Il 20

At the time of procedure, serum was taken for CRP analysis using qualitative and semi-quantitative latex method (Linear, Montgat, Spain). The titer is expressed as the reciprocal of the highest dilution showing macroscopic agglutination, with the normal levels for adults being <6 mg/L. The procedure for CRP measurements was conducted in accordance with the manufacturer’s datasheet.
Remaining serum was frozen in aliquots at −80°C until further work was performed. The cytokine NGAL level was measured after dilution to 1:20, and other cytokines (IL-1α, IL-6, IL-19, IL-20, IL-21, IL-22, and IL-33) were measured directly without dilution of the sample according to the manufacturers’ instructions. Cytokines’ levels in serum were determined using enzyme-linked immunosorbent assay. Procedures of each cytokine immunoassay, including reagent and standard preparations, were conducted following instructions in the manufacturers’ datasheets. OD of each sample was determined using enzyme-linked immunosorbent assay reader at 450 nm wavelength. The manufacturer information is as follows: IL-1α (R&D Systems, Inc., Minneapolis, MN, USA), IL-6 (R&D Systems), IL-19 (R&D Systems), IL-20 (R&D Systems), IL-21 (eBiosience, San Diego, CA, USA), IL-22 (R&D Systems), IL-33 (R&D Systems), and NGAL (RayBio, Norcross, GA, USA).

Patient-derived tumor tissues were treated according to a previously described method.^{16 (link)} We previously confirmed that patient-derived tumor cells plated at 5000 cells per ml yield tumor spheres that are clonally derived from single cells.^{16 (link)} Sphere formation assay was performed as described previously.^{16 (link)} Briefly, cells were plated as single cells on ultralow attachment 24-well plates (2000–5000 cells per well). Spheres were grown in SCM containing 20 ng/ml EGF (Millipore, Billerica, MA, USA), 20 ng/ml basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA), B27 (Gibco, Grand Island, NY, USA) and heparin (Stem Cell Technologies, Vancouver, BC, Canada) or in DMEM/F-12 medium supplemented with 20 ng/ml recombinant human HRG (R&D Systems Inc., Lille, France), 200 ng/ml recombinant human IGF2 (R&D Systems Inc.) and 20 ng/ml AR (R&D Systems Inc.) or 200 ng/ml IL-20 (R&D Systems Inc.), with or without anti-IGF2 antibody (clone S1F2; Millipore) and anti-AR antibody (R&D Systems Inc.). Spheres >75 μm in diameter were counted after 4–7 days. Sphere-forming efficiency was calculated as the ratio of the number of spheres formed to the number of cells originally plated (2000–5000 cells per well).