Reparixin

SUM149 and SUM190 breast cancer cells were obtained from Asterand (Detroit, MI, USA) and cultured in Hams F12 media (Mediatech, Manassas, VA, USA) containing 5 μg/ml insulin, 1 μg/ml hydrocortisone, 10 mM HEPES and antibiotics (penicillin/streptomycin). Medium for SUM149 cells was further supplemented with 5% fetal bovine serum (HyClone, Logan, UT, USA), and for SUM190 cells was further supplemented with 5 mM ethanolamine, 5 μg/ml transferrin, 6.6 ng/ml 3,3',5-triiodo-l-thyronine sodium salt, 8.7 ng/ml sodium selenite, and 1 mg/ml bovine serum albumin. Cells were cultured at 37 °C in a 5% CO₂ incubator. Breast cancer cell line MDA-MB-231 was purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained in DMEM-F12 (1:1) supplemented with 10% fetal bovine serum. Paclitaxel, insulin, hydrocortisone, HEPES, and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Reparixin was purchased from MedChemExpress (MedChemExpress Biotechnology, Monmouth, NJ, USA).

Recombinant human LIF (#300-05, 20 ng/mL), CXCL3 (#300-40, 20 ng/mL) and CXCL8 (#200-08M, 20 ng/mL) were all purchased from Peprotech. LIF neutralizing antibody (AB-250-NA, 2 μg/mL) was purchased from R&D, and CXCL3 neutralizing antibody (PP1014P2, 5 μg/mL) was purchased from Origene. Stattic (#HY-13818), LY3214996 (#HY-101494), PD98059 (#HY-12028), SB 203580 (#HY-10256), LY294002 (#HY-10108), JSH-23 (#HY-13982), BAY 11-7082 (#HY-13453), T-5224 (#HY-12270), TK216 (#HY-122903), TAT-DEF-Elk-1 (#HY-P2262A), SB225002 (#HY-16711), Reparixin (#HY-15251), and EC330 (#HY-100949) were all purchased from Med Chem Express. SB-505124 (#M2250) was purchased from Abmole. A list of the concentrations and targets used for the inhibitors is provided in Supplementary Table 1.

ICA (purity≧94%, cat No. I1286, Sigma-Aldrich, MO, USA) was dissolved in deionized water, and the rats were intragastrically administered with ICA at a dosage of 50 mg/kg/d or 100 mg/kg/d. Both the two dosages were safe for rodents according to previous studies [11 (link)]. The administration of ICA began on the 7^th day after the surgery and ended on the 21st day. Subsequently, two weeks was observed as the washout period. In addition, celecoxib at a dosage of 100 mg/kg/d (Pfizer, Piscataway, NJ, USA) [12 (link)] was administered intragastrically in the positive control group. Reparixin (antagonist of CINC-1 receptor L-lysin salt, MedChem Express, Stockholm, Sweden) or exogenous CINC-1 (cat No.C9709, Sigma-Aldrich, MO, USA) was administered using X-ray-guided percutaneous injection at a dosage of 8 μg/per disc with a 28-gauge needle. A detailed description of the timeline and group assignment is shown in Figure 1.

Transmigration assay was performed to confirm the reactivity of immune cells that migrated using chemotaxis. To prepare the supernatant to be placed in the lower chamber, 2 × 10⁵ of THP-1 cells were treated with MSU crystals (200 μg/ml) for 24 h, with or without PLAG, and then the cells were centrifuged to obtain only the supernatant, which was then placed in the lower chambers and the 5-μm-pore transmigration chamber (Corning, NY, USA) was placed on top. Next, a selective CXCR2 antagonist (SB225002, at 2, 10, or 40 μM; Sigma, USA), an inhibitor of CXC chemokine receptor type 1 (CXCR1, CXCR2) (reparixin, at 2, 10, or 40 μM; Medchem Express, NJ, USA), or an IL-1 receptor antagonist (IL-1RA) (2, 10, or 40 ng/ml; Peprotech, NJ, USA) were treated to differentiated HL-60 (dHL-60, 2 × 10⁵ cells), which mimics neutrophils. dHL-60 cells were placed on the upper chamber in 200 μl of serum-free RPMI 1640 medium and incubated for 24 h at 37°C. The migrated dHL-60 cells to the lower chamber were counted using a hemocytometer with trypan blue staining, and then the total number of cells in the media of the lower chamber was calculated and graphed.

Mouse B16-F10 melanoma cells were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Science (Shanghai, China) and cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. HEK-BLUE hTLR7 cells were purchased from InvivoGen (San Diego, CA, USA) and maintained in selective DMEM supplemented with 10 μg/mL blasticidin (Thermo Fisher Scientific, USA) and 100 μg/mLZeocin (San Diego, CA, USA). 5-Aza was purchased from Apexbio Technology LLC (Houston, TX, USA), polyinosinic–polycytidylic acid (poly: IC) was purchased from Sigma (St. Louis, MO, USA), and reparixin was obtained from MedChem Express LLC (Princeton, NJ, USA).

The human CSCC cell lines Siha, CasKi, and C33a were all purchased from Cell Resource Center (Beijing, China). The cell lines were verified by short tandem repeat (STR) sequencing by the Beijing Microread Genetics Company on July 2018. The cells were cultured in DMEM/F12 medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, USA) and 1% penicillin/streptomycin (PS) (Gibco, Thermo Fisher Scientific, USA) at 37 °C under 5% CO₂ (Thermo Technologies, Vancouver, Japan). The cells were treated with Tri-DAP, MDP (InvivoGen, USA), ML-130 (TargetMol, USA), CXCR1/2 inhibitor (Reparixin, Med Chem Express, USA), NF-κB inhibitor (EVP4593, Sellect, USA), or ERK inhibitor (SCH772984, Sellect, USA) as required. Primary CSCC cells were isolated from patient samples as previously described [58 (link)]. Briefly, the specimens were minced into 1-mm³ pieces in 6-cm petri dishes, and sequentially digested with 0.05% trypsin containing EDTA (Lonza, Walkersville, MD, USA) and 0.2% type I collagenase (Sigma-Aldrich Corp., St Louis, MO, USA) at 37 °C with constant shaking. FBS was added to terminate the reaction, and the cells were washed and re-suspended in DMEM/F12 complete medium with 5% FBS (Gibco). The primary cells were seeded in a petri dish and cultured for 7–10 days.