Epithelial-mesenchymal transition of cancer cells using bioengineered hybrid scaffold composed of hydrogel/3D-fibrous framework

Abstract

Cancer cells undergoing epithelial-mesenchymal transition (EMT) acquire stem cell-like phenotype associated with malignant behaviour, chemoresistance, and relapse. Current two-dimensional (2D) in-vitro culture models of tumorigenesis are inadequate to replicate the complexity of in-vivo microenvironment. Therefore, the generation of functional three-dimensional (3D) constructs is a fundamental prerequisite to form multi-cellular tumour spheroids for studying basic pathological mechanisms. In this study, we focused on two major points (i) designing and fabrication of 3D hybrid scaffolds comprising electrospun fibers with cancer cells embedded within hydrogels, and (ii) determining the potential roles of 3D hybrid scaffolds associated with EMT in cancer progression and metastasis. Our findings revealed that 3D hybrid scaffold enhances cell proliferation and induces cancer cells to undergo EMT, as demonstrated by significant up-regulation of EMT associated transcriptional factors including Snail1, Zeb1, and Twist2; and mesenchymal markers whereas epithelial marker, E-Cadherin was downregulated. Remarkably, this induction is independent of cancer cell-type as similar results were obtained for breast cancer cells, MDA-MB-231 and gastric cancer cells, MKN74. Moreover, the hybrid scaffolds enrich aggressive cancer cells with stem cell properties. We showed that our 3D scaffolds could trigger EMT of cancer cells which could provide a useful model for studying anticancer therapeutics against metastasis.

Figure 1

Study of cell culture growth conditions through infiltration into scaffolds. Depth imaging to examine cells (MKN74) penetration into 3D scaffold by indicated three methods as mentioned in the subheading of ‘Cells seeding and hybrid scaffold development’ under Methods section at depth of 0–4 mm. RFU of scaffold penetration into Method 1, MKN74 cells were pipetted onto scaffolds and immediately gelatinized; Method 2, scaffold were soaked in MKN74 cells for 10 min and gelatinized; Method 3, scaffold soaked in MKN74 cells for 10 min, transferred onto 24-well plate, incubated for 30 min and gelatinized. Finally, method 3 was selected based on the highest fluorescence intensity for subsequent studies.

Figure 2

Proliferation rates of MDA-MB-231 cells in GelMA, scaffold and hybrid scaffold. (A) Fold changes of cell proliferation and (B) Gene expression of proliferation markers, PCNA and Ki67, were assessed at indicated (Day 1, 3, 7 and 14) time points after seeding the cells. β-actin was utilized to normalize gene expression data. Results were shown as mean ± S.D.

Figure 3

Relative transcript and protein expression levels of epithelial and mesenchymal markers using MDA-MB-231 and MKN74 cells in GelMA, scaffold and hybrid scaffold. (A) Transcriptional levels of EMT markers E-Cadherin and N-Cadherin were detected by qRT-PCR at Day1, Day3 and Day7 post culturing MKN74 cells. (B) Protein expression level of N-Cadherin of MKN74 cells at indicated time points, showing the up-regulation of N-Cadherin at D7 in hybrid scaffolds. We performed 3-independent experiments. One representative image has been shown here. Uncropped full images for total protein and western blot gels have shown in Supplementary Fig. S8. (C,D) Transcript levels of EMT markers E-Cadherin, N-Cadherin, Vimentin and Fibronectin in MDA-MB-231 cells at Day1, Day7 and Day14. β-actin was utilized to normalize gene expression data. Results were shown as mean ± S.D. (E) Representative confocal microscopic images of MDA-MB-231 cells stained with an epithelial marker, E-Cadherin (red) and Alexa Fluor 488 phalloidin for actin cytoskeleton (green) in scaffold and hybrid scaffold. Dual immunostaining of 3D hybrid scaffold cultured with cells showing aggregations of cells with minimal expression of E-Cadherin. (F) Representative confocal microscopic images of MDA-MB-231 cells stained with a mesenchymal marker, vimentin (red) and Alexa Fluor 488 phalloidin for actin cytoskeleton (green) in scaffold and hybrid scaffold. Dual immunostaining of 3D hybrid scaffolds showing higher expression level of vimentin.

Figure 4

EMT regulatory transcriptional factors expression in MDA-MB-231 and MKN74 cells using GelMA, scaffold and hybrid scaffold. (A) Transcript levels of EMT regulatory transcriptional factors Snail1 and Zeb1 in MKN74 cells were detected by qRT-PCR at indicated time points. (B,C) Relative transcript expression levels of Zeb1, Zeb2, Twist1, Twist2, Snail1 and Slug in MDA-MB-231 cells. β-actin mRNA was utilized to normalize gene expression data. Results were shown as mean ± S.D.

Figure 5

Relative transcript expression levels of cancer stem cells markers in MDA-MB-231 and MKN74 cells in GelMA, scaffold and hybrid scaffold. (A) Transcript expression levels of CSC markers CD44 and Sox2 in MKN74 cultured cells were measured by qRT-PCR at Day1, Day3, and Day7. (B,C) Transcript expression levels of CSC markers CD44, Sox2 and Oct4 in MDA-MB-231 cells at indicated time points. β-actin was utilized to normalize gene expression data. Results were shown as mean ± S.D. (D) Representative confocal microscopic images of cancer stem cells marker, CD44 (green) and Rhodamine-phalloidin for actin cytoskeleton (red) immunofluorescence using MDA-MB-231 cells in scaffold and hybrid scaffold. Dual immunostaining of 3D hybrid scaffolds showing higher expression level of CD44.