Mcdb107 medium

Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany). The HUVECs were maintained in MCDB107 medium (Funakoshi, Tokyo, Japan), supplemented with 10% FBS, 20 ng/mL bovine brain extract (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan), 10 ng/mL epidermal growth factor (EGF) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 50 μg/mL heparin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). All experiments were performed using HUVECs between passages 5 and 9. The HUVEC culture dishes were coated with type I collagen (Nitta Gelatin Inc., Osaka, Japan). SKOV3 cells were purchased from ATCC (Manassas, VA) and were maintained in McCoy’s 5 A medium containing 10% FBS. All cells were maintained at 37 °C in a humidified 5% CO₂/95% air atmosphere.
Female KCN nu/nu mice (5–6 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan). SKOV3 cells (5 × 10⁶ cells/mouse) were subcutaneously inoculated in the flanks of the mice. In vivo US imaging studies were performed when the tumors reached 100 mm³. Tumor volumes were calculated following the equation: volume (mm³) = (width × width) × (length) × 0.5. The animal use protocol and relevant experimental procedures were approved by the Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Sciences (authorization number: P18-66).

HEK293, BT474, and SKBR3 cells were purchased from ATCC, Manassas, VA, and MCF7 cells from Health Science Research Resources Bank, Osaka, Japan. Human dermal fibroblasts (HDFs) were purchased from Cell Applications, San Diego, CA. HEK293, BT474, and MCF7 cells and HDFs were maintained in DMEM containing 10% FBS, while SKBR3 cells were maintained in McCoy’s 5 A medium containing 10% FBS. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza, Basel, Switzerland, and maintained in MCDB107 medium (Funakoshi, Tokyo, Japan) supplemented with 10% FBS, 20 ng/mL bovine brain extract (Funakoshi), 10 ng/mL EGF (BD Biosciences, Bedford, MA), and 50 μg/mL heparin (Wako, Osaka, Japan). For HUVECs, the culture dishes were coated with 0.3 mg/mL type I collagen (Koken, Tokyo) at 37 °C for 1 h. All cells were maintained at 37 °C in a humidified 5% CO₂/95% air atmosphere.