Mouse breast cancer cells 4T1 (TN) and EMT-6, all syngeneic from Balb/C mice, were obtained from the American Type Culture Collection (ATCC) and were tested regularly for mycoplasma contamination using a Universal Mycoplasma detection kit (ATCC). These cells were maintained in DMEM medium (Dominique Dutscher, Bernolsheim, France) supplemented with 10% fetal bovine serum (FBS, Dominique Dutscher) at 37 °C in a humid atmosphere with 5% CO2. MSC2, a murine immortalized MDSC cell line obtained from Balb/C Gr-1+ splenocytes, was obtained from V. Bronte (Istituto Oncologico, Padova, Italy). They were cultured in RPMI 1640 (Dominique Dutscher) supplemented with 10% FBS and 1% penicillin, streptomycin and amphotericin B (PSA) antibiotic mix (PAN Biotech GmbH, Aidenbach, Germany) at 37 °C under 5% CO2.
For ex vivo experiments, MDSCs and/or anti-CD3/anti-CD28 Dynabeads (Gibco, Thermo Fischer Scientific, Waltham, MA, USA)-activated T lymphocytes were treated or not (Ctrl) for 24, 48 h or 5 days with 100, 250 or 500 nM of doxorubicin +/− 10 or 100 µM of GTN (Merck, Lyon, France) +/− 10 mM of NAC. MSC-2 cells were also treated with 1 mM of Nitrosocysteine (Cys-NO) for 15 min.
For ex vivo experiments, MDSCs and/or anti-CD3/anti-CD28 Dynabeads (Gibco, Thermo Fischer Scientific, Waltham, MA, USA)-activated T lymphocytes were treated or not (Ctrl) for 24, 48 h or 5 days with 100, 250 or 500 nM of doxorubicin +/− 10 or 100 µM of GTN (Merck, Lyon, France) +/− 10 mM of NAC. MSC-2 cells were also treated with 1 mM of Nitrosocysteine (Cys-NO) for 15 min.