Regulation of Epithelial-to-Mesenchymal Transition Using Biomimetic Fibrous Scaffolds

Abstract

Epithelial-to-mesenchymal transition (EMT) is a well-studied biological process that takes place during embryogenesis, carcinogenesis, and tissue fibrosis. During EMT, the polarized epithelial cells with a cuboidal architecture adopt an elongated fibroblast-like morphology. This process is accompanied by the expression of many EMT-specific molecular markers. Although the molecular mechanism leading to EMT has been well-established, the effects of matrix topography and microstructure have not been clearly elucidated. Synthetic scaffolds mimicking the meshlike structure of the basement membrane with an average fiber diameter of 0.5 and 5 μm were produced via the electrospinning of poly(ε-caprolactone) (PCL) and were used to test the significance of fiber diameter on EMT. Cell-adhesive peptide motifs were conjugated to the fiber surface to facilitate cell attachment. Madin-Darby Canine Kidney (MDCK) cells grown on these substrates showed distinct phenotypes. On 0.5 μm substrates, cells grew as compact colonies with an epithelial phenotype. On 5 μm scaffolds, cells were more individually dispersed and appeared more fibroblastic. Upon the addition of hepatocyte growth factor (HGF), an EMT inducer, cells grown on the 0.5 μm scaffold underwent pronounced scattering, as evidenced by the alteration of cell morphology, localization of focal adhesion complex, weakening of cell-cell adhesion, and up-regulation of mesenchymal markers. In contrast, HGF did not induce a pronounced scattering of MDCK cells cultured on the 5.0 μm scaffold. Collectively, our results show that the alteration of the fiber diameter of proteins found in the basement membrane may create enough disturbances in epithelial organization and scattering that might have important implications in disease progression.

Keywords: MDCK cells; epithelial-to-mesenchymal transition; fiber diameter; fibrous scaffolds; hepatocyte growth factor; phenotype.

Fig. 1

Characterization of electrospun fibrous scaffolds by confocal microscope (A, C) and scanning electron microscope (SEM, B, D). Scaffolds were stained with CellTracker^™ Red or sputter-coated with platinum prior to confocal and SEM imaging, respectively. Fiber diameter (E, F) was determined from SEM images using DiameterJ’s histogram analysis. Fibrous scaffolds with an average diameter of 0.5 (A, B, E) and 5 μm (C, D, F) are fabricated employing different electrospinning parameters.

Fig. 2

Modification and characterization of PCL substrates. Cell adhesive peptide (CGGWGRGDSPG: RGD-SH) was covalently conjugated to the surface of PCL fibers (A). The chemical transformations were monitored by Fourier Transform Infrared Spectroscopy (FT-IR, B) and X-ray photoelectron spectroscopy (XPS, C). Spun cast PCL films were used for surface analysis to eliminate the topographical complications. Compared to PCL, aminolyzed PCL (PCL-NH₂) shows characteristic amide and amine peaks (B) and RGD-conjugated PCL (PCL-RGD) shows characteristic S and N peaks (C).

Fig. 3

Characterization of peptide immobilization by confocal imaging based on tryptophan fluorescence (Excitation: 280 nm; Emission: 350 nm). The bright tryptophan signal on the outer surface of the peptide-modified fibers (B and C) is absent on the pristine PCL fibers (A). The cross sectional view of the peptide-modified fibers (C) confirms the uniform peptide conjugation on the fiber surface.

Fig. 4

Characterization of the initial attachment and spreading of MDCK cells on small and large diameter fibers, with or without RGD conjugation. (A): Confocal images of MDCK cells cultured on PCL-based scaffolds (40x). Red: F-actin; Blue: nuclei. (B): Cell spreading as a function of fiber diameter and surface chemistry; (C): Cell circularity as a function of fiber diameter and surface chemistry. The circularity is defined as 4π×area/perimeter². (D): Average number of cells attached to the scaffold per mm² surface area. Quantification was carried out using ImageJ software based on five separate 1,024 × 1,024 μm² confocal images. *Significantly different (p<0.05, ANOVA) from RGD conjugated scaffolds. No significant difference was observed between small and large diameter fibers (p>0.05). Error represents standard error of the mean of 3 repeats.

Fig. 5

Characterization of cell clustering on small and large diameter fibers in the absence or presence of HGF by Live/Dead and confocal imaging. (A): Representative confocal images of MDCK cells stained by Syto 13 (live, green) and PI (dead, red). Circled areas represent clustered cells and boxed regions represent scattered cells. (B): Quantification of the number of clustered cells in both small and larger diameter scaffolds based on five separate 1,417 × 1,417 μm² confocal images. *Statistically (p<0.05, ANOVA) significant compared to HGF-free controls; **Statistically (p<0.05, ANOVA) different between small and large diameter fibers. Error represents standard error of the mean from 3 repeats.

Fig. 6

Fiber diameter affects the adhesion, spreading and scattering of MDCK cells. Cells cluster together, forming a cobble stone network in the absence of HGF on 0.5 μm fibers (A). The addition of HGF induces MDCK cells to adopt a mesenchymal phenotype (B). Cells wrapped around 5 μm fibers and adopted a spread out morphology in the absence (C) and presence (D) of HGF. Solid arrows indicate cell-cell junctions and dotted arrows point to F-actin fibers.

Fig. 7

Expression of E-cadherin by MDCK cells cultured on small and large diameter fibers with or with HGF stimulation. Nuclei (i), E-cadherin (ii) and F-actin (iii) were stained blue, green and red, respectively. The boxed area in the merged image (iv) is shown at a higher magnification in v. Solid arrows indicate cell-cell junctions and dotted arrows indicate extended cell processes developed during EMT.

Fig. 8

Expression of vinculin by MDCK cells cultured on small and large diameter fibers with or with HGF stimulation. Nuclei (i), vimentin (ii) and F-actin (iii) were stained blue, green and red, respectively. The boxed area in the merged image (iv) is shown at a higher magnification in v. Solid arrows indicate cell-cell junctions and dotted arrows indicate extended cell processes developed during EMT.

Fig. 9

Expression of vinculin by MDCK cells cultured on small and large diameter fibers with or with HGF stimulation. Nuclei (i), vinculin (ii) and F-actin (iii) were stained blue, green and red, respectively. The boxed area in the merged image (iv) is shown at a higher magnification in v. Solid arrows indicate cell-cell junctions and dotted arrows indicate extended cell processes developed during EMT.