The Antitumour Efficacy Of A Novel Tumour-targeted Oncolytic Vaccinia Virus VV_△TK_△N1L-RFP For Treatment Of Mouse4T1Breast Tumour Model

BackgroundBreast cancer is the most common cancer in women of the world. Although theadvanced surgery, chemotherapy, radiotherapy, hormonal and targeted therapy, hasimproved relapse-free survival in the adjuvant setting,30-40%of patients with breastcancer still die from a recurrence and metastasis of their breast cancer and makebreast cancer the second leading cause of death from cancer among women in China.Therefore,new therapeutic agents for this disease are urgently needed. The newtherapy should have a better selectivity for the tumor cells and multiple functionalmechanisms. Moreover, it won’t have the antagonism to the existing conventionaltreatments. Tumour-targeted oncolytic viruses (TOVS) is a new class of cancertherapeutics. TOVS can selectively kill cancer cells and replicate in tumor cells butnot in normal cells. We created a new generation of oncolytic vaccinia virus, namedVV△TK△N1L-RFP. This virus might have enhanced antitumour efficacy and a bettersafety. In the present study we aimed to evalute its oncolytic effect and biologicalsafety in vitro and vivo. ObjectiveAssessment of the antitumour efficacy of VV△TK△N1L-RFP and it’s backbonevector VV△TK in vitro and vivo in mouse4T1breast tumor model.MethodsThe work of the topic course is mainly divided into three parts:Part1:Large scal production of the oncolytic vaccinia viruses and validation for thedeletion of TK and N1L gene of oncolytic by Polymerase Chain Reaction(PCR).Part2: The cytotoxicity of the novel oncolytic vaccinia virus VV△TK△N1L-RFPand control virus, VV△TK in breast carcinoma cell line,4T1cells wasdetermined by MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) assay; viralreplication of VV△TK△N1L-RFP and VV△TK was detected by TCID50assay (Tissue culture infective dose50%). For in vivo4T1breast cancer cellswere subcutaneously injected into the mammary fatpad. The xenografts wereintratumorally injected with VV△TK△N1L-RFP, VV△TK and PBS throughfour different directions.Part3: Established the orthopotic implantation and metastatic tumor mice model of4T1breast cancer and the anti-tumor efficacy of a novel oncolytic vaccinevirus VV△TK△N1L-RFP in mouse4T1breast tumor model was detected byobserving the survival of the mice.Results:Part1: A large scale of the two viruses was successfully produced, the virus titerreached to1011pfu/ml. In addition, the deletion of TK and N1L in noveloncolytic vaccine virus was confirmed by PCR. Part2: The EC50value of VV△TK△N1L-RFP and VV△TK were (1.90±0.1)pt/cell and (7.18±2.2) pt/cell,respectively，suggesting that the new virus ismore potent(P<0.05). TCID50results showed that the novel oncolyticvaccinia virus VV△TK△N1L-RFP replication in4T1cells had no statisticallysignificant difference with VV△TK at different time points(24h、48h、72h),(P＞0.05).Part3:In vivo, we successfully established the orthotropic, metastatic and thepost-operative metastatic tumor models. Moreover, there was no significantdifference of tumour size between the intratumor administration of VV△TK△N1L-RFP and VV△TK oncolytic virus and PBS control prior to operation,however，the survival time of administration of VV△TK△N1L-RFP had beenprolonged significantly compared to control virus and PBS.ConclusionThe cytotoxicity of the novel oncolytic vaccinia virus VV△TK△N1L-RFP for4T1breast cancer is more efficacious in vitro and the immunologic response is morepotent in vivo than VV△TK and PBS.This study suggests that the novel vaccinia virusVV△TK△N1L-RFP could have a significant therapeutic impact against metastaticbreast cancer. Administration of VV△TK△N1L-RFP prior to surgery is an effectiveneoadjuvant therapy for breast cancer to enhance the survival.