Therapeutic Effect And Mechanism Of Engineered Salmonella VNP20009-M On Osteosarcoma

Background:Osteosarcoma(OS)is a common malignant bone tumor.Across different age groups,It has the highest incidence of bone tumors among children and adolescents.Once osteosarcoma has progressed to distal metastasis,the 5-year survival rate is only 20-30%.Although the treatment of osteosarcoma has made great progress,the current clinical treatment is still dominated by the use of a large repertoire of chemotherapy drugs.However,the severe adverse reactions caused by these chemo drugs are daunting to many clinicians.However,the small dose of chemotherapy can not achieve the therapeutic effect.Therefore,finding new treatment alternatives for osteosarcoma has always been the focus of the field.As a new type of tumor therapy with relatively few side effects and high efficiency of targeting tumors,bacteriotherapy has attracted more and more attention in recent years.Salmonella typhimurium,a gram-negative facultative anaerobe that can colonize the intestines of humans and animals and multiply in host cells,is one of the most widely studied bacteria in the field of targeting tumors.Among them,VNP20009 is an attenuated Salmonella modified by genetic engineering.The pur I deletion creates a requirement for an external source of adenine,thus limiting growth to areas that have substantial cell turnover,death,and cellular debris,while msb B gene deletion reducesthe endotoxin production,which creates excellent safety profiles.As a result,VNP20009 can only induce mild tumor necrosis factor(TNF)response after delivered to body,which helps avoid the serious inflammatory reaction.VNP20009 showed excellent efficacy in solid tumor models in mice,including melanoma,lung and colon cancers.Phase I of clinical trial conducted in the US proved its safety,but did not show good therapeutic effect or colonization within the tumors.Therefore,genetic engineering on top of the VNP20009 for obtaining a better therapeutic effect has been a focal point in this field.Unlike normal non-cancerous cells,most tumor cells are dependent on methionine for growth.Previous attempts of treating tumors with dietary restriction of methionine intake or injection of recombinant methioninase have not yielded desirable efficacy.Long-term dietary restriction of methionine can cause malnutrition.Mammals do not express methioninase,injection of exogenous methionidase can elicit body's strong immune response.We created the new strain VNP20009-M by transplanting methionidase gene into VNP20009 through genetic engineering.VNP20009-M maintained its excellent tumor targeting characteristics and can specifically hydrolyze methionine in tumors.This study will further explore the therapeutic effect of VNP20009-M on osteosarcoma.Aims:To investigate the therapeutic effect and the underlying mechanisms of genetically engineered bacteria VNP20009-M on osteosarcoma.Methods:1.The attenuated Salmonella typhimurium,VNP20009,was transfected with the plasmid PSV-SPORT or PSV-SPORT-L-methioninase by electroporation to construct genetically engineered strain VNP20009-V(the strain carrying empty plasmid)and VNP20009-M(the strain carrying the methioninase gene).2.Recombinant Salmonella VNP20009-M was co-cultured with a human osteosarcoma cell MNNG-HOS to study its effect on osteosarcoma cell growth;3.Using over-expression of the methioninase gene in three different human osteosarcoma cell lines,MNNG-HOS,U2 OS,Sao S-2,we studied the effects on proliferation and migration of these cells.4.We evaluated the effect of VNP20009-M on the subcutaneous xenografts derived from MNNG-HOS in the nude mice by intratumorally injecting different dosages of VNP20009-M.5.We evaluated the effect of VNP20009-M on the osteosarcoma metastatic model.Results:1.Transfection of the methionase expressing plasmid through PCR showed that the methioninase gene was only found in the target strain VNP20009-M that also displayed high methioninase activity.The recombinant Salmonella,VNP20009-M,was successfully constructed.2.VNP20009-M,after co-cultured with osteosarcoma cell MNNG-HOS,significantly induced tumor cell apoptosis;3.Compared with the control group,overexpression of the L-methioninase gene in the osteosarcoma cells significantly inhibited proliferation and migration.4.VNP20009-M treatment significantly inhibited the growth of subcutaneous tumors in nude mice,but did not change the survival status of the mice.5.VNP20009-M treatment significantly inhibited the occurrence of osteosarcoma bone metastasis and improved the survival of mice.Conclusion:In summary,VNP20009-M can inhibit cell proliferation and migration and induce apoptosis in vitro.It can also suppress the growth of osteosarcoma in vivo,block the bone metastasis,and improve the survival of the tumor-bearing mice.VNP2009-M can be a novel and highly effective drug candidate as a new treatment modality for osteosarcoma in the clinic.