CRISPR/Cas9-engineered Immortalized Breast Epithelial MCF10A Reporter Line for EMT Studies and Anti-cancer Drug Discovery

3/30/2019 — 4/3/2019

AACR Annual Meeting 2019


Metastasis is responsible for most cancer-related deaths. One mechanism of metastasis involves epithelial-to-mesenchymal transition (EMT), a process characterized by the decrease in cell adhesion and increase in cell motility. Cells undergoing EMT often display downregulation of epithelial markers (such as E-cadherin; ECAD) and upregulation of mesenchymal markers (such as vimentin; VIM). Besides metastasis, EMT has also been reported to be associated with other pathological conditions, such as acquired therapeutic drug resistance. Given the roles that EMT plays in the pathological processes, it is of increasing interest as a target for anti-cancer treatment and drug discovery. In vitro reporter models have proven to be a valuable tool for dissecting the signaling pathways that regulate the EMT process and for screening compounds targeting EMT. In previously developed EMT reporter cell lines, the reporter gene was driven by a truncated EMT marker gene promoter. Therefore, the establishment of a more physiologically relevant reporter cell model is critical for advancing our knowledge of EMT.

ECAD, a hallmark of epithelial cells, has been implicated in the onset of metastatic dissemination.6 Using CRISPR/Cas9 knock-in technology, we generated an ECAD-emerald green fluorescent protein (EmGFP) reporter model with immortalized breast epithelial MCF10A (ATCC® CRL-10317™) cells. In the reporter cells, the EmGFP gene was tagged at the C-terminus of ECAD, allowing for real-time monitoring of EMT progression in live cells. The targeted knock-in of the ECAD-EmGFP allele was verified at the genomic DNA, transcript (mRNA), and protein levels. Functional evaluation of the reporter cell line revealed that treatment of ECAD-EmGFP reporter cells with TGF-β led to EMT induction, as demonstrated by a reduction in ECAD-GFP expression and increase in VIM and fibronectin expression. Additional functional characterization revealed that the reporter cells possessed an enhanced migration capacity upon EMT induction with TGF-β. In summary, this MCF10A-ECAD-EmGFP reporter cell line serves as a physiologically relevant in vitro cell model for studying EMT cancer biology and anti-EMT drug discovery.