Study On Anti-Melanoma Mechanism Of Toxoplasma GRA17 Gene Knockout Strain

Background and significanceAt present,prevention and treatment of Chinese cancer is facing severe challenges.The incidence of malignant tumors has increased significantly from 1989 to 2018,especially in the past10 years.Among them,the incidences of tumors are also increasing in young and adults.Moreover,cardiovascular and cerebrovascular diseases of tumors have always ranked the top two mortality rates of various diseases.Therefore,these give us the stimulation that anti-tumor research is becoming urgent.In recent years,major breakthroughs have been made in the research of tumor immunology,and the anti-tumor capabilities of oncolytic viruses and bacteria have been continuously recognized.Thetherapeutic effects been tested in clinical trials of various types of tumors.Currently,the FDA has approved the oncolytic virus therapy T-vec and six monoclonal antibodies targeting PD-1,PD-L1 and CTLA-4 for tumor treatment.The epidemiological study found that Toxoplasma gondii is closely related to the occurrence and development of tumors.Serum antibody analysis of the population has confirmed that serum antibodies against T.gondii have antitumor effects.There are also reports in the literature,which revealed that the single effector molecule GRA15II of T.gondii was able to inhibite tumor proliferation,invasion and metastasis.Recent studies have found that T.gondii nonreplicating avirulent uracil auxotroph vaccine strain(cps),an uracil that lacks carbamoyl phosphate synthase II and blocks the pyrimidine synthesis pathway,which can effectively inhibit the progress of a variety of solid tumors,suggesting that genetic modification of T.gondii strains may be used for anti-tumor,but also it will be a feasible method.Therefore,we used the CRISPR-Cas9 system to transform the GRA17 gene knockout strain of T.gondii,and explored its antitumor effect and studied its specific mechanism through in vitro and in vivo experiments.This study intends to analyze the specific mechanism of immune reactivation of GRA17 gene knockout strains in mouse tumor models through a large number of immunological experiments.Methods(1)Investigate the role ofΔGRA17 strain by constructing a mouse tumor model and draw a survival curve through statistical analysis of tumor size in animal experiments followed byΔGRA17 immunotherapy(2)Verification of the administration mode,biological safety,and immunity ofΔGRA17 strain by using animal experiments.(3)Isolation of tumor lymphocytes by flow cytometry to explore the specific immune mechanism followed byΔGRA17 immunotherapy.(4)To further analyze of the immune mechanism ofΔGRA17 through the constructiion of a mouse bilateral tumor model.(5)RT-qPCR was used to verify the expression of PD-L1 followed byΔGRA17 immunotherapy.(6)Investigate the anti-tumor effect by constructing a mouse tumor model as well as,combination withΔGRA17 and immune checkpoint therapy.Then,the specific immune mechanism of combined therapy wasanalyzed by flow cytometry.(7)To investigate the specific immune mechanism of combined therapy and thus to analyzed whether it depends on the participation of immune cells,such as NK,CD4~+,and CD8~+by blocking in vivo in mice.Results(1)Anti-tumor treatment withΔGRA17 strain significantly slowed tumor growth and prolonged mouse survival time.(2)Animal experiments show that intra-tumorΔGRA17 injection has significant anti-tumor effects,while intravenous and intraperitoneal injections have no obvious anti-tumor effects;Latent infection will not affect the anti-tumor effects ofΔGRA17 strain;inactivatedΔGRA17 has no antitumor effect;ΔGRA17 has no antitumor effect in immunodeficient mice;ΔGRA17 is biologically safe.(3)ΔGRA17 treatment resulted in significant changes in the tumor microenvironment.CD11b~+,NK,and CD8~+T immune cells were rapidly recruited to the tumor site,and the secretion of effector molecules were also increased,such as IFN-γand Granzyme B.(4)Bilateral tumor model showed thatΔGRA17 treatment has systemic immunity and could recruit lymphocytes in systemic tumors.(5)ΔGRA17 treatment caused high expression of PD-L1 in tumor sites.(6)The combination therapy significantly slowed tumor growth and prolonged the survival time of mice,the tumor microenvironment was also rapidly changed,and immune effector cells were quickly recruited and exercised anti-tumor effects.(7)In the early stage of combined therapy,NK and CD8~+T cells were indispensable.Blocking NK and CD8~+cells would make the therapeutic effect ofΔGRA17 disappear.CD8~+T is necessary for treatment in advanced stages.ConclusionsOur results showed that the knockoutΔGRA17 strain has significant antitumor effects.TheΔGRA17 strain is a non-toxic,biologically safe strain,and its antitumor effect is mainly due to the immune stimulation caused by active invasion of cells.The treatment ofΔGRA17 can significantly change the tumor microenvironment,so that immune cells such as CD11b~+,NK,and CD8~+can be quickly recruited to the tumor site,and immune effectors such as IFN-γand Granzyme B are also enhanced.At the same time,PD-L1 was highly expressed followed byΔGRA17 treatment.The mouse bilateral tumor model also stated thatΔGRA17 treatment was systemic immunity.Based on these findingss,we use a combination therapy withΔGRA17 and PD-L1 blockade.The results show that the combination therapy has a significant antitumor effect and prolongs the survival time of mice,and the tumor microenvironment is also changed accordingly.Meanwhile,we found that in the early stage of treatment,the combination therapy relies on NK and CD8~+T cells,and in the later stage of the combination therapy,it mainly depends on CD8~+T.These experimental results show that anti-tumor therapy withΔGRA17 will be an effective method for tumor immunotherapy.