Newcastle Disease Virus Induces Immunogenic Cell Death In Lung Cancer Cells And Its Mechanisms

Lung cancer is the fastest growing rate of morbidity and mortality,one of the most aggressive tumors in the population's health and life.At present,the conventional treatment of lung cancer is surgical resection,radiotherapy,chemotherapy and so on,but these treatments are not suitable for all patients with lung cancer.In recent years,cancer immunotherapy has been listed as one of the four most noteworthy scientific fields.Compared with the previous methods of killing cancer itself,the target of cancer immunotherapy is the autoimmune system,which is used to regulate the body's immune system function to resist the tumor.For example,CTLA-4,PD-1 and PD-L1,the targets of the T cell activation pathway,have been designed with remarkable clinical data.Oncolytic virus immunotherapy is a new method of cancer immunotherapy.Oncolytic virus infection can cause cancer cell lysis and death.At the same time,it can express specific tumor-activating protein and stimulate systemic anti-tumor immune response.Oncolytic virus is a kind of tumor-killing virus with replication ability.It has been reported that many oncolytic viruses induce Immunogenic cell death,such as oncolytic Newcastle disease virus(NDV),oncolytic herpesvirus 1,oncolytic parvovirus H1 and so on.This laid a theoretical foundation for oncolytic virus combined with immunotherapy.This study aims to investigate whether NDV can induce immunogenic cell death(ICD)in lung cancer cells,and whether apoptosis or autophagy play a role in NDV-triggered ICD.To this end,we examined cell surface expression of calreticulin(CRT)on NDV-infected lung cancer cells and measured ICD determinants,high mobility group box 1(HMGB1),heat shock protein 70/90(HSP70/90)and ATP in supernatants following viral infection.Flow cytometry analysis of lung cancer cells infected with NDV by anti-CRT antibody and PI double staining showed that an increase in the number of viable(propidium iodide-negative)cells,suggesting the induction of CRT exposure upon NDV infection.In addition,confocal and Western blot analysis showed that CRT accumulated on the surface of lung cancer cells infected with NDV,indicating the translocation of CRT to the cell membrane upon NDV infection.By examining the concentrated supernatants of NDV-infected cells,we further demonstrated that NDV infected lung cancer cells can induce the release of HMGB1 and HSP70/90.In addition,pre-treatment with either the pan-caspase inhibitor z-VAD-FMK or the necrosis inhibitor Necrostain-1,had no impact on NDV-induced release of ICD determinants in lung cancer cells.In contrast,the release of CRT and HMGB1 induced by NDV was significantly inhibited in lung cancer cells with autophagy-related gene deletion.In a lung cancer xenograft model,the concentration supernatant of lung cancer cells infected with NDV and NDV virus could significantly inhibit the growth of lung cancer in mice.In conclusion,these results suggest that NDV is an effective ICD inducer and autophagy plays a key role in inducing ICD in lung cancer cells.Methods:1.Annexin V/PI double staining of infected and mock-infected NDV lung cancer cells,positive rates of staining was analyzed by flow cytometry.Western blot analysis of related apoptotic proteins.2.NDV infected lung cancer cells,cell supernatants were collected at different time points,the release of HMGB1 was detected by ELISA.CRT on membrane was detected by flow cytometry.Membrane proteins were extracted and the changes of CRT on membrane were detected by Western blot.3.The supernatant and lysates of lung cancer cells infected with NDV were collected at different time points.The supernatant was concentrated and the expression and release of HMGB1,HSP70 and HSP90 were detected by Western blot.4.Lung cancer cells were infected with NDV at different time,translocation of calreticulin(CRT)was assessed by immunofluorescence staining.The ICD inducer,mitoxantrine(MTX),was used as a positive control.DAPI was used for nuclear staining.5.Apoptotic inhibitor Z-VAD-FMK,necrosis inhibitor Nec-1(Necrostain-1),autophagy inducer Rapamycin and BEZ235 were used to treat lung cancer cells infected with NDV CRT on the membrane was detected by flow cytometry.6.Analysis the release of CRT,HMGB1 and HSP70/90 in lung cancer cells knocking down LC3 and ATG5.7.Establishing a subcutaneous tumor model of lung cancer in mice.Intratumorally injecting with NDV/FMW,the concentrated supernatants or PBS and measuring the tumor volume.Results:1.NDV induced lung cancer cells death.2.NDV induced immunogenic cell death in lung cancer cells.3.Autophagy affected NDV-induced immunogenic cell death in lung cancer cells.4.Immunogenic cell death induced by NDV in lung cancer cells displayed anti-tumor effects.Conclusion:1.NDV is an effective ICD inducer for lung cancer cells.2.Autophagy is involved in the process of NDV-induced immune cell death in lung cancer cells.