Sum 159

Five human TNBC cell lines, MDA-MB-231, BT-549, MDA-MB-468, MDA-MB-453 and SUM-159, as well as normal breast epithelial cell line MCF-10A were purchased from ATCC. 293 T and 293 cell lines were conserved in our laboratory. MDA-MB-231, MDA-MB-468, MDA-MB-453, SUM-159, 293 T and 293 cells were cultured in DMEM medium (Gibco, Carlsbad, CA, USA), BT-549 cells were grown in RPMI 1640 medium (Gibco, Carlsbad, CA, USA). MCF-10A cells were maintained in mammary epithelial cell growth medium (MEGM) BulletKit (Lonza, Basel, Switzerland). The cell media contained 10% fetal bovine serum (FBS, HyClone, Invitrogen), 100 U/ml penicillin and 100 mg/ml, cells were maintained in a humidified incubator at 37 °C with 5% CO₂.

MCF-10A, MCF-7 and SUM-159 were purchased from National Infrastructure of Cell Line Resource of China.
MCF-10A cells were maintained in F12 medium (Gibco) supplemented with 5% horse serum (Gibco), 1%(vol/vol) penicillin/streptomycin/L-Glutamin (Gibco), 10 mg/mL insulin, 20 mg/mL EGF, 100 mg/mL cholera toxin and 0.5 mg/mL hydrocortisone. SUM-159 cells were maintained in F12 medium (Gibco) supplemented with 5% fetal bovine serum (GEMINI), 1% (vol/vol) penicillin/streptomycin/L-Glutamin (Gibco), 5 mg/mL insulin and 10 mg/mL dexamethasone. MCF-7 cells were maintained in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% (vol/vol) fetal bovine serum (GEMINI) and 1% (vol/vol) penicillin/streptomycin/L-Glutamin (Gibco). All cells were cultured at 37°C, 5% CO₂ in a humidified atmosphere.

SUM149 and SUM159 were purchased from Asterand Bioscience (Detroit, MI, USA). All other cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). BT474 cells were grown in Hybri-Care Media (ATCC); BT-549, HCC39, HCC70, MDA-MB-231, MDA-MB-453, MDA-MB-468, T47D, ZR-75-10 in RPMI 1640 (Corning, Corning, NY, USA); MCF7 and MDA-MB-453 in DMEM (Corning); and SK-BR-3 in McCoy’s 5A (Sigma-Aldrich, St. Louis, MI, USA). Media was supplemented with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA) and 1% penicillin–streptamycin (Gibco, Waltham, MA, USA). SUM149 and SUM159 cells were grown in Ham’s/F12 (Gibco), supplemented with 5% FBS, 1% penicillin–streptamycin, 1M HEPES (Gibco), and 1 mg/mL hydrocortisone (Sigma-Aldrich). All cell lines were maintained at 37 °C with 5% CO₂. All cell lines were confirmed to be free of mycoplasma contamination (Bimake, Houston, TX, USA or Lonza, Basal, Switzerland) every 2 months.

The human breast cancer cell lines MDA-MB-231, SK-BR3, MCF-7, T47D, SUM159, and ZR-75, the mammary epithelial cell line MCF-10A and the monocytic cell line THP-1 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-231 cells were maintained in L-15 (HyClone, Utah, USA) medium. SK-BR3, MCF-7 and T47D cells were cultured in DMEM medium (GIBCO, CA, USA). SUM159 was cultured in DMEM/F12 medium (GIBCO, CA, USA). ZR-75 and THP-1 cells were cultured in RPMI-1640 medium. All the cells above were cultured in the presence of 10% fetal bovine serum (FBS, GEMINI, USA) with 100 U/mL penicillin G and 100 mg/mL streptomycin sulfate in a 37°C incubator with 5% CO₂. MCF-10A cells were cultured in DMEM/F12 supplemented with 5% horse serum (GIBCO, CA, USA), 0.5 μg/ml hydrocortisone (Sigma, St Louis, USA), 10 μg/ml insulin (GIBCO, CA, USA) and 20 ng/ml recombinant human EGF (PeproTech, NJ, USA). Induction and differentiation of macrophages were performed according to the established method 25 (link). Briefly, to generate M2-like macrophages, THP-1 cells were treated with PMA (200 ng/ml, Abcam, MA, USA) for 6 h and then cultured with IL-4 (20 ng/mL, PeproTech, US) and IL-13 (20 ng/mL, PeproTech, US) for an additional 18 hours.

MDA-MB-231 (a gift from Rolf Brekken, UT Southwestern), HEK293T, SUM159 (gifts from Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA), and HEK293FT (Thermo Fisher Scientific) cells were cultured in high-glucose DMEM or DMEM/Ham’s F-12 (MilliporeSigma) supplemented with heat-inactivated 10% FBS (MilliporeSigma) at 37°C in a 5% CO₂/95% air incubator. Human HUVECs (a gift from Ondine Cleaver, UT Southwestern) were cultured in M200PRF medium with low-serum growth supplement (Thermo Fisher Scientific) at 37°C in a 5% CO₂/95% air incubator. HUVECs within the first 3 passages were used for experiments. Sf9 cells (a gift from Xuewu Zhang, UT Southwestern) were cultured in Sf-900 III SFM medium (Gibco) at 27°C in a shaking incubator.

Cell lines MDA-MB-231 (MDA231, American Type Culture Collection (ATCC)), MDA-MB-231-LM2 (MDA231-LM2, RRID:CVCL_5998, provided by J. Massagué)^{10 (link)} and 4T1, HEK293T and RAW264.7 (all from ATCC) were cultured in DMEM (GlutaMAX, Thermo Fisher Scientific) with 10% fetal bovine serum (FBS, Gibco). SUM159 (Asterand Bioscience) and SUM159-LM1 cells⁵¹ were maintained in DMEM/F12 medium (Thermo Fisher Scientific) with 5% FBS and 5 μg ml^–1 human insulin (Sigma-Aldrich). E0771 (CH3 BioSystems) and HL60 (ATCC) cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific) with 10% FBS; E0771 medium also included 10 mM HEPES (Sigma-Aldrich). Primary human pulmonary ECs (Lonza) were cultured in EBM-2 medium with the EBM-2 bullet kit (Lonza). The human pulmonary EC line ST1.6R^{57 (link)} was provided by R. E. Unger and C. J. Kirkpatrick, and cultured on collagen type I-coated plates with M199 (Thermo Fisher Scientific) containing 20% FBS, 25 μg ml^–1 EC growth supplement (Corning) and 25 μg ml^–1 heparin (Sigma-Aldrich). Bone marrow-derived macrophages were isolated from 8–10-week-old BALB/c mice and cultured in DMEM with 10% FBS and 20 ng ml^–1 macrophage colony-stimulating-factor (M-CSF, Peprotech). All media contained 50 U ml^–1 penicillin and 50 μg ml^–1 streptomycin (Sigma-Aldrich). All cancer cell lines were authenticated using Multiplex Cell Authentication by Multiplexion Heidelberg.