Eribulin mesylate

Eribulin mesylate (Eisai, Research Triangle Park, NC), paclitaxel and vinblastine (Sigma-Aldrich, St Louis, MO) were dissolved in DMSO and stored as a 5 mM stock in glass vials at −20°C. Serial dilutions of drugs were performed in 50% DMSO in glass vials and stored as 100-fold stock at −20°C. Wells of 96-well plates were coated with 25 μg/ml rat tail collagen I (Roche Diagnostics, Mannheim, Germany) in PBS, prior to seeding with 5000 cells per well. Effect of the drugs on cell viability was assessed with a sulforhodamine B assay [37 (link)] after 72 h of drug exposure. The final concentration of DMSO (0.5%) was the same for both drug-treated and control cells.

Primary human umbilical vein endothelial cells (HUVECs) were either purchased from Lonza (Walkersville, MD) or isolated from a single umbilical cord by a method described previously
[23 (link)] and maintained in endothelial cell growth medium EGM-2 supplemented with EGM-2 SingleQuots except for hydrocortisone (Lonza,). Human brain vascular pericytes (HBVPs) were obtained from ScienCell Research Laboratories (Carlsbad, CA), and were grown in Pericyte Medium (ScienCell). To confirm their authenticity, cultured HBVPs were examined for expression levels of 6 key pericyte markers grown on plastic (Additional file
1). Both HUVECs and HBVPs were grown on collagen type I-coated plastic ware. For cell proliferation and gene expression experiments, cells were used at <5 passages.
Green fluorescent protein (AcGFP)-expressing HUVECs were established by infection with a retrovirus for gene transfer of AcGFP followed by collecting high level AcGFP-expressing HUVECs by fluorescence activated cell sorting. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.
Paclitaxel was purchased from Sigma-Aldrich (Saint Louis, MO) and Wako Pure Chemical (Osaka, Japan). Eribulin mesylate was manufactured by Eisai Co., Ltd (Ibaraki, Japan). Both compounds were dissolved in DMSO to yield a stock concentration of 1 mmol/L.

Eribulin mesylate (hereafter, eribulin) was supplied by Eisai Inc. (Cambridge, MA, USA) as laboratory-grade dry powder active pharmaceutical ingredient (API) obtained from the same manufacturing stream used for Eisai’s branded clinical product, Halaven^®. Paclitaxel and vinorelbine tartrate (hereafter, vinorelbine) were obtained as dry powders from Selleckchem (Shanghai, China). Eribulin, Paclitaxel and vinorelbine were prepared as 10 mM stock solutions in 100% (v/v) DMSO, aliquoted into small volumes and stored at −20 °C until day of use. Although not a designated test agent for this study, cisplatin was included in all assays as an internal reference control for assay performance; cisplatin was obtained as a laboratory grade liquid formulation from Hospira Australia Pty Ltd. (Melbourne, Australia), with storage per manufacturer’s instructions and dilutions for cell culture studies on day of use.