The Feasibility Evaluation And Oncolytic Mechanism Of Live Attenuated Swine Pseudorabies Virus As A Novel Oncolytic Virus

Cancer is the main killer of human health,causing high mortality,posing a serious threat to human health.The most recent data show that thirty-four people in world are diagnosed with cancer and seventeen people are died of cancer every minute.In recent years,with the rise of immunotherapy,its remarkable therapeutic activity and minimal side effects bring the dawn of tumor treatment.Among these,oncolytic viral therapy has enough safety and strongly therapeutic activity,which brings a novel insight to the research of antitumor drugs.Studies have demonstrated that the types and the clinical research of oncolytic virus do not inadequately prepare to meet the challenge of tumor heterogeneity and tumor complexity.Thus,it is necessary to develop more kinds of novel oncolytic virus and explore the oncolytic potential and mechanism,which provide scientific evidence for the oncolytic treatment and more candidate oncolytic agents.In this study,firstly,we cultured and isolated vaccine strains from 17 kinds of veterinary or avian live attenuated vaccines in market.The cytopathic effects and cell viability were observed and performed,after 5 kinds of cancer cells have been infected with attenuated viruses for 48 hours.The results confirmed pseudorabies virus live attenuated vaccine strain induced strong cytopathic effects in 5 kinds of cancer cells,and it didn’t induce the CPEs in human normal cells.In addition,most of human beings had not or low special antibodies against PRV.Thus,PRV-LAV was a potential candidate of oncolytic virus.Secondly,the safety of oncolytic virus is the most evaluation metric.PRV-LAV exhibited the an excellent safety profile in the tail-vein injection rat and mouse model at the dose of 1×108pfu by the analysis of body weight,blood hemogram and blood biochemistry.In addition,PRV-LAV also shown enough safety in the mouse nasal-injection model and rat intracranial injection model at the dose of 1×107 pfu.Thirdly,to perform the tumor-killing activity of PRV-LAV,the percent of viable cells were performed,after 47 kinds of cancer cell lines and 3 kinds of the normal cell lines were infected with different MOIs of PRV-LAV.These results confirmed PRV-LAV had the high tumor tropism,can effectively lyse the cancer cells,and no effects on normal cells.In the evaluation of therapeutic activity of PRV-LAV in vivo,PRV-LAV exhibited the excellent therapeutic activity in GBM,HepG2 and A549 bearing nude mouse models,and can absolutely clear the 100mm3 GBM and HepG2 tumor.PRV-LAV can also clear the 200mm3 or 400mm3 HepG2 tumor.PRV-LAV can clear 100mm3 tumor tissues in the mouse liver cancer Hepa1-6,kidney cancer Renca and colon cancer CT26 bearing mouse models,and prolong the survival time.PRVLAV showed more strongly therapeutic activity compared with sorafenib and antiPDL1 antibody,and prolong the survival time.Fourthly,in the research of the oncolytic mechanism of PRV-LAV,we found the proliferation of PRV-LAV was associated with EGFR//PI3K/AKT/mTOR signaling pathway,after we high-thoughoutly screened the inhibitors against the proliferation of PRV in PK15 and GBM cells from kinase inhibitor library.EGFR inhibitor afatinib also can suppress the proliferation of PRVLAV.PRC-LAV can interact with EGFR to boost the proliferation of PRV-LAV in cancer cells.Over-expression of EGFR can increase the proliferation of PRV-LAV.Knock-down of EGFR can suppress PRV-LAV.EGFR commonly overexpressed in cancer cells,and normal cells had no or low expression,which led to the high tropism of PRV-LAV.Fifthly,the effective tumor-killing activity of PRV-LAV was associated with multiply death pathways induced by PRV-LAV.PRV-LAV can induce cancer cells apoptosis via ER stress pathway.It also can induce the splice of GSDME to lead cancer cells pyroptosis.The infection of PRV-LAV can specially downregulate the expression of GPX4 and SLC3A2/SLC7A11,which led to the imbalance between oxidation and reduction system and ferroptosis.This interesting oncolytic mechanism was never reported.Sixthly,PRV-LAV can activate the tumor microenviroment and increase the number of T cells,B cells,NK cells and macrophages.Combination with anti-PD1 antibody can boost the therapeutic activity of PRV-LAV.The multimechanism of PRV-LAV had huge potential to form a novel anti-tumor drug.Seventhly,in order to achieve precise regulation of oncolytic treatment,it was necessary to screen the small molecular drugs to inhibit the proliferation of PRV-LAV.Adefovir dipivoxil exhibited the strong inhibitory activity against PRV in vitro and in vivo.lastly,the cancer cell sensitive spectrums of PRV-LAV and OVH exhibited partial complementarity.Combination with PRV-LAV can enlarge the therapeutic range of oncolytic virus,and benefit more patients.In this study,we prove that PRV-LAV is a novel safe and effective oncolytic virus.It interacts with the high expression of EGFR in cancer cells,and it can induce cancer cell death via apoptosis,pyroptosis and ferroptosis pathways,which boost the tumorkilling activity of PRV-LAV.These studies form a basis for the development and the personalized precision of oncolytic virus.