Enhancement Of Anti-liver Cancer Effect And Mechanism Of Recombinant Oncolytic Influenza Virus Combined With PD-1 Antibody

Objective Liver cancer is the most common cause of cancer death in the world,one of the five cancers with the highest mortality rates.Despite various treatment methods,the therapeutic effect is not ideal.In recent years,immune checkpoint inhibitors have made great breakthroughs in the field of tumor therapy.In contrast to PD-1 inhibitors,oncolytic viruses can specifically infect tumor cells,replicate in large numbers within tumor cells,and eventually kill tumor cells without damaging normal tissue.In addition to its direct killing effect,oncolytic viruses can further present antigens to stimulate local or systemic anti-tumor immunity and inhibit tumor progression.In this study,the combined effect of recombinant oncolytic virus del NS1-GM-CSF and immune checkpoint inhibitor PD-1 on hepatocellular carcinoma and its molecular mechanism of killing tumor were investigated.Methods 1.The recombinant oncolytic influenza virus del NS1-GM-CSF was comprehensively identified,and the stable passage and virus titer of recombinant oncolytic influenza virus were determined by hemagglutination test and TCID50.The morphology,size and distribution of virus particles were observed by electron microscopy.2.Establish H22 liver cancer tumor bearing nude mouse model to explore the replication ability and safety of oncolytic virus;The protein expression of exogenous gene GM-CSF was detected by ELISA.3.Using the H22 Balb/c mouse model,the targeting of tumor was verified by PT-q PCR and TCID₅₀.The tumor volume was measured to verify the efficacy of combination therapy in killing tumor.The contents of CD3⁺,CD4⁺,CD8⁺,CD45⁺and CD69⁺in mouse spleen cells were detected by flow cytometry.The content of CD4+and CD8+cells and the proliferation of tumor cells were detected by immunohistochemical staining of tumor tissues.HE staining was used to evaluate its safety.4.Transcriptomic sequencing of mouse tumor tissue was used to analyze the signaling pathway of its action,and Western-blot was used to verify the action pathway at the cell level and tissue level.5.The distal effect of combined treatment with del NS1-GM-CSF and PD-1 antibody was explored using H22 bilateral tumor bearing Balb/c mouse model.Results In this study,the hemagglutination titer of recombinant oncolytic virus del NS1-GM-CSF was stable in the range of 2⁸～2⁹,and the virus titer could reach 10⁷～10⁸TCID50/m L.Recombinant oncolytic virus del NS1-GM-CSF could be passed statically,and the size and sequence of recombinant plasmid were in line with expectations.The morphological structure and size distribution of del NS1-GM-CSF were consistent with the typical characteristics of influenza virus,and GM-CSF protein could be successfully expressed in vivo.In the nude mouse model of H22 hepatocellular carcinoma,it was observed that the recombinant oncolytic influenza virus was targeted and showed a time-dependent growth.In a single H22 hepatocellular carcinoma Balb/c mouse model,the combined treatment group with del NS1-GM-CSF and PD-1 antibody showed stronger inhibition of tumor growth than that with single virus or single antibody（P<0.001）,the number of spleen T cells was significantly increased after combined treatment compared with PBS group,and T lymphocytes were significantly activated,showing stronger antitumor activity compared with PBS group（P<0.001）;Compared with PBS group,the tumor tissue of mice in the combined treatment group was more enriched with T lymphocytes,the tumor showed serious necrosis and the tumor proliferation was significantly inhibited.Transcriptome data analysis showed that the JAK2-STAT3 pathway was significantly enriched in combination therapy,which was verified in Hep G2 liver cancer cells and tumor tissues.Phosphorylated JAK2 and STAT3were significantly inhibited in the combination therapy group compared with the control group.Compared with PBS group,the combined treatment of del NS1-GM-CSF and PD-1antibody could significantly kill the untreated tumor on the opposite side（P<0.01）.Histopathologic sections of each group of mice showed that recombinant oncolytic influenza virus del NS1-GM-CSF was safe.Conclusion The combined treatment of del NS1-GM-CSF and PD-1 antibody has a significant effect on the treatment of liver tumor in mice,which can activate the systemic immune response of mice and target the killing of tumor tissue,with good safety.Hope to provide a new idea for clinical immunotherapy of liver cancer.