Gastrointestinal stromal tumors (GIST) are the most frequent mesenchymal malignancies of the gastrointestinal tract. Pathogenesis of GIST is mostly associated with KIT tyrosine kinase gene mutations. Therefore, tyrosine kinases inhibitor imatinib mesylate (Gleevec) is the first-line GIST treatment. Nevertheless, GIST patients are facing secondary resistance to imatinib within 2 years of ongoing treatment. One of the possible mechanisms related to imatinib resistance is the FGF/FGFR autocrine loop, thus, FGF and FGFR might be prospective targets for imatinib-resistant GIST treatment. One of the possible approaches to identify the role of FGF/FGFR autocrine loop in both GISTs’ pathogenesis and imatinib sensitivity is the genome editing tool CRISPR/Cas9 used for target gene knock-out. Thereby the aim of the study was to generate imatinib resistant GIST cell line T1-IM-R expressing doxycycline-inducible endonuclease Cas9 which could be used as in vitro model for gene knockout studies in GIST.
Doxycycline-inducible endonuclease Cas9 gene transfer into imatinib resistant GIST cell line T1-IM-R was performed by lentiviral transduction using pCW-Cas9 plasmid (#50661, Addgene). Providing lentiviral transduction was successful, T1-IM-R cells were selected in the presence of 1 ug/ml puromycin and were used for further clonal selection. The level of Cas9 expression was detected by Western Blot analysis after 6 days of cultivation in the presence of 1 ug/ml doxycycline. The mouse monoclonal antibody against Cas9 (MA1-202, Invitrogen, USA) was used to detect endonuclease Cas9. The b-tubulin was used as loading control.
Totally 32 clonal sublines were obtained after transduction of the T1-IM-R cell line by Cas9 gene containing lentiviral particles. Out of them 12 clonal sublines (T1-IM-R/Cas9) demonstrated higher levels of Cas9 expression. Clones T1-IM-R/Cas9-p4-E9 and T1-IM-R/Cas9-p4-C10 were selected for the further research.
Thus, we obtained 12 imatinib resistant GIST cell line T1-IM-R clones expressing Cas9 endonuclease. T1-IM-R/Cas9-p4-E9 and T1-IM-R/Cas9-p4-C10 clones might be used as the in vitro models for the further investigation of the effects of FGF/FGFR autocrine loop on the GIST imatinib sensitivity.
Research Laboratory \"Biomarker\", Institute of Fundamental Medicine and Biology, Kazan Federal University.
This paper has been supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030) (VS, DF, IL, DR, RK) and Russian Science Foundation (RSF) 20-15-00001 (PD, SB).
All authors have declared no conflicts of interest.