The Antitumor Efficacy And Potential Functional Mechanisms Of The Vaccinia Viruse In Murine Colorectal Cancer

Background and objective:Colorectal cancer(CRC)is the third leading cause of cancer deaths around the world,with nearly two million new cases and one million fatalities in 2018.Laparoscopic surgery,chemotherapy,radiotherapy have limited impacts on the cure rate and long-term survival rate,so it is urgent to develop new promising treatment regimens.Obesity,physical inactivity,alcohol and tobacco have been identified as important risk factors of CRC.Besides,gene aberration is also an important factor of colorectal cancer.In addition to the mutations of proto-oncogenes such as APC,KRas and suppressor genes such as p53,an important feature of colorectal cancer is that it has a large proportion of microsatellite instability(MSI).Microsatellite instability leads to mutation accumulation in colorectal cancer,which makes it easier for CRC to produce neoantigens that can be recognized by the immune system,making patients with colorectal cancer positive response to immunotherapy.Oncolytic viruses have the characteristics of selectively replicating in tumor cells,lysing tumor cells directly,and inducing anti-tumor immunity.Oncolytic virus interacts with the immune system of the host by different pathways to alter tumor microenvironment,facilitate infiltration of immune cells and enhance tumor-specific immune response.Activation of anti-tumor immunity is a major aspect of function mechanism of oncolytic virus,and also a significant advantage of oncolytic therapy over surgery,chemotherapy and radiotherapy.Anti-tumor immunity induced by oncolytic virus not only target the virus treated-tumor,but also has effects on distant metastatic tumor,which can effectively prevent tumor recurrence.Three oncolytic viruses to date have been approved for clinical use for treatment of cancer patients,including Rigvir for treating different kinds of tumors including melanoma,Oncorine(H101)for advanced head and neck tumors,and talimogene laherparepvec(T-Vec)for melanoma.Vaccinia virus,as a promising agent for the treatment of tumors,has been studied extensively and made important progress in the past 20 years.A variety of genetically engineered oncolytic vaccinia viruses have been successively entered into clinical trials,among which JX-594(pex-vec),a potent drug for treating advanced liver cancer,has entered phase?clinical trials.Unfortunately there was no significant benefit compared to standard comprehensive care treatment.It is needed to develop next generation of oncolytic vaccinia virus.Thymidine kinase(TK)gene is considered to be associated with various neoplastic and TK gene is essential for virus replication in normal resting cells,but not necessary in mitotic cells.Therefore,deletion of TK gene can make the virus selectively replicate in tumor cells.Vaccinia virus N1L protein is an apoptosis inhibitor and deletion of N1L gene can reduce the virulence of vaccinia virus.Studies on a disease model of vaccinia virus infection showed that absence of N1L did not affect the proportion of CD4~+T and CD8~+T cells infiltrating to lung inflammation sites,nor did it affect the CTL activity.Vaccinia virus deletion of the TK gene and N1L gene can enhance the tumor selectivity of vaccinia virus and reduce the virulence of vaccinia virus.Interleukin 21(IL-21)can enhance cellular immunity through multiple pathways.IL-21 can enhance the antigen affinity of CD8~+T cells in vitro,up-regulate the expression of co-stimulating signal CD28 on CTL,stimulate the expansion of memory cells and naive CD8~+T cells,induce the expression of granules and perforins through T-bet activated by STAT1 signal pathway.In addition,IL-21 can promote NK cells in mice to differentiate into effector cells and expressing high levels of IFN-?,perforins and other cell-killing molecules.In this study,we constructed an oncolytic vaccinia virus VV?TK?N1L-mIL21with TK/N1L genes deletion and ectopic expression of mIL21.Its replication and killing ability of mouse colon cancer cells CMT93,CT26,and lung cancer cells CMT64,CMT167,CMT170,LLC were measured.Efficacy to treat subcutaneous tumors of CMT93,CT26,and CMT64,as well as liver metastatic tumors of CT26were evaluated.The potential functional mechanisms of the treatment in subcutaneous tumors of CMT93 and metastatic tumors of CT26 were investigated.Part ? The Therapeutic Efficacy of Vaccinia Virus in CMT93, CT26 and CMT64 Subcutaneous Tumor ModelsObjectiveTo investigate the replication ability of VV?TK?N1L-mIL21 in tumor cells and its therapeutic efficacy on the subcutaneous tumor.Methods1.The killing ability of VV?TK?N1L-mIL21 on mouse colon cancer cell lines and lung cancer cell lines was evaluated by cell killing assay(MTS).2.The replication ability of VV?TK?N1L-mIL21 in mouse colon cancer cell lines and lung cancer cell lines was measured by virus replication assay.3.Subcutaneous tumor models of mouse lung cancer cell line CMT64,colon cancer cell lines CMT93 and CT26 were established to test treatment efficacy of VV?TK?N1L-mIL21.Results1.Both VV?TK?N1L-mIL21 and VV?TK?N1L-RFP can replicate in cell lines of CMT93,CMT64,CMT167,CMT170 and LLC efficiently,and further lyse these cells.VV?TK?N1L-mIL21 can produce and secrete mIL21 to the outside of the CMT93 cell when replicating.2.Killing ability of