Fedratinib

The mouse macrophage cell line RAW264.7, the human colorectal adenocarcinoma cell line Caco-2, and the human normal colon epithelial cell line NCM460 were obtained from ATCC. All cells were maintained in DMEM medium (Gibco, USA) with 10% fetal bovine serum (Procell, China) in a humidified atmosphere containing 5% CO₂ at 37 ℃. RAW264.7 cells were seeded in a 24-well culture plate at a density of 5.0 × 10⁵ cells/mL and stimulated with 1 µg/mL lipopolysaccharide (LPS from Escherichia coli serotype 055:B5, Sigma‒Aldrich, USA) for 6 h to induce M1 macrophage polarization. To evaluate the effect of the STAT3 inhibitor S3I-201 (Abcam, UK) and the JAK2 inhibitor fedratinib (SAR302503, Selleck, China) on JAK/STAT3/FOXO3 signaling, cells were incubated with 7-mix (2^− 2, namely 2.5 × 10⁷ CFU), mix-sup (2^− 4), S3I-201 (50 µM), fedratinib (10 µM), or S3I-201 + fedratinib for 4 h.

DNase I (HY‐P72974, MedChemExpress, Monmouth Junction, NJ, USA) was used to degrade NETs and NEi (GW‐311616; HY‐15891, MedChemExpress) was used to inhibit neutrophil elastase. Fedratinib (S2736, Selleck, Houston, TX, USA) was used to inhibit JAK2, C188‐9 (S8605, Selleck) was used to inhibit STAT3, and fisogatinib (BLU‐554; S8503, Selleck) and BLU9931 (A8706, APExBIO, Houston, USA) were used to inhibit FGFR4. Clodronate liposomes (40337ES10, YEASEN Biotechnology, Shanghai, China) was used for macrophage depletion, and diphtheria toxin (D0564, Sigma‐Aldrich, St. Louis, MO, USA) was used for dendritic cell depletion.
Recombinant human FGF19, IL‐1α, CXCL11, MIF and PAI‐1 were purchased from R&D Systems (Minneapolis, MN, USA). Recombinant human IL‐1β, IL‐18, and complement C5a were purchased from Novoprotein (Shanghai, China).
Human FGF19 neutralizing antibody (AF969, R&D Systems), human IL‐1β neutralizing antibody (AF‐201‐NA, R&D Systems), and human complement C5a neutralizing antibody (MAB2037, R&D Systems) were used in this study. Mouse Ly‐6G antibody (16‐5931‐85, eBioscience, San Diego, CA, USA) was used for neutrophil depletion, mouse asialo GM1 antibody (16‐6507‐39, eBioscience) was used for NK cell depletion, and mouse CD20 antibody (152104, Biolegend, San Diego, CA, USA) was used for B cell depletion.

Docetaxel and JAK inhibitors (baricitinib, ruxolitinib and fedratinib) were purchased from Selleckchem, Houston, TX, USA. For drug combination studies, cells were seeded overnight in 96‐well plates at a density of 5000 cells/well. Cells were treated with docetaxel and/or JAK inhibitors (baricitinib, ruxolitinib and fedratinib) alone or in combinations at dose‐response matrix format. Plates were then incubated at 37°C in a humidified 5% CO₂ incubator for 72 hours. The plates were terminated by MTT cell proliferation assay at 72 hours after treatment. Calcusyn 2.1 software (Biosoft, Cambridge, UK) was used to generate combination index (CI) based on Chou‐Talalay method, in which CI <1, = 1 and >1 indicates synergism, additive and antagonism effect, respectively (Table S1).²¹ (link), ²² (link), ²³ (link), ²⁴ (link), ²⁵ (link) The dose‐response surface curves with levels of highest single agent (HSA) synergy were plotted by Combenefit software (Cancer Research UK Cambridge Institute).²¹ (link), ²² (link), ²⁶ (link)