The Generation of a NSCLC EMT Reporter Cell Line for Metastatic Lung Cancer Drug Discovery and Development

Cancer metastasis continues to be the leading cause of mortality in cancer patients around the world. One of the prominent biological processes implicated in cancer metastasis is epithelial to mesenchymal transition (EMT). EMT has also been shown to have roles in many aspects of cancer initiation and progression, including tumorigenesis and drug resistance. Despite the accumulation of a large body of data on the association of EMT with cancer, EMT has not been an active target for therapeutic development partly due to the lack of appropriate in vitro models. Utilizing some of the basic biology of EMT, we have created a novel advanced in vitro model for use in both basic research and discovery of new anti-EMT drugs.

In lung cancer, vimentin (VIM) intermediate filament proteins are associated with EMT and the metastatic spread of cancer. Vimentin expression  is  generally  upregulated when epithelial cells transition to the mesenchymal phenotype. We capitalized on this biological phenomenon using CRISPR/Cas9 gene editing to generate a VIM-red fluorescent protein (RFP) fusion reporter cell line in the A549 non-small cell lung cancer (NSCLC) cell line, which is one of the most widely used and recognized lung cancer cell lines. The A549 VIM RFP cell line harbors a C-terminal RFP tag on the vimentin gene, which enables end-point or real-time tracking of the EMT status as cells transition from epithelial to mesenchymal phenotype under  defined  conditions.  The EMT reporter cell line was verified at the nucleic acid (genomic and mRNA) and protein levels and functional levels by cell-based assays. Bio-functional evaluation of the A549 VIM RFP cell line shows sensitivity to metastatic NSCLC drugs PP1 (SRC inhibitor) and A 83-01 (ALK5 inhibitor).

These results provide the foundation for the use of this cell line in high-throughput screening (HTS) applications including the identification of new anti-EMT drugs for metastatic NSCLC. Furthermore, the A549 VIM RFP reporter cell line is also a convenient and sensitive model for basic science research on the mechanisms of metastasis.