VV?TK?N1L-mIL21 on CT26 cells was weak and the average of EC50 is 57 PFU/cell.This result demonstrated that the replication of VV?TK?N1L-mIL21 in CT26 cells was limited.3.Oncolytic viruses of VV?TK?N1L-mIL21 and VV?TK?N1L-RFP can effectively delay the growth of subcutaneous tumors in CMT64 and CT26 models.4.The subcutaneous tumors in CMT93 cell model were partially cleared and most tumors were completely cleared in VV?TK?N1L-mIL21 treatment group with a rate of 85.7%(6/7),while the rate was 42.9%(3/7)in the treatment group of VV?TK?N1L-RFP.ConclusionVV?TK?N1L-mIL21 treatment can inhibit the subcutaneous tumor growth in CMT64,CT26 cell models and partly eradicate the subcutaneous tumors in CMT93cell model(with 6/7 mice).Part ? Potential Functional Mechanisms of a Vaccinia Virus in the Treatment of Subcutaneous CMT93 cell Tumor ModelObjectiveTo explore the potential mechanism of VV?TK?N1L-mIL21 in the treatment of subcutaneous tumor of CMT93.Methods1.CMT93 subcutaneous tumor model was established,and the mice were randomly divided into three groups when the average tumor volume reach the same tumor size to 150 mm~3.The day of mice grouping was denoted as D1.On D1,D3 and D5,I.T injection of PBS 100?L/mice?VV?TK?N1L-RFP 1×10~8PFU/100?L/mice and VV?TK?N1L-mIL21 1×10~8PFU/100?L/mice into CMT93subcutaneous tumors.2.On D7,D14 and D20,three mice in each experimental group were randomly selected and their lymph nodes,spleens,subcutaneous tumors and serum were collected.3.In the frozen sections of subcutaneous tumors,the infiltration of CD4~+T and CD8~+T cells was detected by immunohistochemistry(IHC),and also the capsid protein of vaccinia virus and mIL21 protein were also detected.4.Flow cytometry analysis was performed to sort for T cell subgroups,macrophages,dendritic cells and NK cells in lymph nodes and spleen.5.IFN-?release assay was conducted to detect the response of spleen cells to different stimuli in mice with different experimental groups.Results1.After three doses of virus treatment,on D20,part subcutaneous tumor of CMT93(2/3)were cleared in VV?TK?N1L-mIL21 group.2.Viral treatment can influences the microenvironment of subcutaneous tumor of CMT93.The number of infiltrated CD4~+T and CD8~+T cells in subcutaneous tumor was significantly higher in the two viral treatment groups than that of the PBS group.3.On D7,the proportion of CD4~+T cells in the spleen of VV?TK?N1L-mIL21 treatment group was significantly reduced,while the proportion of CD8~+T was significantly increased.These results indicate that VV?TK?N1L-mIL21 treatment of subcutaneous tumor can lower the ratio of CD4~+T/CD8~+T in mice spleen compared with that of VV?TK?N1L-RFP and PBS treatment groups respectively.4.On D7,the numbers of Tem-CD4 and Tem-CD8 cells in two viral treatment groups were significantly increased than those in the PBS group.On D14 and D20,the number of Tcm-CD8 cells in VV?TK?N1L-mIL21 group was significantly increased compared with that of Tcm-CD8 cells in VV?TK?N1L-RFP and PBS groups.5.On D20,the levels of IFN-?stimulated by CMT93-MMC and B8R in the spleen of VV?TK?N1L-mIL21 treatment group were significantly increased compared to that of VV?TK?N1L-RFP and PBS treatment groups.ConclusionVV?TK?N1L-mIL21 treatment can change the immune microenvironment in the subcutaneous tumor of CMT93 animal model and further stimulate the differentiation of immune memory cells in spleens.Part ? Vaccinia Virus Treatment Efficacy and Potential Mechanism on CT26 Liver Metastasis ModelObjectiveTo investigate treatment efficacy of VV?TK?N1L-mIL21 on liver metastases of CT26 and the potential mechanism.Methods1.Liver metastasis model of CT26 was established by splenic injection.HE staining of liver tissues on D5,D7 and D9 was employed to analyze CT26 liver colonization.Tumor growth was analyzed by B-mode ultrasound at 3,4 and 5 weeks after model established.2.On D7,D9,D11,D21,D23,D25 of CT26 liver metastasis model established,PBS 125?L/mice,VV?TK?N1L-RFP 2×10~8PFU/125?L/mice and VV?TK?N1L-mIL21 2×10~8PFU/125?L/mice were injected intravenously.The mice survival of each group were recorded and the survival rates were analyzed.3.CT26 liver metastasis model was established.The day of model establishment was recorded as D0,and mice in each group were treated on D7,D9and D11 with the same regime as before.Livers,spleens,lymph nodes,serum and blood of mice were collected on D14 and D18.4.The infiltration of CD4~+T and CD8~+T in frozen sections of mouse liver tissues were detected by IHC,and the capsid protein VV of vaccinia virus was also detected.5.Flow cytometry was employed to analyze the changes of T cell subsets in lymph nodes and spleens of experimental mice.6.mIL21 secretion in mice serum was detected by ELISA.Results1.The liver metastasis model of CT26 was well establised.2.VV?TK?N1L-RFP and VV?TK?N1L-mIL21 therapy significantly delay liver tumor metastasis compared to PBS treatment.3.VV?TK?N1L-RFP and VV?TK?N1L-mIL21 can survive in the blood and successfully infect live tumor cells.4.VV?TK?N1L-RFP and VV?TK?N1L-mIL21 therapy reshaped the microenvironment of CT26 liver metastases,increased infiltration of CD4~+T cells and CD8~+T cells,and altered T-cell subsets in mouse lymph nodes and spleen.ConclusionVV?TK?N1L-mIL21 treatment delayed the development of the CT26metastatic tumors,reshape the CT26 liver tumor microenvironment.