Rmp1 14

C57BL/6AnCrl and BALB/cAnCrl female mice were injected subcutaneously with 2.10⁵ TC1, CT26, MC38, B16F10, or B16OVA cells in 100 µL of phosphate buffered saline (PBS 1×, Gibco, France) into the left flank. Tumor growth was monitored every 3 days with a caliper and groups were formed when tumor reached a mean diameter of 50–60 mm². All CT procedures were conducted in the Pharmacy Department of the University Hospital of Besançon (France). Mice were administered a single dose of intraperitoneal cisplatin (5 mg/kg) and 5-fluorouracil (25 mg/kg). After 2 days, mice were anesthetized by injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and received single-fraction beam photon irradiation (8 Gy) using Clinac 2100 CD (Varian Medical System) with a maximal energy of 6 MV and in 2D with one beam. Mice from control group were injected with the solvent used to dilute the drug (the Pharmacy Department of the University Hospital of Besançon, France). Anti-mouse CTLA-4 antibody (9H10, Euromedex) and anti-PD-1 (RMP1-14, Euromedex) injections (200 µg/mouse two times a week for 2 weeks) started 2 days prior to CT and 3 days after RT, respectively. All experimental studies were approved by the local ethics committee in accordance with the European Union’s Directive 2010/63.

All chemotherapy procedures were conducted in the Department of Pharmacy, University Hospital of Besançon (France). FOLFOX regimen was administered with an intraperitoneal (IP) single dose of 5-fluorouracil (50 mg/kg) and oxaliplatin (6 mg/kg). The control group received the solvent used to dilute the drug and the appropriate isotype antibodies anti-rat IgG2a (2A3, Euromedex) and anti-mouse IgG1 (MOPC-21, Euromedex). Anti-PD-1 (RMP1-14, Euromedex) and anti-TGF-β1 (1D11, Euromedex) mouse antibodies IP injections were started concomitantly with the chemotherapy treatment: 200 µg per mouse twice a week for two weeks.