Establishment And Characterization Of An Immortalized Human Gastrointestinal Stromal Tumor Cell Culture Model

Objective: Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the digestive tract that originate from gastrointestinal pacemaker cells(interstitial cells of Cajal,ICC)or related stem cells.GIST cell lines are essential tools for studying the molecular pathogenesis and drug resistance mechanisms of the disease,as well as for developing neoadjuvant therapeutic regimens.Foreign GIST cell lines are widely used as cell models for in vitro experimental studies because of their comprehensive information and clear availability of mutation site data.The majority of domestic GIST cell lines,however,have not been widely used because they were established with unclear backgrounds and lack strict quality control.This study aimed to establish a primary culture model of immortalized human GIST cells and to characterize them entirely using primary GIST cells as controls.Methods:1.GIST cells were isolated from surgically resected gastric stromal tumor tissue,and primary culture and identification were performed.2.Primary GIST cells were transfected with Simian Virus 40 Large T Antigen to establish immortalized human GIST cell culture model.The culture conditions were optimized during the cell culture process.3.Immunoblotting(western blot)and quantitative real-time polymerase chain reaction(q RT-PCR)were used to detect the expression of C-KIT,SV40,and P53 in immortalized GIST cells and primary GIST cells for the identification of immortalized cells.4.C-KIT and PDGFRα mutations were detected in immortalized GIST cells and primary GIST cells using a gene mutation detection kit(Fluorescence PCR Capillary Electrophoresis Sequencing Analysis).5.Immortalized GIST cells and primary GIST cells were identified by Short Tandem Repeat(STR).6.Karyotype analysis was conducted on immortalized GIST cells.7.Immortalized GIST cells were characterized in terms of cell cycle,cell proliferation,drug sensitivity,and other aspects with primary GIST cells as controls.Results:1.Primary GIST cells showed spindle morphology and fused growth with the same positive markers as the GIST pathological tissue.The primary cells have shown slow proliferation and enlarged deformation with the increase of passaged culture.2.Compared with primary GIST cells,the spindle morphology of immortalized GIST cells became shorter and changed to neuronal cell-like irregular radioactive growth.The characteristics of fusion growth still existed.The immortalized GIST cells have been cultured in vitro for six months,passed through more than 35 generations,and maintain strong proliferation ability.3.Western blot results showed no significant difference in CD117 expression between immortalized GIST cells and primary GIST cells.Western blot and q RT-PCR results showed that SV40 was not expressed in primary GIST cells but was highly expressed in immortalized GIST cells;The expression of P53 in primary GIST cells was significantly higher than in immortalized GIST cells.4.Both immortalized and primary GIST cells showed C-KIT 11 mutation.5.The STR typing results of immortalized GIST cells and primary GIST cells were consistent and sound,without multiple alleles and human cross-contamination.6.Immortalized GIST cells had a clonal deletion of the long arm of chromosome 6,deletion of the short arm of chromosome 12,deletion of chromosome 14 and deletion of chromosome 22.7.Immortalized GIST cells showed a significantly higher proliferation index and increased proliferation rate compared with primary GIST cells.Immortalized GIST cells were sensitive to imatinib(IM).Conclusion:1.This study successfully isolated and identified primary human GIST cells.2.This study successfully established and characterised a high-purity immortalized human GIST culture model.3.The immortalized GIST cells can be cultured stably for a long time,which express the signature proteins of GIST cells and maintain the biological properties and genomic characteristics of normal primary GIST cells.