Bibf1120

All animal work adhered to the Agency for Science, Technology and Research (A*STAR; Singapore), Institutional Animal Care and Use Committee (IACUC) guidelines on the use and handling of animals. SKOV3-Luc-D3 cells (Xenogen Co., Alameda, CA, USA) at a density of 3.5×10⁶ in 100 μl of PBS were injected into the intraperitoneal cavity of 4-week-old female BALB/c nude mice. At 6 weeks post-implantation, the mice were randomly divided into control and treatment groups (n = 5 animals per group). For the treatment group, mice were administered via oral gavage with 50 mg/kg AZD0530, 50 mg/kg BIBF1120, or 25 mg/kg AZD0530 plus 25 mg/kg BIBF1120 (Selleck Chemicals, Houston, TX) for 5 days a week for 2 weeks. The drug was re-suspended in 0.5% hydroxypropyl methycellulose (Sigma-Aldrich) and 0.1% polysorbate buffer (Sigma-Aldrich). The control group received the vehicle buffer alone. The growth of tumor xenografts was monitored by bioluminescence using the IVIS system 2000 series (Xenogen Co.). The xenografts were harvested at 8 weeks post-implantation for gene expression microarray analysis (Affymetrix GeneChip Human Gene 1.0ST Array, Santa Clara, CA, USA) to ascertain the EMT scores, and then subjected to paraffin embedding followed by immunohistochemical staining for E-cadherin (#3195S, Cell Signaling Technology, Beverly, MA).

Further six bone marrow samples (4M/2F median age 68) were processed, as described above, to obtain MPC culture in BIOW2 sera batch with or without adding 4 nM of Erlotinib-HCl (OSI-744, SelleckChem, Houston, USA-TX), a potent EGFR inhibitor, and 100 nM of Nintedanib (BIBF 1120, SelleckChem) to simultaneously inhibit VEGF, FGF, and PDGF receptors. Cell cultures were performed in duplicate in six-well culture plates for suspension cultures, media were changed after 48 and 72 h restoring inhibitors concentration and at day 6 cultures were detached for flow cytometry analysis to quantify mesenchymal CD73⁺CD90⁺ population. Similarly and in parallel, MPC cultures were performed applying LZM sera batch in presence or absence of 50 nM of Linsitinib (OSI-906, SelleckChem) as IGF-1R inhibitor. Significant differences were revealed by one-way ANOVA test, and the inhibition index was calculated as difference in CD73⁺CD90⁺ percentages in treated and no-treated cultures divided by the percentage in the non-treated. Data were collected in duplicate and reported as median values ± standard error (SE).

Mouse embryonic fibroblasts (MEFs) were previously stably transfected with the wild‐type β6 subunit (MEF‐β6) by this group (Xu et al. 2009). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM), 4 mmol/L l‐Glutamine, 10% foetal calf serum (FCS) containing 5 μg mL⁻¹ Blastocidin (InVivoGen, San Diego, CA). The transformed mink lung reporter cells (TMLC) stably expressing firefly luciferase under the control of a TGF‐β‐sensitive portion of the plasminogen activator inhibitor‐1 (PAI‐1) promoter (Abe et al. 1994), were a gift from Dan Rifkin (New York University, New York), and were cultured in DMEM, 4 mmol/L l‐Glutamine, 10% FCS, and 250 μg mL⁻¹ G‐418 sulphate (Sigma, Dorset, UK).
The antibodies used were mouse monoclonal anti αVβ6 ‐clone 10D5, (Millipore, Billerica, MA), F(ab')2 fragment of goat anti‐mouse IgG conjugated to R‐phycoerythrin (Life Technologies, Paisley, UK), and mouse monoclonal anti‐TGF‐β1, β2, β3 –clone 1D11 (R&D systems, Abingdon, UK).
The Alk5 inhibitor (SB525334A) was obtained from GSK (Stevenage, UK). Pirfenidone, NAC, and dexamethasone were purchased from Sigma (Dorset, UK) and BIBF1120 purchased from Selleckchem (Munich, Germany). TGF‐β1 was obtained from (R&D systems).