Egm 2 bulletkit media

Human ovarian cancer cells (SKOV‐3) from American Type Culture Collection (ATCC) were grown in 10% foetal bovine serum‐Dulbecco's modified Eagle's medium (FBS‐DMEM) (Hyclone Laboratories). Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and used between passages 4 and 6 for all experiments. Cells were cultured in EGM‐2^® BulletKit media, according to the manufacturer's instructions (Lonza).

Human umbilical vein endothelial cells (HUVECs) from Lonza (Walkersville, MD, USA) were cultured in EGM-2® BulletKit media and used in the passage ranges 4–6 for all experiments, according to the manufacturer's instructions (Lonza) [32] (link). Human non-small cell lung cancer cells (NSCLC: A549, H1299) from the American Type Culture Collection (Manassas, VA, USA) were grown in 10% fetal bovine serum (FBS)-Dulbecco's modified Eagle's medium (DMEM) (Hyclone Laboratories, Logan, UT, USA).

MCF7, CN34-LM1, MDA-MB-231 and their metastatic derivatives were kindly provided by Dr Joan Massague (Memorial Sloan Kettering Cancer Center), and Dr Sohail Tavazoie (Rockefeller University)6 (link). Human umbilical vein endothelial cells (HUVEC) were purchased from LONZA (Basel, Switzerland) and cultured in EGM-2 bullet kit media (LONZA)5 (link). 4T1 derivatives and RAW 264.7 cells were provided by Drs Fred Miller (University of Michigan) and Suk-Jo Kang (KAIST), respectively. CN34-LM1 cells were cultured in M199 medium containing 2.5% FBS, 10 μg ml⁻¹ insulin, 0.5 μg ml⁻¹ hydrocortisone, 20 ng ml⁻¹ EGF and 100 ng ml⁻¹ cholera toxin. All the other cells were cultured in in DMEM supplemented with 10% FBS. The cell lines were recently authenticated by DNA fingerprinting analysis and regularly tested for mycoplasma contamination.

Human umbilical vein endothelial cells (HUVECs) from Lonza (Walkersville, MD, USA) were grown in EGM‐2^® BulletKit media and used between passages 4 and 6 for all experiments, according to the manufacturer's instructions (Lonza). Human non–small cell lung cancer (A549 and H1299) and ovarian cancer (SKOV‐3) cells from the American Type Culture Collection (Manassas, VA, USA) were cultured in 10% foetal bovine serum‐Dulbecco's modified Eagle's medium (FBS‐DMEM, Hyclone Laboratories, Logan, UT, USA).

For HUVEC adhesion assay, HUVEC cells were plated into 12-well gelatin coating plates and cultured in EGM-2 bullet kit media (LONZA) for 2 days. When HUVEC cells reached a >99% confluent monolayer, cancer cells were added into same plates. For matrigel adhesion assay, 12-well ultra-low binding plates (Corning) were coated with 4% growth factor reduced matrigel (BD Biosciences) at 37 °C for 3 h, followed by plating cancer cells onto matrigel-coated plates. After 30 min incubation at 37 °C, non-adherent cancer cells were washed by PBS washing. Relative numbers of adherent cancer cell were determined by using Luciferase assay system (Promega) according to the manufacturer's instructions.