Cilengitide

2D films or 3D scaffolds were seeded with tumor cells, cultured for 24 h, and then treated with 10 μM Cilengitide (Selleck Chemicals, Huston, TX, USA), 10 μM GANT 58 (Santa Cruz Biotechnology, Dallas, TX, USA), 100 nM SD208 (Sigma-Aldrich, St. Louis, MO, USA), or DMSO as a control in serum-free media. After 48 h of drug treatment, RNA was harvested for qRT-PCR analysis.

The function-blocking anti-α_Vβ₆ antibody 6.3G9 (henceforth referred to as 3G9) has been described previously [21 (link)]. The TGF-β receptor type II-murine Fc:IgG_2A chimeric protein (Fc:TβRII) that functionally inhibits TGF-β1 and -β3, as well as control IgG₁ clone 1E6, were all prepared sterile and pyrogen-free at Biogen and used as solutions of the purified proteins in PBS and stored at − 80 °C until use. The c-RGD peptides cyclo-(Arg-Gly-Asp-D-Phe-Val), a selective inhibitor of the α_Vβ₃ integrin [22 (link)], was from Bachem (Bubendorf, Switzerland) (H-2574) and Cilengitide, a selective inhibitor of the α_Vβ₃ and α_Vβ₅ integrins [23 (link)] was from Selleckchem (Munich, Germany). The ROCK inhibitor Y-27632 was from Sigma-Aldrich. Human fibronectin and vitronectin were purified from fresh human plasma as described [24 (link), 25 (link)].

The isolation of peritoneal MCAs of exfoliated GC cells from ascites/peritoneal lavage fluid were referred to in the literature^{25 (link)}. Human GC cell lines SGC7901 and BGC823 (originally purchased from ATCC) were authenticated using STR method and maintained in RPMI1640 medium (containing 10% FBS, Gibco) at 37 °C in 5% CO2 and 100% humidity. 2 × 10⁴ SGC7901 or BGC823 cells were seeded in 100-mm ultra-low adhesion dishes and cultured in the stem cell medium, i.e. serum free DMEM/F12 medium (1:1, Gibco, Grand island, USA) containing 20 μg/L epidermal growth factor (EGF, Sigma, USA), 20 μg/L basic fibroblast growth factor (Sigma) and B27 supplement (1X, Invitrogen, USA). The primary reagents used in this study were as follows: Integrin α_vβ₃ inhibitor Cilengitide (Cat#.S7077, Selleck) or Integrin α_vβ₃ co-stimulator ligand RGD (Arg-Gly-Asp) (Cat#.ab142698, Abcam) or ERK1/2 inhibitor PD-184161 (Cat#.ab143847, Abcam) or GLI1/2 inhibitor GANT61 (Cat.no.ab120904, Abcam) or Hedgehog/smoothen inhibitor Cyclopamine (Cat#.ab120392, Abcam).

All reagents and chemicals used in this study were purchased from Sigma (St. Louis, USA) unless otherwise specified. For cell culture, RPMI-1640 medium was purchased from PAA Laboratories GmbH (Austria). Tenascin C protein (Cat. No. CC065) was obtained from EMD Millipore (Billerica, Massachusetts, USA). Cilengitide was purchased from Selleckchem (Houston, USA). Rabbit anti-human integrin α_v and β₃ were from Cell Signaling Technologies, Inc. (Danvers, MA, USA). HRP goat anti-rabbit IgG secondary antibody was from Zymed (San Francisco, California, USA). Sequencing-grade trypsin was from Promega (Madison, MI, USA). Human Pan Monocyte Isolation Kits were purchase from Miltenyi Biotec GmbH (Germany) and 5-Carboxyfluorescein N-Succinimidyl ester (CFSE) fluorescent dye was purchase from Cayman Chemical (Michigan, USA).

For chemical perturbations, suspended fibroblasts were pre-incubated with the inhibitors: SMIFH2 (20 μM, Merck Millipore, Billerica, MA), Y27632 (10 μM, Sigma-Aldrich), H1152 (1.6 nM, Merck Millipore), CK666 (200 μM, Tocris Bioscience, Bristol, UK), CK869 (200 μM, Tocris Bioscience, Bristol, UK), C8 inhibitor50 (1 μM, kind gift of William deGrado, UCSF), blebbistatin (20 μM, Sigma-Aldrich), ML-7 (50 μM, Tocris Bioscience, Bristol, UK) and STC (2 μM, Sigma-Aldrich) for 30 min in SCFS media at 37 °C. To inhibit RhoA, fibroblasts were incubated with C3 toxin (2 μg ml^–1, Cytoskeleton, Denver, USA) or Y12 (30 μM, Merck Millipore) for 2 or 3 h, respectively, before the experiments. All reagents were dissolved in dimethylsulphoxide (DMSO) except cell permeable C3 toxin, which was dissolved in 50% (v/v) glycerol. As control we used 0.1% (v/v) DMSO. To block β1-integrins, trypsinised fibroblasts were incubated with α5β1 blocking antibody MAB2575 (Millipore, USA) for 30 min, before the experiments. To block β3-integrins, trypsinised fibroblasts were incubated with 1 μM cilengitide (Selleck Chemicals, Houston, TX, USA) for 30 min, before the experiments. SCFS was conducted in the presence of the respective drug/antibody in the stated concentrations.