Pha 665752

Gastric tumors (2-cm diameter) were aseptically resected from established patient-derived gastric cancer xenografts with MET amplification and overexpression, then minced into 3×3×3 mm pieces. Host mice were then anesthetized with isoflurane and a section of tumor was implanted into the left flank of each mouse. Each gastric model that developed tumors reaching 150–200 mm³ in size were randomized into the following four treatment groups (10 mice per group): Group 1, once-daily dose with vehicle by intravenous (i.v.) tail injection; and groups 2, 3 and 4, once-daily dose with 10, 20 and 30 mg/kg PHA665752 by i.v. tail injection, respectively. PHA665752, a selective MET inhibitor, was purchased from Selleck Chemicals (Houston, TX, USA). In a subsequent experiment, the CNGAS028 model was also treated with vehicle, 15 mg/kg PHA665752, the pan-fibroblast growth factor receptor (FGFR2) selective inhibitor NVP-BGJ398 (15 mg/kg once-daily, oral administration; Selleck Chemicals) or 30 mg/kg PHA665752 in combination with 15 mg/kg NVP-BGJ398, respectively. All treatments were continued for 21 days and the mice were sacrificed by CO₂ inhalation 2 h after the last treatment.

PHA-665752 (PHA), SU11274 (SU), Criz, Baf A1, HCQ, and MHY were purchased from Selleck Chemicals (Houston, TX). Voli was kindly provided by AstraZeneca (Cambridge, UK). HGF was purchased from Life Technologies (Frederick, MD). Reagents were formulated and stored following the manufacturer’s protocols for in vitro and in vivo experiments. Voli was formulated in a 0.5% (v/v) carboxymethylcellulose sodium solution for mice^{6 (link)}. Primary antibodies against Met, pMet (Y1234/1235), p62, LC3, mTOR, pmTOR (S2448), pULK1 (S757), Atg5, Beclin-1, NBR1, and secondary horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology (CST, Danvers, MA). Anti-β-actin primary antibody was purchased from Sigma-Aldrich (St. Louis, MO).

For testing the impact of the two tyrosine kinase inhibitors, 10,000 cells per well were seeded in 96-well plates and on the next day were kept untreated or were treated in triplicate with increasing concentrations of foretinib and PHA-665752 (C_max = 24 µM and C_max = 60 µM, respectively, Selleck Chemicals, Munich, Germany). After 48 h, the supernatant was discarded, and the cells were incubated for 10 min with 50 µL 0.5% (w/v) crystal violet solution and washed three times using distilled water. Crystal violet was dissolved in 100 µL 98% methanol per well, and absorption at 595 nm was measured (Photometer Infinite F50, Tecan, Männedorf, Switzerland). For determination of IC₅₀ values, data were normalized on a scale of 0%–100% viability and fitted with the software Prism (GraphPad, San Diego, United States) using nonlinear regression, log(inhibitor) vs. normalized response (variable slope).
HGF-dependent proliferation was also determined using a crystal violet assay as described above. The cells were seeded at a density of 4000 cells/well in 96-well plates and incubated overnight. The next day, HGF was added, and the cells were incubated for another 72 h until the crystal violet test was performed.