Study On The Inhibitory Effect And Mechanism Of Orf Virus On Tumor

Neoplastic disease is a major public health problem in the world.It is not only an important disease threatening human health,but also causes great harm to animal health.In most countries around the world,tumor has been ranked as the first cause of death in dogs and cats.Dogs spontaneously formed tumors and most human malignant tumors(such as lung cancer,breast cancer,osteosarcoma melanoma)have many similar characteristics in terms of tumor pathologic morphology,tumor genetics and response to drug therapy.The study of human tumorigenesis mechanism and prevention and treatment technology based on animal model has been paid more and more attention.Therefore,it is of great significance to carry out research on anti-tumor drug screening and related mechanism in pet clinic and medical clinic.As a new type of anti-tumor protocol,the oncolytic virus(OV)has been widely concerned.Traditional view believes that OV can induce tumor apoptosis,but the theory of"immune resting"state during apoptosis contradicts the phenomenon that OV can trigger relatively strong anti-tumor inflammatory response,which is an unelucidated scientific problem in this field.In this study,Orf virus(ORFV)with oncolytic properties was used as the research object to investigate the antitumor mechanism of ORFV.In this study,a tumor-bearing mouse model was constructed and ORFV was used for intratumoral therapy.It was found that ORFV could significantly inhibit tumor growth.Frozen slices of tumor tissue showed that ORFV intratumoral treatment could significantly improve the level of necrotic tumor cells,the expression level of pro-inflammatory factors,and the transcription level of pyroptotic genes.Moreover,ORFV infection could induce tumor cells with plasma membrane swelling,release of inflammatory contents such as LDH/HMGB1,Annexin V/7-AAD double staining positive and other pyroptotic markers.The above results indicate that ORFV can inhibit tumor growth and induce pro-inflammatory pyrosis in tumor tissues and cells.Further studies showed that ORFV could activate the caspase-3/GSDME signaling pathway after infecting tumor cells,tumor tissues of tumor-bearing mice,and clinical tumor samples.The knockout of Gsdme in tumor induced the transformation of tumor cells from pyroptosis to apoptosis after ORFV infection,which proved that GSDME was the key factor of ORFV triggering pyroptosis of tumor cells.It has been reported that GSDME is poorly expressed or even not expressed in most tumor cells.In order to further explore the killing and inhibiting effect of ORFV on this kind of tumor,tumor cells with low expression of GSDME in mouse breast cancer cells(4T1),mouse melanoma cells(B16),and human non-small cell lung cancer cells(A549)were selected as the research objects to conduct oncolytic study of ORFV.The results showed that ORFV could also trigger pyroptosis of tumor cells with low GSDME expression.Detection of ORFV-infected tumor cells showed that ORFV did not significantly improve the level of Gsdme transcription in the cells.However,early infection with ORFV could significantly upregulate the protein level of GSDME.Subsequently,it was found that ORFV was able to pre-stabilize the GSDME protein by reducing the ubiquitination level of GSDME during the initial stage of infection,and then trigger GSDME cleavage,activation,and cell pyroptosis during its replication process.Subsequently,this study further explored the impact of ORFV-triggered pyroptosis of tumor cells on the tumor immune microenvironment.ORFV was found to recruit tumor specific CD8~+T cells and cytotoxic T lymphocytes into the tumor.Knockout of tumor cell Gsdme can significantly inhibit ORFV induced apoptosis and lymphocyte infiltration.It was subsequently found that the ability of ORFV to inhibit tumor was also weakened when CD8~+T cells were functionally blocked in mice.These results suggested that ORFV could activate functional T cells to perform tumor killing by inducing GSDME mediated tumor cell pyroptosis.Immunohistochemical results showed that ORFV could induce PD-1 positive T cells to infiltrate into the interior of the tumor.It is suggested that ORFV could transform immune"cold"tumors into"hot"tumors,thereby improving the tumor immune microenvironment.Subsequently,immune checkpoint blocking therapy combined with ORFV had synergistic action,and it was found that immune checkpoint blockade could significantly enhance the anti-tumor effect of ORFV and prolong the survival time of mice.Moreover,the addition of Etoposide,a chemotherapeutic agent with the ability to induce cell pyroptosis,could further enhance the tumor inhibition effect.The above results indicate that ORFV combined with immune checkpoint blocking therapy and chemotherapeutic therapy has a stronger therapeutic effect on tumors.Virulence gene deletion of ORFV can significantly reduce its potential pathogenicity and improve its biosafety.This study further constructed clinically acceptable recombinant ORFV strains ORFV△120 and ORFV△120-121 by performing virulence gene deletion on wild-type ORFV,and their targeting and lethality were visually detected.The results showed that recombinant ORFV can trigger pyroptosis of tumor cells in vivo and in vitro.Among them,the recombinant ORFV,which is systematically delivered through veins,can target and enrich in lung metastatic tumors and inhibit the development of metastatic lesions.In summary,this study demonstrated that ORFV can inhibit tumor growth and trigger inflammatory cell death in tumor tissues and cells,which is mainly characterized by cell membrane rupture,and is considered as pyroptosis.It was also found that ORFV up-regulated the protein expression of GSDME in low-level GSDME tumor cells by inhibiting the ubiquitination of GSDME,and then triggered GSDME dependent pyroptosis of tumor cells.At the same time,GSDME is also an important factor for ORFV to recruit and activate killer T lymphocytes and activate anti-tumor immunity.ORFV improved the tumor immune microenvironment,made the immune"cold"tumor become"hot",and combined with immune checkpoint blockade or chemical therapy produced a stronger anti-tumor effect.The in vivo test also further proved the targeting of ORFV.ORFV can target and enrich in tumor tissues,induce pyroptosis in tumor tissues,and inhibit tumor growth and metastasis.The research results not only help to reveal the anti-tumor mechanism of oncolytic virus,but also lay a theoretical foundation for the development of anti-tumor drugs and universal tumor vaccines,which are cutting-edge biotechnology.