The Effect And Mechanism Of Immune Checkpoint VISTA On Choriocarcinoma Xenografts In Nude Mice

BACKGROUNDGestational trophoblastic neoplasms originate from trophoblastic cells including invasive mole,choriocarcinoma and other rare types,which has different features compared to somatic cell originated tumors.Multi-agent chemotherapy is the foundation of choriocarcinoma treatment,such as EMA-CO,FAEV regimens.But a certain part of patients suffered from chemoresistance and relapse,which reduce their 5-year survival rate.Since the first approval of CTLA-4 inhibitor in 2011,various immune checkpoint inhibitors have entered the guidelines of different cancer treatment,changing the landscape of malignancies treatment.Research of biopsies of normal placental and gestational trophoblastic neoplasms have found the high expression of PD-L1 in them.Further research demonstrated PD-L1 has played an important role in maintaining the immune tolerance of placenta and choriocarcinoma.Thus,applying immune checkpoint inhibitors in choriocarcinoma have achieved promising remission rate.However,some cancer patients face primary or secondary resistance to PD-1/PD-L1 inhibitors,which implies that there may be other immune escape mechanisms.Now scientists have found that there are other negative immune checkpoints participating in this process,including TIM3,B7H3 and so on.VISTA is expressed on both cancer and immune cells,functioning as immune suppressor.It can inhibit the activity of antigen presenting cells and T cells through different ways.Although VISTA is negative in most normal tissues and cancers except immune cells,it is found to be strongly expressed in placenta and gestational trophoblastic neoplasms.The function of VISTA in choriocarcinoma formation and development is unknown.Our team has demonstrated knockdown of VISTA can interfere proliferation,cloning,invasion,and migration of JEG-3 cell line in vitro.However,it is unknown how VISTA influences JEG-3 cell line in vivo.OBJECTIVEThis research aimed to investigate the influence of VISTA to JEG-3 cell line tumor formation by engrafting VISTA-knockdown and wild JEG-3 cell lines to nude mice.Detect change of relevant molecule pathways.Further explore the impact of VISTA to proliferation,invasion of JEG-3 cell line in vivo.Provide more evidence of VISTA function in choriocarcinoma.METHODS1.Dilute the VISTA-knockdown JEG-3 cell line that have been constructed by lentiviral transfection and wild-type JEG-3 cell line to different concentrations and inoculate them below the axilla of nude mice to observe the optimal concentration for tumor formation and observation.After diluting at the optimal concentration,two cell lines were respectively inoculated under the bilateral axilla of nude mice to establish subcutaneous tumor xenograft models.Growth status of the nude mice,incubation period of the engrafted tumors,the growth curves were observed;2.Inject VISTA-knockdown and wild-type JEG-3 cell lines into the tail vein of nude mice with optimal concentration,observe the growth of nude mice,and dissect the lungs of nude mice every week to observe the status and growth of lung metastases;3.The subcutaneous xenograft nude mice of each group were euthanized and then the xenografted tumors were taken out.The specimens were used for western blot and immunohistochemical staining to verify the source of the metastatic choriocarcinoma cell line,VISTA knockdown status,and detect P53,HSP90,NF?B,and ?-catenin expression.RESULTSAfter preliminary experiments,the optimal inoculation concentration of VISTA-knockdown and wild type choriocarcinoma cell line in nude mice is 2.5×107/mL.After inoculation at the optimal tumor concentration,the engrafied tumors grow rapidly under the bilateral axilla of nude mice.Tumor hemorrhage can be seen in the late stage.The growth speed of VISTA-knockdown JEG-3 cell line is slower than wild-type cell line.Injecting two types of cell lines into circulation system of nude mice through tail vein,formation of metastatic tumors in lung of VISTA-knockdown cell line has lower success rate and longer latency.After pathological anatomy,the engrafted tumor was full of heterotypic cells,hCG staining was strongly positive,and VISTA in knockdown cell line was negative.Both western blot and IHC staining demonstrated that the expression of NF?B and ?-catenin in the knockdown cell line engrafted tumor was significantly reduced,and the expression of P53 and HSP90 did not change.CONCLUSIONThis research investigated the influence of VISTA to choriocarcinoma cell line in nude mice subcutaneous xenograft and lung metastases models.It validated the result of in vitro experiment that knockdown of VISTA interferes cloning and proliferation of choriocarcinoma cell line and provided further evidence of VISTA reducing migration and invasion ability.This research also detected several related pathways that may change while VISTA changes,including NF?B and ?-catenin.