Fig 3. Simulated VEGF-A and TGF−β1/2 exposure promoted phenotype heterogeneity.

Simulated response to TGF−β1/2 and VEGF-A exposure with and without axis specific inhibitors. Vimentin and E-cadherin abundances (in nM) were used to quantify the shift in population at 48 hrs. (A-C) VEGF-A (50 a.u.) treatment resulted in a population with enhanced epithelial properties. This was contrary to the addition of TGF−β2 (10 a.u.), which shifted the population towards a mesenchymal phenotype. Interestingly, the combined effects of TGF−β2 and VEGFA was found to increase both ecadherin and vimentin levels, creating a heterogeneous population (black arrow), which can also be seen in a minority of untreated cells (gray arrow). (D-F) To isolate the effect of NFAT, we inhibited NFAT de-phosphorylation in combination with VEGFA. This negated the increase in ecadherin expression and shifted the population towards a mesenchymal phenotype. Likewise, combining NFAT inhibition with TGF−β mitigated all VEGF enhanced ecadherin expression. Lastly, combination of TGF−β2, VEGFA, and NFAT inhibition nearly mitigated all effects of VEGFA, shifting the heterogeneous population towards a mesenchymal phenotype. In whole, high levels of phosphorylated-Sp1 correlated with vimentin expression, while NFAT was responsible for maintaining E-cadherin expression in the presence of other factors, although neither were mutually exclusive.