Actemra

The mouse colon cancer cells (CT26) were injected intraperitoneally (1 × 10⁶ CT26 cells per mouse, 100 μl) as described above. After 4 days, the tumor‐bearing mice were randomly divided into four groups (each group contained 12 mice, a total of 48 mice), injected with OH2 virus (1 × 10⁶ CCID50 per mouse, 250 μl), and added tocilizumab (named Actemra from Roche, a monoclonal anti‐IL6R neutralizing antibody) by 10 mg/kg IP once. Some mice were inspected every 3 days, and killed on day 9, and others continued to be raised and followed up for survival time.

Adult male rats were randomly selected and divided into five groups: the CN (healthy rats, n = 6), OA (oleic acid administration, n = 6), OA + TCZ-2 (oleic acid and tocilizumab at 2 mg/kg, n = 6), OA + TCZ-4 (oleic acid and tocilizumab at 4 mg/kg, n = 6), and OA + DEX-10 (oleic acid and dexamethasone at 10 mg/kg, n = 6) groups. Oleic acid (50 µL) was dissolved in 250 µL of 1% bovine serum albumin and administered via the tail vein in all groups except the CN group. DEX (Dekort Ampul 8 mg/2 mL, Deva Ilac, Turkey) and TCZ (Actemra, Roche, Germany) were administered intraperitoneally twice 6 h after OA injection at an interval of 12 h using an insulin injector. The rats were anesthetized by administering intraperitoneal ketamine hydrochloride (60 mg/kg b.w) and xylazine hydrochloride (5 mg/kg b.w) [23 (link)]. Subsequently, cervical dislocation was applied to the animals. Systemic autopsies were performed and lung tissues were taken for biochemical and pathological analysis. Lung tissues were stored at − 20 °C for biochemical analysis and were kept in NBF for pathological analysis.

Preosteoclasts, prepared like the BMDMs, were plated in 96-well plates (7,000 cells per well) in standard medium supplemented with 20 ng/ml M-CSF and 50 ng/ml Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL) (R&D Systems, Minneapolis, MN, USA). On the 3rd day (after 48 h), the medium was replaced by the CM of BMDM, supplemented with RANKL and M-CSF. Where indicated, we also added 2 μg/ml of neutralizing antibodies (Ab) against IL1β (Kineret, anakinra, SOBI, Stockholm, Sweden), IL6-receptor (Actemra, tocilizumab, Roche, San Francisco, CA, USA), or TNFα (Humira, adalimumab, AbbVie Inc., Chicago, IL, USA). These neutralizing Ab, effective in both humans and mice (15 (link)–19 (link)), are referred to as anti-IL1β, IL6, and TNFα Ab, respectively. On the 4th day, cells were stained using a TRAP kit (Sigma-Aldrich, St. Louis, MO, USA), and multinucleated (>3 nuclei) TRAP-positive cells were defined as osteoclasts. Images were acquired at an original magnification of × 4 (Evos FLC, Life Technologies, MS, USA). The number of osteoclasts and the total osteoclast area were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).