Equine serum

Breast cancer cell lines BT549, MCF-7, SK-BR-3, T47D, MDA-MB-231, and normal mammary epithelial cell MCF-10A were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Breast cancer cells were cultured in a DMEM medium with 10% FBS (Gibco, Grand Island, NY, USA) and penicillin-streptomycin antibody (Biyuntian, Shanghai, China). MCF-10A cells were cultured with DMEM/F12 (1:1) medium, 5% equine serum (Gibco), 10 μg/mL insulin (Wanbang Biochemical, Jiangsu, China), epidermal growth factor (20 ng/ml; Sigma, Shanghai, China), and 0.5 μg/mL hydrocortisone. The culture condition was an incubator containing 5% CO₂ at 37°C.

Intraepithelial and lamina propria (LP) lymphocytes from small intestinal tumors and unaffected intestine were isolated as follows. Briefly, Peyer’s patches were removed and tumors were cut out; the remaining tumor-free small intestine was cut into small pieces (3–5 mm). Epithelial cells were removed by HBSS medium containing EDTA (5 mM, Invitrogen), equine serum (10%, Hyclone GE Healthcare), and HEPES (15 mM, Invitrogen) for 3 × 15 min at 37°. This fraction also contained intraepithelial lymphocytes (IEL), which were used for further analysis. The remaining tissue was digested with collagenase D (0.03 U/mL, Roche) in RPMI-1640 (Gibco) media supplemented with equine serum (20%) and HEPES (15 mM, Gibco) for 1 h. Subsequently, the released cells were recovered and the remaining tissue was mixed twice in the same media for 1 min with gentleMACS™ Dissociator (Miltenyi). Dissociated tissue was filtered through a 70-µm nylon mesh to recover remaining lymphocytes and reduce debris content.

MCF10A cells were obtained from the American Type Culture Collection (ATCC) and authenticated using short tandem repeat (STR) profiling. Cells were maintained in DMEM/Ham's F12 supplemented with 5% equine serum (Gibco), 10 mg/mL insulin, 500 ng/mL hydrocortisone (Sigma-Aldrich), 20 ng/mL EGF (R&D Systems), and 100 ng/mL cholera toxin (Sigma-Aldrich). Cells were passaged for no more than 6 months and routinely assayed for mycoplasma contamination.

Cells used in this study were obtained from ATCC. MCF-10A cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Thermo Scientific) supplemented with 5% equine serum (GIBCO), 1% penicillin-streptomycin (GIBCO), 20 ng/ml EGF (Sigma-Aldrich), 10 μg/ml insulin (Sigma-Aldrich), 0.5 mg/ml hydrocortisone (Sigma-Aldrich) and 100 ng/ml cholera toxin (Calbiochem). MCF-7 and T47-D cells were cultured in RPMI (GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO), 1% L-glutamine (GIBCO) and 1% penicillin-streptomycin (GIBCO). NMuMG-NZEB1 and NMuMG-Vector cell lines were cultured in DMEM supplemented with 10% FBS, 1% L-glutamine, and 400 μg/ml G418 (Sigma-Aldrich). Other cell lines (HEK-293T; BT-549; MDA-MB-231; MDA-MB-468; SKBR-3; MDA-MB-361 and BT-474) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. All the cell lines used in this work were negative for mycoplasma contamination.

The MCF 10 A cell line is a non-tumorigenic human epithelial cell line (ATCC). Cells were grown in DMEM /F12 Ham's Mixture (Invitrogen) supplemented with 5% Equine Serum (Gibco), EGF 20 ng/mL (Sigma), insulin 10 μg/ml (Sigma), hydrocortisone 0.5 μg/mL (Sigma), cholera toxin 100 ng/mL (Sigma), 50 μg/mL of gentamycin (Sigma) at 37°C, 5% CO₂.

PC12 cells (adrenal gland; pheochromocytoma) were purchased from the American Type Culture Collection. Human HepG2 hepatoma cells were purchased from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Dimethyl Sulphoxide (DMSO), Bicyclol, Okadaic Acid, 3-(3,4-dimehylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and equine serum were purchased from Gibco BRL (New York, NY, USA). All other chemicals were of analytical grade and were commercially available.