Genetical engineered lung cancer cell for analyzing Epithelial-Mesenchymal transition

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological processes that also exert important roles in disease progression Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the future in vivo.


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The ability of cells to temporally acquire different characteristics, also known as cell plasticity, plays  animal models and patients [8,9]. Recently, a hybrid stage between epithelial and mesenchymal 40 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted September 23, 2019. ; https://doi.org/10.1101/778316 doi: bioRxiv preprint 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].

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In order to study the transitory stages of EMT, MET

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The oligonucleotides used to generate the gRNA were gRNA-F and gRNA-R (Table S1). The template 76 plasmid used for inserting DYKDDDDK-tagged (FLAG) mCardinal fluorescent protein gene after 77 VIM was designed as following (gRNAsite-800nt of Vim-P2A-mCardinal-FLAG-800nt of 3'VimUTR-78 gRNAsite) and was synthetized by Thermo. The Cas9-gRNA and the template plasmids were both 79 nucleofected to cells at the same time and the cells were selected 2 days upon nucleofection for 80 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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The vectors harbouring miR-145, miR-200b, miR-200c and miR-205 genomic fragments were created 104 by inserting each PCR amplified microRNAs gene into the 3'UTR of mNeon fluorescent protein 105 expressing vector (pmR-mNeon). All listed above genomic fragments were amplified using tiHybrid 106 DNA polymerase (EURx) from the DNA, which was purified from the blood of healthy volunteer 107 with the use of GeneAll Exgene Blood SV kit (GeneAll). The sets of primers used for amplification of

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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The genotyping was further confirmed by PCR using the same set of primers (170 and 249), tiHybrid 123 DNA polymerase and high quality genomic DNA, purified from single-cell clones with the use of       (Table S1).       The targeting sequence in H2170 cells was confirmed by sequencing ( Figure S1). The H2170 lung 212 cancer cells were nucleofected with the CRISPR and template (vKIT, Figure 1A) plasmids, following 213 enrichment using puromycin. vKIT contained the sequence of self-cleaving T2A peptide followed by

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Two days after nucleofection, the cells were selected using puromycin (1 µg/ml) for another two days.

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Single cell clones were obtained by dilution, further they were genotyped by PCR with the efficiency

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To further characterise VRCs, they were then sorted according to the fluorescence intensity, resulting 246 in two populations (dim-VRCs and bright-VRCs) that differ about 3 times in mCardinal fluorescence 247 intensity ( Figure S4). That difference was reduced upon culturing to less than 2 fold mCardinal 248 intensity between dim-VRCs and bright-VRCs after 48 h ( Figure 2G). VIM as well as mCardinal 249 expression measured at the transcriptional level confirmed that those two genes are expressed at 250 equal levels ( Figure 2H and S5).

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The dim-VRCs where then exposed to a series of EMT-inducing factors, to analyse the correlation 252 between mesenchymal conversion and mCardinal expression.  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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EMT might be cell type specific and more work needs to be done to clarify this. Our data is in 423 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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In conclusion, our data uniquely illustrate a reporter line, VRCs, as a reliable reporter model for

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. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made