Screening Of Oncolytic Viruses And Anti-tumor Mechanism Of NDV/HK84 In The Treatment Of Primary Hepatocellular Carcinoma

Oncolytic virus(OV)has been used in tumor therapy for a hundred years.With the rapid development of tumor immunotherapy and gene editing technology in recent years,OV becomes a hotspot in tumor therapy at present and in the coming decades.Althogh the recombinant herpes simplex virus T-VEC has been approved for the treatment of advanced melanoma in 2015,OV has a slow progress in the treatment of solid tumors,especially advanced tumors with high incidence and short survival time,such as primary hepatocellular carcinoma(HCC).The lack of oncolytic virus strains with high efficiency and low virulence is the reason for it,which is the bottleneck of relevant work.As an oncolytic virus,Newcastle disease virus(NDV)could be used in the treatment of leukemia,which was reported in the New England Journal of Medicine in 1964.Over the next60 years,gene editing techniques have matured,and modified NDV strains have been shown to be effective and safe in cancer treatment both in vivo and in vitro.However,the clinical trials on NDV were mostly terminated in phase I/II due to their failure to prolong the survival period of patients.This reminds us that searching for suitable natural strains of oncolytic virus is the top priority of current research.Based on the above considerations,we initially screened 10 NDV strains from the database of hundreds of natural NDV strains identified by the Joint Institute of Virology of Shantou University and Hong Kong University,in order to find out potential natural NDV strains that have specifically oncolytic effect on HCC.10 NDV strains with high-efficiency oncolytic effect were selected from the candidate strains in our study.The inhibition of NDV on tumor cells was detected by Cell Counting Kit-8(CCK-8).The results showed that NDV strains had difference on oncolytic effect at different multiplicity of infection(MOI).At high multiplicity of infection(MOI=20),the oncolytic activities of nine NDV strains exceeded 80% except DK/JX/21358/08.With the decrease of MOI,the oncolytic activities of most virus strains decreased.However,NDV/HK84 maintained good oncolytic activities at low MOI,and its oncolytic activities were more than80% at different MOI(MOI=20,2,0.2).In conclusion,NDV/HK84 has the potential of high oncolytic efficiency and has a high safety threshold due to its oncolytic effect at low viral titers.On this basis,we focused on NDV/HK84 to investigate its effect on HCC cells.We found that compared with cisplatin and NDV La Sota strain in the positive control group,NDV/HK84 infection resulted in significant cytopathic effect(CPE)in HCC cells,promoted apoptosis of cancer cells and inhibited the formation of cell clones.In wound healing experiment,NDV/HK84 reduced the migration distance and the number of migrating cells,suggesting that NDV/HK84 has inhibitory effect on migration and invasion of hepatocellular carcinoma cells.Therefore,hepatocellular carcinoma cell line SK-HEP-1-LUC was used to construct subcutaneous transplanted tumor model in nude mice.After NDV/HK84 injection into tumor-bearing mice,the data tracked by in vivo luciferase imaging system showed that subcutaneous tumors in the right flank of some nude mice(6/10)completely disappeared,and almost no intact tumor cells could be seen under the light microscope,which indicates good therapeutic effect.In vitro and in vivo experiments demonstrated the biosafety of NDV/HK84.These results indicate that NDV/HK84 strain has a highly specific inhibitory effect on liver tumor cells and does not affect the function of normal cells in mice.Finally,in order to explore the mechanism of high-efficiency oncolytic effect of NDV/HK84,we analyzed the involved signaling pathways and differential gene expressions at the transcriptome level by high-throughput RNA sequencing,and verified them by real-time quantitative PCR.We found that type I interferon(IFN)pathway-related genes were enriched in liver cancer cells treated by NDV/HK84,and the oncolytic effect may be highly dependent on the activation of type I IFN signals.In conclusion,NDV/HK84,a new natural strain of oncolytic virus with high efficiency and low virulence that can specifically target HCC was screened out in this study,and its efficacy and safety were verified by in vitro and in vivo experiments.These results suggest that NDV/HK84 may be a “new star” specifically targeting HCC for a long time in the future,though further researches on NDV/HK84 are needed to be done.