Three-dimensional culture promotes reconstitution of the tumor-specific hypoxic microenvironment under TGFβ stimulation

In vitro tumor growth in a three-dimensional (3D) architecture has been demonstrated to play an important role in biology not only for developmental organogenesis and carcinogenesis, but also for analyses on reconstitution and maintenance in a variety of biological environments surrounding the cells. In addition to providing architectural similarity to living organisms, 3D culture with a radial flow bioreactor (RFB) can also closely mimic the living hypoxic microenvironment under which specific organogenesis or carcinogenesis occurs. The findings of the present study under the RFB culture conditions show that cancer cells underwent a shift from aerobic to hypoxic energy metabolism, in addition to protein expression to maintain the 3D structure. In RFB-cultured cells, protein stability of hypoxia-inducible factor 1 (HIF1) alpha, a subunit of HIF1, was increased without upregulation of its mRNA. Under these conditions, PHD2, HIF-prolyl-4-hydroxylase 2 and a HIF1 downstream enzyme, were stabilized without affecting the mRNA levels via downregulation of FK506-binding protein 8. PHD2 accumulation, which occurred concomitant with HIF1 stabilization, may have compensated for the lack of oxygen under hypoxic conditions to regulate the HIF levels. 3D-culture-induced overexpression of carbonic anhydrase (another representative HIF downstream enzyme) was found to occur independently of cell density in RFB-cultured cells, suggesting that the RFB provided an adequately hypoxic microenvironment for the cultured cells. From these results, it was hypothesized that the key factors are regulatory molecules, which stabilize and degrade HIF molecules, thereby activating the HIF1 pathway under a hypoxic milieu.