Figure 2

TGF-β promotes tube formation in pancreatic cancer cell lines. (A) TGF-β1 treatment (10 ng/mL) alters the sphere-forming capacity of the YamaPaca-6.12 clone toward tube formation. The sphere-forming YamaPaca clones were cultured in collagen for 8 days with subsequent stimulation; mock treatment was used as a negative control (100 ng/mL Activin-A, 100 ng/mL Nodal, 100 ng/mL BMP-2/BMP-7, 100 ng/mL BMP-4/BMP-7, and 10 ng/mL TGF-β1). (B) Dose dependency was evident for the YamaPaca-6.12 clone in the 3D culture condition. (C) An increasing number of tubes was observed with increasing TGF-β1 concentration in the 3D culture of YamaPaca-6.12 clones. TGF-β1 was administered to 1000 cells per 20 μL of collagen for 8 days at the following concentrations: mock, 0.1, 1, 10, and 100 ng/mL (n = 3). Multiple comparison (Williams’ method) was used for statistical analysis. *Static ≥ rejection value (5%) and ** static ≥ rejection value (2.5%) compared with the controls (mock treatment). Horizontal bars, averages. (D) YamaPaca-6.12 cells form tube structures by TGF-β1 stimulation. A subpopulation of cells formed tubes by TGF-β1 stimulation, while the large majority of cells did not. (E) The speed of the growth of sphere structures was decreased by TGF-β1 stimulation. The diameter of sphere structures under the TGF-β1 stimulation was smaller than that of the structures that received mock treatment. **P < 0.01, t-test. (F) Treatment with a TGF-β-signaling inhibitor (LY-364947 or SB-431542) alters the tube-forming YamaPaca-6.2 clone toward the formation of spheres. Increasing concentrations of the inhibitors diminish the tube-forming capacity of the YamaPaca-6.2 clone and antagonize the treatment with 10 ng/mL of TGF-β1, whereas the sphere-forming capacity of the YamaPaca-6.12 clone is not affected by treatment with TGF-β-signaling inhibitors. (G) The number of spheres and tubes in the 3D culture of YamaPaca-6.2 cells was counted (n = 3). ^†1Uncountable because of tube-network generation. ^†2Common.