Genetical engineered lung cancer cell for analyzing Epithelial-Mesenchymal transition

Abstract

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible 12 manner, is a physiological processes that also exert important roles in disease progression Two forms 13 of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, 14 mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome 15 of different types of neoplasias as well as drug resistance development. Since EMT/MET are 16 transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red 17 fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) 18 in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT 19 and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform 20 to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the 21 future in vivo.22 Keywords: epithelial-mesenchymal transition (EMT); mesenchymal-epithelial transition (MET); 23 cancer cell line; vimentin; reporter; 24 25 29 transition (EMT) and mesenchymal-epithelial transition (MET), respectively. EMT has been 30 correlated to the metastasis and invasion potential of many types of malignancies [2]. The fact that 31 the poor outcome of many types of neoplasias correlates with EMT/MET, makes these molecular 32 phenomena important focus for research and drug targeting [3-6]. Despite the clinical association,33 the role of EMT/MET in metastasis is inconclusive, for example mesenchymal-like prostate cancer 34 cells survive in circulation but, unlike epithelial or cells undergoing EMT, they are unable to form 35 macrometastases [7]. In fact, breast cancer cell metastasis to lung tissue in mice was not affected by36 decreasing EMT -by targeting EMT-triggering transcription factors such as SNAI1 (Snail) and SNAI2 37 (Slug) by overexpressing miR-200 [8]. An additional role of EMT in tumorigenesis might be the 38 development of apoptotic tolerance and increased resistance to chemotherapy as has been found in 39animal models and patients [8,9]. Recently, a hybrid stage between epithelial and mesenchymal 40 2 of 19 phenotypes (hybE/M) has been recognized, and such hybE/M cells migrate outside the primary 41 tumors displaying some mesenchymal features such as spindle-like morphology, increased nuclear 42 levels of ZEB1 transcription factor, and epithelial ones such as cell-cell adhesion potential [10,11]. 43In order to study the transitory stages of EMT, MET and hybE/M, in particular at in vivo settings, 44 there is need to generate reporter cells to visualize phenotypical changes. There have been different 45 approaches to this, for example the use of heterologous fluorescent proteins driven under stage-46 specific promoters such as mesenchymal (ZEB1) and epithelial (CDH1) [12], mesenchymal snai1 and 47 epithelial sox10 in zebrafish [13], or mesenchymal Vimentin (Vim) in mice [14]. Nevertheless, 48 hete...