Therapeutic Effect And Mechanism Of Oncolytic Adenovirus Encoding LIGHT Inhibits Tumor Growth In 4T1 Mouse Mammary Tumor Model In Immune Competent Syngeneic Mice

Breast cancer is the most common cancer in women and the main cause of cancer death in women.At present,drug resistance exists in the treatment of breast cancer,and there is still lack of effective treatment for metastatic,advanced and refractory breast cancer.Adenovirus（AD）is one of the most commonly used vectors in gene therapy for tumor,which is mainly divided into conditional replication adenovirus and replication defective adenovirus.Oncolytic adenovirus is a kind of conditional replication adenovirus,which can selectively replicate in tumor cells and dissolve them without affecting normal cells.However,there exists immunosuppressive cells in tumor microenvironment,which can counteract the immune effect of oncolytic virus after lysis of tumor cells and inhibit oncolytic adenovirus.Using oncolytic adenovirus to carry immune gene can activate the immune system and enhance the anti-tumor effect of oncolytic adenovirus.LIGHT is a member of tumor necrosis factor ligand family,which plays a key role in T cell homeostasis and proliferation.It can selectively induce apoptosis of some tumor cells simultaneously expressing lymphotoxinβreceptor（LTβR）and TR2/HVEM receptor,such as 231 cells,HT-29 cells and TIL1200200 cells.At present,the effect of LIGHT gene on breast cancer has not been studied.Therefore,the purpose of this study is to evaluate the therapeutic effect of r Ad.light on breast cancer and to elucidate its mechanism.First of all,we prepared four kinds of viruses:r Ad.Light,r Ad.Null,Ad.light and Ad.Null.They have been constructed formerly in the laboratory,and there are two control viruses,r Ad.Null and Ad.Null.On this basis,we amplified r Ad.Light and Ad.Light viruses,and purified them by cesium chloride density gradient method.The titer of virus particles（VP）was determined by spectrophotometry.r Ad.Light was1.2×10¹³vps/m L,r Ad.Null was 1.3×10¹²vps/m L,Ad.Light was 1.4×10¹³vps/m L,Ad.Null was 1.4×10¹² vps/m L.The titer of virus infection（IU/ml）was determined by TCID50.r Ad.Light was 1.8×10⁹ IU/m L,r Ad.Null was 8×10⁹ IU/m L,Ad.Light was2.5×10⁹ IU/m L,Ad.Null was 8×10⁹ IU/m L.The above results showed that we have successfully obtained the virus whose purity and titer meet the requirements of subsequent experiments.Secondly,we tested the effect of LIGHT on the proliferation and apoptosis of breast cancer cells.The expression of LIGHT protein was detected by flow cytometry48 hours after infection with Ad.Light and r Ad.Light,respectively.The results showed that both viruses could well mediate the expression of LIGHT in 4T1 cells.4T1 cells were infected with r Ad.Light and Ad.Null,respectively.After 48 hours of infection,CCK8 method was used to detect the effect of LIGHT on the proliferation of 4T1 cells.The results showed that the survival rate of 4T1 cells in r Ad.Light group was lower than that in Ad.Null group,indicating that LIGHT could inhibit the proliferation of 4T1cells.4T1 cells were infected with r Ad.Light and Ad.Null with different particle titers.48 hours after infection,apoptosis was detected by flow cytometry.The results showed that the apoptotic rate of Ad.Null group was higher than that of r Ad.Light group,and the apoptotic rate increased with the increase of virus particle titer.Therefore,we speculate that LIGHT may play an anti-tumor role by inhibiting tumor growth and promoting apoptosis.Then,the effect of r Ad.Light on transplanted tumor model of breast cancer was evaluated.We used 4T1 cells of mouse breast cancer and subcutaneous injection on the back of mice to establish the transplanted tumor model of breast cancer.The injection amount was 1×10⁶ cells/mouse.Mice were randomly divided into buffer control group,r Ad.Null group and r Ad.Light group on the 7th day after tumor bearing.On the 7th and10th day,the dose of virus in the treatment group was 2.5?10¹⁰VPs/mouse each time,and the buffer group was injected with 200μL PBS.The changes of tumor volume were recorded on the 7th,14th,18th,21st and 25th day after tumor bearing.On the 25th day after tumor bearing,mice were killed.Then the tumor tissues were taken,and tumor weight was measured.HE staining method was used for histopathological analysis.The results showed that r Ad.Null or r Ad.Light could inhibit the growth of tumor in 4T1transplanted tumor model.Compared with r Ad.Null group,r Ad.Light group had a stronger inhibitory effect on tumor growth.After HE staining,the tumor necrosis area increased in the treatment group,and the effect of r Ad.Light group was more obvious.These results indicated that r Ad.Light could promote tumor cell necrosis and inhibit tumor growth.Finally,we studied the mechanism of r Ad.Light in the treatment of breast cancer.On the 15th and 19th day after tumor bearing,peripheral blood was collected to detect the phenotype of T lymphocytes.The mice were killed on the 25th day.The expression of CD3 and caspase-3（including CD3,CD4,CD8,Treg and T memory）was detected by immunohistochemistry.RNA was extracted and the expression of LIGHT receptor（including LTβR and HVEM）,Th1 cytokines（including IL-2,IFN-γand TNF-α）and Th2 cytokines（including TGF-βand IL-10）were detected by real-time quantitative PCR.Immunohistochemical analysis of tumor tissue showed that the proportion of CD3⁺T cells infiltrated and caspase-3-positive apoptosis cells increased in the treatment group,and the proportion of r Ad.Light group was higher.The results of real-time quantitative PCR showed that the expression of LTβR and HVEM of LIGHT receptor increased significantly in r Ad.Light group compared with rad.Null group.The expression of Th1 type cytokines was up-regulated and Th2 type cytokines were down regulated.Compared with r Ad.null group,r Ad.Light group had stronger regulatory effect.The proportion of CD4⁺T lymphocytes was down regulated and the proportion of CD8⁺T lymphocytes was up regulated after the treatment of oncolytic virus.Compared with r Ad.null group,r Ad.Light group appeared earlier and had stronger effect.The proportion of CD4⁺CD25⁺Foxp3⁺Treg cells decreased after r Ad.Light treatment.In addition,we also found that the percentage of CD62L^HighCD44⁺memory T cells was up-regulated after treatment with oncolytic adenovirus,and the effect of r Ad.Light group was earlier and stronger.The above results showed that r Ad.Light could regulate the balance of Th1/Th2 cytokines by promoting the infiltration of CD3⁺cells,apoptosis of tumor cells,expression of LIGHT and receptor,and then regulate the local microenvironment of tumor.Through increasing the proportion of CD8⁺T lymphocytes,promoting CD4⁺T memory cells and reducing the number of regulatory T cells,it could enhance the anti-tumor immune response.In conclusion,both LIGHT and oncolytic adenovirus can induce apoptosis and death of breast cancer cells.In the subcutaneous transplanted breast cancer model of mouse,r Ad.Light can induce apoptosis and death of tumor cells,bringing strong anti-tumor immune response,including inducing down-regulation of Tregs,activating memory T cells,enhancing the expression of Th1 cytokines and inhibiting the expression of Th2 cytokines.Therefore,r Ad.Light,as an effective treatment for breast cancer,has a broad prospect.