Pazopanib

Stock solutions of sunitinib and pazopanib were obtained from LC Laboratories (Woburn, MA) and dissolved with dimethyl sulfoxide (DMSO). Stock solutions were diluted with phosphate buffered saline (PBS) to achieve a final concentration of 10 µM for sunitinib (to achieve a targeted intratumoral concentration of 10 µmol/L^{32 (link)}), and 150 µM for pazopanib (pazopanib is clinically dosed at 800 mg daily in comparison to sunitinib at 50 mg daily). Vehicle control was 1% DMSO diluted in PBS. Rhodamine lectin was diluted at a 1:10 ratio with PBS immediately prior to intravascular injection. Stock Hoechst solution was diluted at a 1:1000 ratio with PBS.

The compounds doxorubicin, methotrexate, cisplatin, and etoposide were purchased from Sigma Aldrich (St. Louis, MO, USA), mafosfamide from Toronto Research Chemicals Inc. (TRC) (Toronto, Canada), and regorafenib, pazopanib, and cabozantinib from LC Laboratories (US, Canada). All the compounds were diluted in dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA), except cisplatin, which was diluted in N,N-dimethylformamide (DMF) (Sigma Aldrich, St. Louis, MO, USA), and stored at −20°C in a 10 mM stock solution.

ICR-SS-1 (2000/well), HS-SY-II (3000/well), and SYO-1 (2000/well) cells were seeded in clear 96-well, flat-bottom plates (Corning Inc., Corning, NY, USA). Plates were incubated for 24 h before replacing media with a panel of small molecule inhibitors at a concentration of 500 nM for all drugs except NVP-AUY922, which was at a concentration of 50 nM (details and source of inhibitors are shown in Supplementary Table S1). After 72 h, cell viability was determined using CellTitre-Glo (Promega, Madison, WI, USA), following the manufacturer’s instructions. Dose response assays were conducted by seeding ICR-SS-1 (2000/well), HS-SY-II (3000/well), and SYO-1 (2000/well) cells in clear 96-well, flat-bottom plates (Corning). Plates were incubated for 24 h, after which the medium was replaced with increasing concentrations of doxorubicin hydrochloride (Sigma Aldrich) or pazopanib (LC Laboratories) at the indicated dose. Data points from dose response assays were used to fit a four-point non-linear regression curve via Graphpad Prism and the drug screen data were subjected to hierarchical clustering using Perseus software [35 (link)] with Euclidean distance as the distance metric.

1,2,4,5-Benzenetetraamine tetrahyrdrochloride (Y15) is an allosteric FAK inhibitor and was ordered from Sigma. Y15 was dissolved in 1× PBS at a concentration of 25 mM and stored at −20 °C. Pazopanib, Sunitinib, and Sorafenib were ordered from LC Laboratories and reconstituted in DMSO at a concentration of 25mM. Cabozantinib was ordered from Selleck Chem and solubilized in DMSO at 25mM. PF-04554878 was obtained from Adooq Bioscience and dissolved in DMSO to concentration of 25mM.