Cell lines, culture and reagents
The urothelial carcinoma cell lines SW780 (FGFR3-BAIP2L1 fusion) and RT4 (FGFR3-TACC3 fusion) were obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM/F-12, plus glutamine and sodium bicarbonate (Invitrogen, Carlsbad, CA, USA) supplemented with 1% GlutaMax, 1% HEPES, 1% Penicillin/Streptomycin, and 10% Fetal Bovine Serum (Invitrogen), and incubated at 37°C in 5% Carbon Dioxide. BGJ398 was obtained from Novartis (Basel, Switzerland). AZD8931, R428 and erdafitinib were purchased from Selleck Chemicals (Houston, TX, USA).
Establishment of drug resistant cell lines
Resistance to BGJ398 in SW780 and RT4 bladder cancer cell lines was established by either gradual dose escalation of drug (referred to as SW780 RD and RT4 RD) or by sustained exposure to 1 mM BGJ398 (labelled SW780 RS and RT4 RS). Gradual dose escalation (RD cell lines) began with treatment of 3 nM BGJ398 with stepwise concentration escalation until cells were able to proliferate in 1 µM of drug. Comparatively, SW780 RS and RT4 RS cell lines were continuously maintained in 1 µM BGJ398, with fresh drug added each time the cells were passaged. Parental cell lines were passaged in parallel in equivalent concentrations of DMSO. Parental and resistant cells were regularly assessed for Mycoplasma contamination and the authenticity of the cell lines verified using the Promega StemElite ID System.
Cell viability and apoptosis assays
Cell viability was measured using either the CellTitre-Glo luminescent cell viability assay (Promega, Madison, WI, USA) or the MTS assay (Promega). For CellTitre-Glo assays, cells were seeded in 96 flat bottom well plates at a density of 1500-5000 cells per well, then treated the following day with drug for 72 hours. Luminescence was measured using a SpectraMax L Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) and compared to DMSO treated cells. MTS assays were performed using the same protocol, except cell viability was assessed after 72h using the CellTiter 96Ò Aqueous MTS reagent (Promega) as per manufacturer’s instructions, and measurement of absorbance at 490 nm (MTS) and 630 nm (background absorbance) using a SPECTROstar Nano microplate reader (BMG labtech, Germany).
Apoptosis was assessed following 72 hours of drug treatment by Propidium Iodide staining as described previously (28), followed by FACS analysis using a BD FACS Canto II flow cytometer (BD Biosciences San Jose, CA). The percentage of apoptotic cells was determined by calculating the percentage of cells with a sub-diploid DNA content using the FLOWJO software V10.0 (FlowJO LLC, Ashland, OR, USA).
Phospho-receptor tyrosine kinase (RTK) arrays
RT4-RS and SW780-RS cells were cultured in fresh media without drug for 24 hours prior to collection to negate effects induced by acute drug exposure. Control and resistant lines were then lysed in Radio immunoprecipitation assay buffer (Sigma-Aldrich, St Louis, MO, USA) containing complete Protease Inhibitor Cocktail Tablets (Roche, Basel, Switzerland) and PhosSTOP (Roche). 200 mg of lysate was then incubated with human Phospho-RTK Arrays (ARY001B, R&D Systems, Minneapolis, MN, USA) as per manufacturer’s instructions, and blots were read using the ChemiDoc X Imaging System (Bio-Rad, Hercules, CA, USA).
Protein lysates were prepared as above, denatured using 10x NuPage Sample Reducing Agent (ThermoFisher, Waltham, MA, USA), and run on NuPAGE Novex 4-12% Bis-Tris precast gels (Invitrogen) in MES (2-(N-morpholino)ethanesulfonic acid) buffer (ThermoFisher). Proteins were transferred using the iBlot® Dry Blotting System (Invitrogen) and signal detected using the Li-Cor® Odyssey Infrared Imager (Li-Cor, Lincoln, NE, USA). The following antibodies were obtained from Cell Signaling Technologies (Danvers, MA, USA): pERK p44/42 MAPK T202/Y204 (9106); t-ERK p44/42 MAPK (9107), p-ERBB3 (2842S), ERBB3 (4754S), EGFR (2232), pEGFR Tyr1068 (D7A5), AXL (8661) and p-AXL (5453). Anti- b-tubulin (ab6046) was obtained from Abcam (Cambridge, UK).
Statistical analyses were performed using Prism v5 (GraphPad Software, La Jolla, CA, USA). Data shown is mean±SEM from 3 technical replicates from a representative experiment unless stated otherwise. Biological replicates were performed for the majority of experiments and are stated in the figure legends. Groups were compared using parametric unpaired Student’s t-test with Welch’s correction. P-values ≤0.05 were considered to be statistically significant. Synergy was determined using the synergy ratio formula whereby, SR<0.8 is considered synergistic, SR 0.8 – 1.2 is considered additive, and SR > 1.2 considered antagonistic.