspension were added into 12-well plates. Following incubation for 4 h, cells were treated with paclitaxel (5 nM), ticagrelor (20 µM) or paclitaxel (5 nM) + ticagrelor (20 µM). Cells in the control group were treated with DMSO. The medium was replaced every 4 days. After culture for 12 days, the medium was removed and cells were washed with PBS for three times. Finally, for Giemsa staining, cells were fixed with methanol.
Wound healing assay. For wound healing assay, 2x105 cells/well were seeded into 6-well plates. The next day, wounds were created by scratching using 1-ml pipette tip. Following washing twice with PBS, the wells were supplemented with 2 ml DMEM (with 2% FBS). To evaluate cell migration, images were captured at three fields from each wound at 0 and 48 h under a microscope.
Transwell assay. For Transwell assays, the membrane of the 24-well Transwell plates was pre-coated with 100 µl Matrigel for 2 h at 37℃. Cells in culture flasks were harvested using 1 ml trypsin/EDTA solution and resuspended in DMEM containing 1% FBS to stop digestion. Subsequently, 100 µl (1x105 cells) of the cell suspension was added into the upper chamber of the 24-well Transwell plate. The lower chamber was supplemented with 700 µl DMEM and incubated for 48 h at 37℃. Following incubation, the membrane was removed from the 24-well plate, and the invasive cells were fixed with methanol and stained with 0.2% crystal violet solution after washing with PBS.
Phosphatidylserine (PS) externalization assay. Isolated platelets (1x107) were treated with paclitaxel (5 nM), ticagrelor (20 µM) or paclitaxel (5 nM) + ticagrelor (20 µM). Following treatment, the cells were mixed with Annexin V-binding buffer (1X) and Annexin V-FITC (50:50:1) for 1 h. The cell samples were shaken gently and analyzed by flow cytometry after 15 min.
Western blot analysis. Platelets were lysed by RIPA buffer (cat. no. WB3100), the isolated protein samples were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were then incubated with primary antibodies against Bad, Bax, caspase-3 and GAPDH (reference gene). Images of the protein bands were captured using the Tanon-5200 Multi system (Tanon Science and Technology Co., Ltd.). Data were semi-quantified using the Image J software V1.8.0 (National Institues oof Health).
Tumor mouse models. In the current study two tumor models were established in C57BL/6 mice. Briefly, 3x105 B16F10 or 5x105 LLC cells in 50 µl PBS were injected into the tail vein of 5 mice/group. Mice were treated with paclitaxel (10 mg/kg) or ticagrelor (10 mg/kg) every 2 days by tail vein injection for 14 days for the B16F10 model or 20 days for the LLC model. On day 15 (B16F10 model) or 21 (LLC model), mice were sacrificed by cervical dislocation. Subsequently, the lungs were collected and the number of metastatic lung nodules, with a size of 0.2-1.5 mm, was recorded.
Statistical analysis. All statistical analyses were carried out utilizing Student's t-test or one-way ANOVA with the SPSS 19.0 software (IBM Corp.). All data are expressed as the mean ± standard deviation (SD; n≥3). All P-values were two-sided and P<0.05 was considered to indicate a statistically significant difference.