Figure 3. Three dimensional cell culture increases expression of β3 integrin and enhances FGF2 signaling.

(A) Control (CNTRL) and FGFR1 expressing NMuMG cells were grown under 3D culture conditions in the presence of exogenous FGF2 (20 ng/ml) for 10 days. Brightfield images showing the resulting hollow acinar structures (top) and filled structures (bottom) were collected using an EVOS FL microscope. (B) Control NMuMG cells or cells expressing FGFR1 and/or β3 integrin (β3-Int) were cultured in the presence or absence of exogenous FGF2 as in panel A and overall cell growth was quantified by bioluminescence. Data in panel B are the mean ± SD of triplicate wells. Data in panels A and B are representative of at least three independent experiments yielding similar results. (C) Metastatic D2.A1 cells were grown on tissue culture plastic or in 3D culture conditions containing basement membrane extract (BME) alone or supplemented with additional Fibronectin or Collagen I as described in the materials and methods. Photomicrographs represent typical growth morphologies of these cells when grown in these various conditions. These brightfield images were collected using a TS100 Nikon microscope at 100x magnification. (D) Following 10 days of culture in the conditions shown in panel C (Fibronectin = FN; Col I = Collagen I) cells were lysed and analyzed by immunoblot for expression of β3 integrin (β3-Int). Actin served as a loading control. (E) D2.A1 cells were cultured on tissue culture plastic or in 3D culture conditions as shown in panel C. These cells were stimulated with FGF2 (20 ng/ml) for 30 minutes and analyzed for downstream phosphorylation of Erk1/2. All data in panels C–E are representative of at least three independent experiments yielding similar results.