Training and validation simulations. The population of EMT models qualitatively captured TGF−<i>β</i>-induced EMT signaling.

<p>(A-I) The population was generated using JuPOETs and trained using 11 different objective functions (41 data sets) taken from Medici <i>et al.</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005251#pcbi.1005251.ref007" target="_blank">7</a>]. The model captured the simulated experiments for all cases when compared to randomized controls. (J-L) The model populations were also compared against untrained temporal data to measure the effectiveness as a pure prediction. The western blot data was reproduced from Medici <i>et al.</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005251#pcbi.1005251.ref007" target="_blank">7</a>]. The intensity of each band was estimated using the ImageJ program. These blot intensities were then used as the training data for the EMT parameter estimation studies.</p

Simulated VEGF-A and TGF−<i>β</i>1/2 exposure promoted phenotype heterogeneity.

<p>Simulated response to TGF−<i>β</i>1/2 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−<i>β</i>2 (10 a.u.), which shifted the population towards a mesenchymal phenotype. Interestingly, the combined effects of TGF−<i>β</i>2 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−<i>β</i> mitigated all VEGF enhanced ecadherin expression. Lastly, combination of TGF−<i>β</i>2, 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.</p

Analysis of underlying signaling responses.

<p>(A) We examined the distribution of NFATc1 and AP1/SP1 in cells containing the hybrid phenotype (VEGF-A + TGF−<i>β</i>2 case), showing the potential for cells to express both SP1 and NFATc1 in a non exclusive manner. (B) In the absence of YREG1, most of the population failed to consistently to retain a stable epithelial state. (C) We identified a novel regulator of LEF1 called YREG1 that allows Snail/Slug to emulate an inducer by repressing YREG1, which was required to stabilize the untreated population. YREG1 overexpression revealed an enhanced epithelial phenotype, while some inherently transformed cells moved towards a hybrid phenotype.</p

Ductal branching is dependent upon phenotype heterogeneity within MCF10A in 3-D culture.

<p>MCF10A and DLD1 were formed into spheroids overnight and explanted to a collagen gel for 72 hrs. For compaction and tubular assays, cells were embedded into collagen gels for 72 hrs, and the extent of tubulogenesis was measured at 7 days. (A-D) Within MCF10A, TGF−<i>β</i>2 (10ng/ml) enhanced invasion and contractile properties while, VEGFA (50ng/ml) promoted increased migration. TGF−<i>β</i>2 with VEGFA significantly increased migration, while limiting with compaction. VIVIT (10<i>μ</i>M) in combination with VEGFA and TGF−<i>β</i>2 decreased migration and compaction, while increasing invasion. (D) Likewise, cell morphology (circularity index) correlated with both invasion and compaction in MCF10A. (E-F) The size of tubular structures (acini) also increased significantly upon addition of VEGFA, while the number of ductal branches was most significant upon simultaneous TGF−<i>β</i>2 and VEGFA treatment (Red-Ecadherin, Green-Factin, Blue-Nuclear). DLD1 cells followed a similar trend, although the degree of migration, invasion, and compaction was less significant. In addition, no tubular structures were identified during the 7 day tubulogenesis endpoints. Scale bars: 500<i>μ</i>m, 1000<i>μ</i>m, 250<i>μ</i>m, and 80<i>μ</i>m, respectively. C = Control, T = TGF−<i>β</i>2, V = VEGFA, VI = NFAT inhibitor (VIVIT). Asterisks signify statistical differences from each other according to a one-way ANOVA with Tukey’s post hoc (p≺0.05). Boxes in the left-most panel identify regions identified by arrows that were then imaged in greater zoom in the panel immediately below. The box diagram was not repeated for arrows in the other panels for clarity, but the same method was applied.</p

Simultaneous TGF-<i>β</i>1/2 and VEGF-A treatment induced phenotype heterogeneity and is dependent upon NFAT activity <i>in-vitro</i>.

<p>(A) In MCF10A, treatment with (10ng/ml) TGF−<i>β</i>2 increased Slug and vimentin, while ecadherin expression was inhibited at both the gene and protein level at 48 hrs. Conversely, VEGFA alone increased both NFATc1 and ecadherin gene expression. Simultaneous TGF−<i>β</i>2 (10ng/ml) and VEGFA (50ng/ml) treatment increased Slug, NFATc1, and vimentin expression, while also increasing ecadherin levels via qPCR. (B-C) Immunofluorescence confirmed these results and nuclear co-localization of both phospho-Sp1 and NFAT were found dependent upon TGF−<i>β</i>2 and VEGFA, respectively. (D) To isolate the effect of NFAT, treatment of VEGFA (50ng/ml) and VIVIT (10<i>μ</i>M) reduced ecadherin expression at 48hrs (control-dashed line). Similarly, combined TGF−<i>β</i>2, VEGFA and VIVIT treatment increased Slug and vimentin expression, while inhibiting ecadherin levels via qPCR. (E) These findings were confirmed via immunofluorescence as the VIVIT peptide inhibited ecadherin and nuclear localization of NFATc1 in all three cases. (F) Quantitative flow cytometry also confirmed this trend. Similar experiments in DLD1 followed a similar trend (supplement). Magnification, 40x. Scale bars: 50<i>μ</i>m. C = Control, T = TGF−<i>β</i>2, V = VEGFA, VI = NFAT inhibitor (VIVIT). Asterisks signify statistical differences from each other according to a one-way ANOVA with Tukey’s post hoc (p≺0.05).</p

Model connectivity recreates the core architecture during EMT.

<p>The EMT network contains 97 nodes (proteins, mRNA, and genes) interconnected by 169 interactions. Central to EMT induction, activation of the MAPK cascade occurs through TGF-<i>β</i>1/2 binding which activates the AP-1/Sp1 transcriptional axis. AP-1/Sp1 drives an autocrine response of TGF-<i>β</i>3, which activates the Smad cascade, leading to phenotypic change. Conversely, VEGF-A binding can stabilize an epithelial phenotype through NFAT activation. Downstream activation of <i>β</i>-catenin signaling due to E-cadherin loss and GSK3 inactivation of <i>β</i>-catenin confinement is critical to the complete activation of the EMT program. The complete list of molecular interactions that comprise the model is given in the supplement.</p