| BackgroundAs the frontier field of tumor biotherapy,oncolytic virotherapy has shown broadly prospect in recent years,and provides a new direction for immunotherapy of tumor.Oncolytic virotherapy can trigger local and systemic immune response by injecting oncolytic virus directly into the tumor,so as to tumor cells were split,tumor related antigens were released,and then tumor specific T cells were activated.A variety of oncolytic viruses have shown good safety,targeting and exciting therapeutic effects in preclinical and clinical trials.Talimogene laherparepvec(t-vec),a oncolytic virus based on herpes simplex virus type ?,has completed phase ? clinical trials and has been approved by the U.S.Food and Drug Administration(FDA)for the treatment of unresectable melanoma recurrence after initial surgery.To understand the changes of immune system and its influence on tumor microenvironment caused by oncolytic virus treatment is helpful to improve the therapeutic effect of oncolytic virus immunotherapy in the future.Therefore,in this study,we used oHSV2,an oncolytic virus based on herpes simplex virus type ?,to investigate its direct oncolytic effect and anti-tumor immune response in the tumor bearing mice with complete immunity and immune deficiency(CT26 cell line of colorectal cancer).ObjectiveThe main purpose of this study is to investigate the direct antitumor effect of oHSV2 in tumor bearing(CT26 cell line of colorectal cancer)model and its antitumor immune response.By establishing a double-sided tumor bearing model of mice with complete immunity and immune deficiency,detecting the phenotype of lymphocytes in the spleen and their killing effect on tumor cells,analyzing the pathological characteristics of tumor microenvironment and the expression of immune genes in the microenvironment of bilateral tumors,we can provide evidence for that the oncolytic virus oHSV2 can stimulate specific and lasting anti-tumor immune response and change the immune state of tumor microenvironment.Methods(1)CCK-8 proliferation assay was used to detect the antitumor activity of oHSV2 on mouse colon cancer cell line CT26 in vitro.(2)In order to investigate the therapeutic effect of oHSV2 in vivo and its anti-tumor immune response,a unilateral and bilateral tumor bearing(CT26 cell line)model of BALB/c mice was established by subcutaneous inoculation.(3)The specific immune response induced by oHSV2 was studied by the rechallenge and immunological killing experiments.(4)To explore the role of innate immune response in the treatment of tumor by oHSV2,bilateral tumor bearing model of immunodeficient mice was established by subcutaneous inoculation.(5)Flow cytometry was used to detect the phenotype of spleen lymphocytes in each treatment group,and the pathological characteristics of bilateral tumors and the immune related genes expression profile were analyzed to explore the changes of whole immunologic state of the body and local tumor microenvironment after oHSV2 treatment.Results(1)oHSV2 has significant antitumor effect on CT26 cell line of mice colon cancer,which is time-and dose-dependent.(2)The antitumor effect of oHSV2 was observed in unilateral and bilateral colon cancer models.oHSV2 treatment can effectively inhibit the growth of unilateral and bilateral tumors,prolong the survival time of mice,and has no obvious side effects.(3)oHSV2 treatment can effectively prevent tumor formation after rechallenge with the same tumor cell line.For bilateral tumor bearing models,oHSV2 treatment in a single focus can effectively inhibit the growth of bilateral tumors.(4)The nonspecific immune response caused by NK cells,that is,the nonspecific recognition and killing effect on target cells(tumor cells infected by virus),also plays a certain role in inhibiting tumor growth in the process of oHSV2 treatment of tumor.(5)It can effectively reduce the level of immunosuppressive cells(such as Tregs and MDSCs)in the peripheral immune system,which is conducive to breaking the immune tolerance;at the same time,it can improve the level of positive immune cells(such as NK cells,DCs and CD8+T cells)in the peripheral immune system,which is conducive to strengthening the non-specific and specific anti-tumor immune response;following the specific immune response,the body has formed an immune memory,which is conducive to effectively prevent tumor metastasis and recurrence.(6)oHSV2 treatment changed the immune status of tumor microenvironment.This effect is not limited to the local tissues under treatment,but will cause similar changes to the microenvironment of the same tumor in other parts at the same time.ConclusionsThe first anti-tumor effect played by oncolytic virus oHSV2 is to kill tumor cells directly.On the basis of the first effect,tumor associated antigen(TAAs)is released along with tumor cell lysis,so as to show the second effect of oHSV2 treatment,that is,to cause innate immune response and specific immune response.Following the specific immune response,the body has formed immune memory,which is conducive to effectively prevent tumor metastasis and recurrence.The application of oHSV2 can reconstruct the immune state of mouse spleen and tumor microenvironment(including tumors not directly treated by virus),suggesting that the local treatment mode of oHSV2 can be transformed into a systemic treatment mode.Therefore,oHSV2 has a good clinical application potential. |
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