Sapitinib

Gentamicin sulfate, dimethyl sulfoxide (DMSO), Triton X-100 (used at 1%), mTOR inhibitor rapamycin (used at 1 μM) and bafilomycin A1 (used at 100 nM) were all purchased from Sigma-Aldrich (Merck, Darmstadt, Germany). Sapitinib (AZD8931) and lapatinib were ordered from Selleck Chemicals (Houston, TX, USA) and SignalChem (Richmond, BC, Canada), respectively. Kinase inhibitors from the published kinase inhibitor sets (PKIS)1 and PKIS2 were kindly provided by GlaxoSmithKline Global Health Medicines R&D (GSK) and the Structural Genomics Consortium of the University of North Carolina at Chapel Hill (SGC-UNC) (Elkins et al., 2016 (link); Drewry et al., 2017 (link)). Larger quantities of GW296115X were obtained from AOBIOUS (Gloucester, MA, USA). All PKIS compounds were dissolved in DMSO at 10 mM. Adenosine monophosphate-activated protein kinase (AMPK) activator compound 991 (ex229) (Xiao et al., 2013 (link)) (Spirochem, Basel, Switzerland; used at 25 μM) was kindly shared by Eline Brombacher and Dr. Bart Everts (Leiden university Medical Center, Leiden, Netherlands). Chemical structures were generated from published simplified molecular-input line-entry specification (SMILES) using ChemDraw (PerkinElmer, Waltham, MA, USA).

Hydrostatic pressure was restored daily in the high-throughput platform, while medium was changed every 2 days. After culturing for 4–5 days to allow a perfused vasculature to form within each VMT, culture medium was replaced with medium containing the drugs at the desired concentration. Drugs were delivered to the tumor through the vascular bed via gravity-driven flow. Sapitinib (reversible, ATP-competitive inhibitor of EGFR, ErbB2 and ErbB3), MK-2206 2HCl (Akt 1/2/3 inhibitor), and paclitaxel (microtubule stabilizer) were purchased from SelleckChem. Razuprotafib is a VE-PTP inhibitor that was purchased from MedChemExpress. MDA-MB-231 VMT were randomly assigned to one of four conditions: control (vehicle only), 5 nM paclitaxel, 10 nM razuprotafib, or combination 5 nM paclitaxel with 10 nM razuprotafib. VMT receiving razuprotafib were treated on day 4 for 24 h, followed by 48-h paclitaxel treatment for the combination condition or complete media for the single-agent condition. Single-agent paclitaxel was also treated for 48 h. HCC1599 VMT were treated on day 4–5 with one of the following: control (vehicle only), 100 nM Sapitinib, 100 nM MK-2206 2HCl, or combination 100 nM Sapitinib with 100 nM MK-2206 2HCl. Complete medium was replaced after 48 h. Fluorescent micrographs of VMT were taken every 48 h for 6 days post-treatment and growth of the tumor was quantified.

Eleven GSK compounds in addition to sapitinib, erlotinib and gefitinib (Selleckchem) were sent for biochemical IC₅₀ determination against EGFR, ERBB2 and ERBB4 (Reaction Biology Corp., Malvern, PA, USA) (see supplementary material, Table S3).