Mcf 7 adr cells

HPLC-grade chloroform, methanol, acetonitrile and formic acid (Fisher Scientific, Loughborough, UK) were used. Methyl-tert-butyl ether (MTBE) was purchased from Aladdin Co., Ltd. (Shanghai, China). Ammonium acetate was taken from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was produced by a Milli-Q plus (Milford, MA, USA) water purification system. MCF-7/ADR cells were purchased from Keygen Biotech Co., Ltd. (Nanjing, China) and parental MCF-7/S cells were purchased from Chinese Academy of Medical Science. Adriamycin was purchased from Hisun Pharm Co., Ltd. (Taizhou, China). RPMI-1640 medium was taken from Hyclone, China. Fetal bovine serum was purchased from Hangzhou Sijiqing Co., Ltd. (Hangzhou, China). Penicillin and streptomycin were purchased from Beijing Dingguo Co. Ltd. (Beijing, China). Leucine enkephalin was obtained from Waters (Milford, USA).

Human breast cancer cell lines MCF-7 and MDA-MB-231 cells were obtained from ATCC (Manassas, Virginia, USA), and respectively cultured in high glucose DMEM (Gibco, Grand Island) and L15 medium (Gibco). Adriamycin resistant MCF-7/ADR cells were purchased from KeyGen BioTECH (Nanjing, China), and cultured in RPMI-1640 medium (Gibco). HEK-293 T cells were preserved in our laboratory and cultured in high glucose DMEM. The three media were supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island), 80 U/ml penicillin (Sangon Biotech, China), and 0.08 mg/ml streptomycin (Sangon Biotech, China) at 37 °C under humidified atmosphere with 5% CO₂. MCF-7/ADR cells were cultured with 1 μM adriamycin to maintain adriamycin resistance. PI3K inhibitor (LY-294002) and ERK1/2 inhibitor (VX-11e) were purchased from APExBIO. Adriamycin and human recombinant PD-1 (rPD-1) were purchased from the Zhongda Hospital Southeast University and BioVision, respectively. BMS-202 (synonyms: PD-1/PD-L1 inhibitor 2) and Human IFN-gamma were purchased from the MedChemExpress and Pepro Tech, respectively.
Four pairs of fresh breast cancer and adjacent mammary gland epithelial tissue samples were randomly selected from the Tumor Hospital of Jiangsu Province. Written informed consent from all patients and approval of the hospital ethics review committees were obtained.