TGFβ signaling regulates epithelial–mesenchymal plasticity in ovarian cancer ascites-derived spheroids

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   Figure 1

   EMT is induced during EOC ascites cell spheroid formation and reversed upon re-attachment. (a) Quantitative RT-PCR analysis of CDH1, SNAI1, TWIST1, TWIST2 and ZEB2 mRNA in monolayer/adherent cells, spheroid cells, and 72 h re-attached spheroid cells using primary ascites-derived EOC patient samples (n=6). (b) Western blot analysis of E-cadherin and Snail protein in adherent (A), spheroid (S) and 72 h re-attached spheroid samples (R) samples in primary ascites-derived EOC patient samples (n=7). Tubulin was used as a loading control.
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   Figure 2

   TGFβ induces EMT in EOC ascites-derived cells but spheroid formation alone is a more potent inducer of EMT. (a) Quantitative RT-PCR analysis of CDH1, SNAI1, TWIST2, ZEB2 and VIM mRNA in control and TGFβ1-treated adherent EOC ascites-derived cells (n=4) at 2 h, 24 h and 72 h (*P<0.05 as determined by Student's t-test). (b) TGFβ1 treatment of EOC ascites-derived cells induces transformation of epithelial cells towards a fibroblastic mesenchymal morphology. Representative image from EOC57 patient sample at 72 h post treatment (scale bar=200 μm). (c) Western blot analysis of E-cadherin, Claudin1 and Snail protein in adherent (A) and spheroid (S) samples treated with TGFβ1 or not in ascites-derived EOC patient samples (n=2). Tubulin was used as a loading control. Panels represent separate lanes from the same blot and same length of exposure.
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   Figure 3

   Inhibition of TGFβ signaling in spheroids blocks EMT and compromises EOC spheroid morphology. (a) Quantitative RT-PCR analysis of CDH1, SNAI1, TWIST2 and ZEB2 mRNA in control and SB-431542-treated spheroids (n=7; *P<0.05 as determined by Student's t-test). (b) Western blot analysis of E-cadherin and Snail protein in spheroids formed from EOC ascites-derived patient samples (n=6). Actin was used as a loading control. (c) SB-431542 treatment of EOC ascites-derived cells at the time of seeding to ULA forms smaller and less cohesive spheroids compared with DMSO controls. Representative image from EOC75 patient sample at 72 h post treatment (scale bar=500 μm). (d) Cell viability was determined using CellTiter-Glo assay following 3 days of SB-431542 treatment, or DMSO control across EOC patient ascites samples (n=8) cultured as adherent cells and spheroids. Data is represented as mean±s.e.m. and Student's t-test for statistical significance (****P<0.001).
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   Figure 4

   Inhibition of TGFβ signaling in spheroids decreases migration of cells across a Transwell. (a) SB-431542 treatment of EOC ascites-derived cells at the time of seeding to ULA and transfer of day 3 spheroids to Transwell inserts decreases cell migration as compared with DMSO-treated controls. Representative EOC154 patient sample showing fewer migrated cells in SB-431542 treated spheroids compared with DMSO controls (scale bar=100 μm). (b) Transwell cell migration of treated spheroids from patient-derived samples (n=9) quantified using ImageJ software and averaged among five different fields per image (*P<0.05 as determined by Student's t-test).
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   Figure 5

   Inhibition of TGFβ signaling in re-attached spheroids decreases motility of dispersing cells and enhances their epithelial phenotype. (a) SB-431542 treatment of EOC spheroids started at the time of re-attachment to standard tissue culture plates disrupts the spheroid core and decreases cell dispersion area. Representative image from EOC154 patient sample at 24 h post treatment (scale bar=500 μm). (b) Dispersion area was quantified using ImageJ software and averaged amongst 12 replicates per treatment condition (SB-431542 or DMSO control) for each EOC patient sample (n=7). Dispersion area was calculated 24 h after spheroids had been re-plated to standard tissue culture plastic (*P<0.05 as determined by Student's t-test). (c) SB-431542 treatment of spheroids started at the time of re-attachment to standard tissue culture plates disrupts EOC spheroid core and changes morphology of dispersing cells to a more cuboidal epithelial phenotype. Representative image from EOC154 patient sample that was Hema-3-stained at 72 h post treatment (scale bar=500 μm). (d and e) Immunofluorescence images of cells dispersing out of SB-431542 treated re-attached spheroids shows enhanced β-catenin staining and reduced stress fiber formation (by rhodamine–phalloidin staining) compared with DMSO controls (scale bar=100 μm).
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   Figure 6

   TGFβ signaling in EOC ascites cells protects spheroids from platinum-induced cell death. (a) EOC ascites-derived cells (n=4) were treated with either DMSO or SB-431542 at the time of seeding to 24-well ULA cluster plate to form spheroids. Three days later, spheroids were treated with carboplatin (100 μM) for 72 h. Representative EOC209 patient sample showing reduced spheroid formation potential (fewer spheroids, more single cells) with combined carboplatin and SB-431542 treatment (scale bar=1 mm). (b) Cell viability was determined using CyQUANT NF assay. Data is represented as mean±s.e.m. and one-way ANOVA with Tukey's Multiple Comparison test (*P<0.05; **P<0.01; ***P<0.001).

• © 2016 Society for Endocrinology