Lll12

Regorafenib was provided by Bayer and administered by oral gavage daily at a dose of 5, 10 and 20 mg/kg in 34% 1,2-propandiol (Sigma-Aldrich), 34% PEG400 (Sigma-Aldrich), 12% pluronic F68 (Thermo Fischer Scientific, Waltham, Massachusetts, USA), and 20% water, as per manufacturer’s recommendation. Mouse anti-PD1 antibody (clone RMP-014) was purchased from BioXcell (Lebanon, New Hampshire, USA) and Mouse IgG isotype control was purchased from Thermo Fischer Scientific. The STAT3 inhibitor LLL12 was purchased from BioVision (Milpitas, California, USA). Antimouse PD1 antibody or IgG (control) were given intraperitoneally at a dose of 10 mg/kg thrice weekly, as described.11 (link) Daily gavage of vehicle and intraperitoneal injection of 10 mg/kg of isotype-matched IgG were given as control treatments for anti-PD1 antibody. For the survival studies, moribund status was used as the end point and moribund was defined as symptoms of prolonged distress, >15% of weight loss compared with the starting date, body condition score >2 and tumor size of >24 mm in diameter.

The STAT3 phosphorylation (Tyr705) inhibitor, 5-hydroxy-9,10-dioxo-9,10-dihydroanthracene-1-sulfonamide (LLL12; BioVision Milpitas, CA, USA), a non-peptide, cell-permeable small molecule, was commercially obtained. The LLL12 stock was dissolved in dimethyl sulfoxide (DMSO; Mallinckrodt Chemical Works, St Louis, MO, USA) at 0.01 M.

The human epithelial ovarian cancer line HEY3 was obtained from the Royal Women’s Hospital Melbourne, A2780 from Deakin University, while PA1, SKOV3, and ES2 were obtained from American Type Culture Collection (Rockville, MD). Cell lines were grown as monolayers in 25 or 75 cm² flasks (BD, Australia) in complete growth medium consisting of Basal Medium Eagle for PAI, McCoy’s 5a for SKOV3 and ES2, Dulbecco’s Modified Eagle’s Medium (DMEM) HEY3, and RPMI1640 for A2780, in each case supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 2 mM glutamine (Invitrogen Corporation, Melbourne, Australia) in the presence of 37°C with 5% CO₂. Recombinant human leptin (Sigma Aldrich, Melbourne, Australia) was used at 0-100 ng/ml, along with recombinant mouse LEPR-Fc chimera (Leptin inhibitor, 0.5 μg/ml) (R&D Systems, Minneapolis, MN, USA), WP1066 (JAK2 inhibitor, 2.5 μM) and LLL12 (STAT3 inhibitor, 2.5 μM) (BioVision, Mountain View, CA, USA), cisplatin (20 μg/ml) (Pfizer, Perth, WA, Australia), sodium azide (0.5%) and hydroxyurea (50 μM) (Sigma Aldrich), as required. Grade 3 serous ovarian carcinomas were collected from patients after obtaining written informed consent under protocols approved via the Victorian Cancer Biobank and approved by the Deakin University Human Research Ethics Committee (DUHREC#2010-104). All patients had undergone chemotherapy with standard agents.