br Both cells cultured under normoxic
Both LY 500307 cultured under normoxic condition on random fiber substrates (R-PLLA and R-PCL) produce less spread and more
Fig. 4. Quantification of the cellular morphologies of cytoplasm roundness ((a) and (a’)), cytoplasm elongation factor ((b) and (b’)), nuclear elongation factor ((c) and (c’)), and AN/AC ratio ((d) and (d’)) as boxplots for MCF-7 cells cultured under normoxic and hypoxic conditions at day 7. PLLA, poly(L-lactic acid); PCL, poly(ε-caprolactone).
Fig. 6. Relationships between gene expression of TGFB and morphological parameters (cytoplasm roundness (a), cytoplasm elongation factor (b), nuclear elongation factor (c), and nuclear area to cytoplasm area ratio [AN/AC] (d)) for MDA-MB-231 cells cultured on the three type of PLLA substrates under normoxic and hypoxic conditions at day 7. TGFB, transforming growth factor beta; PLLA, poly(L-lactic acid); GAPDH, glyceraldehyde-3-phosphatase dehydrogenase.
231 cells cultured under hypoxic condition on A-PCL and MCF-7 cells cultured under hypoxic condition on A-PLLA exhibited less spread and more circular morphologies (Fig. 3(a’) and (d’) and Fig. 4(a’) and (d’)).
For both MDA-MB-231 and MCF-7 cells cultured on R-PCL under both oxygen concentration conditions (1% and 20% O2), smaller value of the nuclear elongation factor was observed. This indicates that the cell stretching is restricted with R-PCL substrate, and the more circular morphology of the cells is overwhelming. As seen in Figs. 3 and 4, the hypoxic treatment had no remarkable effect on both cell's morphologies.
Furthermore, MDA-MB-231 cells can be more deformed than MCF-7 cells owing to an enhanced metastatic potential. This interpretation is supported through detailed cellular migration analysis under both normoxic and hypoxic conditions at day 7 (see section 3.5).
3.3. Cellular morphologies and induced EMT
The cellular morphologies were strongly influenced by the substrate topography and stiffness for both breast cancer cells. Thus, the gene expression changes were investigated to under-stand the role of ECM for malignant phenotype. In this study,
typical epithelial cell marker of CDH1 and mesenchymal marker of vimentin and CDH2 [39e43] were chosen because EMT is a critical phenomenon inducing cancer metastasis [44e53]. To know the detail of EMT and metastasis, TGFB [39,54,55] and SNAI2 [39,56,57] and ZEB1 [54,56e59] were added to the analysis. TGFB is known to induce EMT. SNAI2 and ZEB1 are potent repressor of CDH1 gene expression. Cancer cells respond to the hypoxic microenvironment through the activity of HIF1A [60e62] that behaves as a promotor of EMT. During tumor progression, the ECM is remodeled to produce a stiffer and aligned architecture. Daniele M. Gilkes et al. reported that in hypoxia, cells produced ECM with collagen fiber alignment, which influences the cellular morphology and mortality . Because the aligned topography is favorable for cellular migration , EMT might also be induced for more efficient migration.
Fig. 5 shows the gene expression of CDH1, TGFB, and HIF1A for MDA-MB-231 and MCF-7 cells cultured on different substrates under both oxygen concentration conditions at day 7. For CDH1 expression, significant repression was observed for MCF-7 cell culture under hypoxic condition on R-PLLA substrates (Fig. 5(a’)), whereas no significant changes were observed for MDA-MB-231 cells (Fig. 5(a)). For comparison, the expression levels at each substrate were normalized to the data of F-PLLA. MDA-MB-231 cells incubated on A-PLLA under hypoxic condition showed a significant increase in TGFB expression (Fig. 5(b)), indicating that the cells are undergoing EMT transition because of the EMT-promoting gene [39,54].
The HIF1A level under hypoxic condition was markedly increased in MDA-MB-231 cells incubated on A-PLLA (3-fold) in
Fig. 7. Relationships between gene expression of HIF1A and morphological parameters (cytoplasm roundness (a), cytoplasm elongation factor (b), nuclear elongation factor (c), and nuclear area to cytoplasm area ratio [AN/AC] (d)) for MDA-MB-231 cells cultured on the three type of PLLA substrates under normoxic and hypoxic conditions at day 7. HIF1A, hypoxia-inducible factor 1a; PLLA, poly(L-lactic acid); GAPDH, glyceraldehyde-3-phosphatase dehydrogenase.
comparison with that of normoxic condition but not on both Fe and R-PLLA substrates under hypoxia (Fig. 5(c)). Similar results were obtained when MCF-7 cells were treated with hypoxic condition (Fig. 5(b’) and (c’)).
For vimentin expression, this surface protein is expressed by MDA-MB-231 and MCF-7 cells with differences on all substrates (Fig. S6: Supplementary data). The MDA-MB-231 cells incubated on F-PLLA, F-PCL, and R-PLLA substrates (Fig. S6(a) and (c): Supple-mentary data) in the hypoxic group express more vimentin in comparison with those on A-PLLA and A-PCL substrates (Fig. S6(b): Supplementary data). Similar changes were obtained in MCF-7 cells on F-substrate as shown in Fig. S6(a’) (Supplementary data). All the results suggest that hypoxia is the driving force of EMT in both cancer cells.