Particularly

the effect of sorafenib on the interaction between TAM and NK cells remains elusive. In this work we studied sorafenib-triggered activation of polarized Mϕ, which show a TAM-like phenotype. Sorafenib-dependent Mϕ induction eventually affected NK cells, which displayed enhanced activity against tumor cells. selleck chemical This interaction with NK cells was confirmed for autologous TAM isolated from human HCC tissue. The observed sorafenib-triggered NK cell stimulation was dependent on NF-κB activation and cytokine induction in polarized Mϕ. AFP, alpha-fetoprotein; C57BL/6wt, C57BL/6 wildtype; Cr, [51Cr]chromium; CSF-1, colony stimulating factor-1; DMSO, dimethylsulfoxide; EGTA, ethylene glycol tetraacetic acid; ELISA, enzyme linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hank’s balanced salt solution; IFN, interferon; IL, interleukin; JAK, Janus kinase; LPS, lipopolysaccharide; L-SIGN, liver/lymph node-specific ICAM-3-grabbing nonintegrin; LTα/β, lymphotoxin α/β; Mϕ, macrophage; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor “kappa-light-chain-enhancer” of activated B-cells; NK, natural killer; PMA, phorbol myristate acetate; RAF, rat fibrosarcoma; RAS,

rat sarcoma; STAT, Signal Transducers and Activators of Transcription; tg, transgenic; TNF, tumor necrosis factor; UV, ultraviolet. C57BL/6 wildtype mice (C57BL/6wt), hepatitis B virus replicating HBV1.3.32 (HBV-tg),13 or albumin-promoter-controlled lymphotoxin-α/β buy GW-572016 transgenic mice (LTα/β-tg)14 were maintained under pathogen-free conditions. C57BL/6wt mice were used for experiments at the age of 6 months, LTα/β-tg mice at 14-28, and HBV-tg mice at 21-25 months. Animal experiments were performed in accordance with the German legislation governing animal studies and the Principles of Laboratory Animal Care guidelines (National Institutes of Health, NIH). NK cells (CD3−/CD56+) were sorted from blood leukocytes this website with a MoFlow (Beckman Coulter, Krefeld, Germany) or were purified untouched using magnetic beads (Miltenyi Biotech, Bergisch-Gladbach, Germany). Circulating CD14+ monocytes were enriched by

positive magnetic isolation (Miltenyi) and were cultured in the presence of 10 ng/mL colony stimulating factor-1 (CSF-1) (Peprotech, Hamburg, Germany) for 1 week to generate polarized macrophages (Mϕ). Also, 3 × 104 NK cells, Mϕ, and TAM per well were cultured in flat-bottom 96-well plates with RPMI-1640 medium (Gibco, Carlsbad, CA), supplemented with fetal calf serum (10%), L-glutamine (1%), penicillin (1%), and streptomycin (1%) (all Sigma-Aldrich, St. Louis, MO). K562, Raji, and HepG2 cells were maintained under equal conditions. Then 3 × 104 NK cells and TAM per well were used for coculture at the day of isolation. Following patient informed consent and local Ethics Committee approval, TAM were obtained from histological confirmed HCC tissue.