The Establishment Of A GIST Cell Line And Its Low-sensitivity To C-KIT Pathway Inhibition

OBJECTIVE As the diagnose rate of GIST(gastrointestinal stromal tumor)keep on growing and effective treatment remains to be explored,there is an urgent require for research data on the effects of target inhibitors.However,available GIST cell model at home is barely enough.This study aims to establish a gastrointestinal stromal tumors cell model for in-depth research.METHODS Peritoneal metastatic GIST tissue from surgery was primarily cultured and sub-cultured up to Passage 20 in vitro before fully characterized.The expression of CD117 and DOG-1 were detected by immuno-cytochemistry.Chromosome karyotype along with mutations in C-KIT and PDGFRA have been analyzed.The tumorigenicity was tested by subcutaneous inoculation in BABL/c and B-NSG nude mice.Mycoplasma detection and species identification were detected by PCR.STR profiling was employed for cell identity.Plasmid contains Large-T-eGFP gene fragments was constructed.After being packaged,the lentivirus was used to infect the human-derived ESF cell,and plasmid with TK gene was also transferred into it to establish a new feeder cell line ESF-LT-TK.The growth of ESF-LT-TK and the growth-arresting effect of ganciclovir on it and the supporting effect of ESF-LT-TK for GIST was observed.Different types of medium were used to explore better in vitro culturing for GIST cells.The GIST was further transfected with LT-eGFP lentivirus for longer and easier culture.The growth inhibition rates of single and combined inhibitors for C-KIT downstream pathways on GIST were evaluated.RESULTS The cell line was consecutively passaged more than 20 times.The cell line was designated as PUMC-GIST-1.Morphologically the cells arrayed like nerve cell and showed positive expression of CD117 and DOG-1.Its chromosomes ranged between 38-48,KIT Exon 11 V559D mutation and PDGFRA Exon 12 CCG-CCA mutation were detected.No tumor mass could be palpated in BABL/c and B-NSG nude mice at the end of 3 months.The STR profile of PUMC-GIST-1 was same with that of the original tumor tissue,no mycoplasma contamination was detected.Plasmid with LargeT-eGFP has been constructed.The feeder cell line ESF-LT-TK has been established and could express Large-T,eGFP and TK stably.It has been sub-cultured over 66 passages.When the concenration of ganciclovir ranged 4-8μg/mL,ESF-LT-TK could remain alive for 4-5d after lost its proliferation ability.The best medium condition for PUMC-GIST-1 was D/F12,10%FBS and 1%ITS.PUMC-GIST-1-LargeT cells have been established which could express LargeT-eGFP continuously.Compared with untreated P24 PUMC-GIST-1,the ESF-LT-TK supporting PUMC-GIST-1 showed happier morphology and could grow faster.CCK assay showed the inhibition rate of GDC-0941 on PUMC-GIST-1 was 13.42%(5μmol/L group 0.445±0.024 vs control group 0.514±0.034,F=8.956,P<0.001).The single use of AZD-6244 could improve the proliferation ability of PUMC-GIST-1(10μmol/L AZD-6244 group 0.58±0.02 vs control group 0.56±0.03,F=9.775,P<0.001)and inhibit the growth of GIST-430(10μmol/L GDC-0941 group 1.40±0.07vs control group 1.51±0.04,F=6.613,P<0.001).The combined inhibition rate of these two inhibitors on PUMC-GIST-1 was 9.68%(5μmol/L AZD-6244+5μmol/L GDC-0941 group 0.373±0.030 vs control group 0.413±0.0125 F=3.298,P=0.007).And the inhibition rate for PUMC-GIST-1 is far behind that of GIST-430 which is 93.83%.That shows the low-sensitivity of PUMC-GIST-1 to combined C-KIT downstream pathways inhibition.The results of Q value method showed that the q value(5μmol/L AZD and 5μmol/L GDC)of PUMC-GIST-1 was 3.08 and for GIST-430 that was 3.91,both of them are higher than 1.15,which means 5μmol/LAZD and 5μmol/LGDC had synergistic inhibition effect on the growth of GISTs.CONCLUSION A GIST cell line with low sensitivity to C-KIT pathway downstream targets inhibition as well as a motalized feeder cellline ESF-LT-TK were established.With clear biological properties and gene mutation background the cell lines would provide a base for future GIST research.