Effects Of RAAV Mediated Target Delivery Of SPD1 On Proliferation And Migration Of Triple Negative Breast Cancer 4T-1 Cells In Mice

Background Triple negative breast cancer(TNBC)is characterized by no or low expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER-2),Therefore,hormonal and anti-HER-2 targeted therapies are not suitable for TNBC.In addition,TNBC has a high degree of malignancy and a poor prognosis.The risk of recurrence and metastasis is significantly higher than that of other breast cancer subtypes,and cancer cell metastasis is the main cause of clinical death.Immunotherapy can prolong the survival of solid tumors,but it also brings problems such as autoimmune toxicity and drug resistance.Gene therapy based on adeno-associated virus(AAV)is a promising therapeutic strategy,which can circumvent the limitations of drug therapy.EC 1-AAV2M vector is a novel adeno-associated virus vector that can target 4T-1 tumor cells in vitro and in vivo.Soluble PD1 is the extracellular domain of PD1,which can bind with PD-L1 on the membrane surface to release T cell inhibition and stimulate anti-tumor immune response.Currently,there is an urgent need to develop new therapeutic strategies to inhibit tumor growth and metastasis of TNBC.Objective The sPD1 expression plasmid was constructed and the tumor-targeting recombinant adeno-associated virus EC 1-AAV2M-sPD 1 was packaged.The virus was injected into 4T-1 triple-negative breast cancer bearing mice through tail vein.sPD1 was produced in the tumor microenvironment,and sPD1 bound to PD-L1 on the surface of the tumor membrane,thereby relieving its inhibitory effect on T cells and finally alleviating tumor growth and metastasis in mice.Methods(1)The pAAV-sPD1 expression plasmid was constructed by inserting the extracellular segment sequence of mouse PD1 into the AAV expression vector(containing Luciferase sequence)by enzymatic ligation.The 293T cells transfected with pAAV-sPD1 were then examined by Western Blot and in vitro bioluminescence.(2)The binding of sPD1 to PD-L1 on the surface of 4T-1 cells was assessed by cellular immunofluorescence.(3)Assessment of EC1-AAV2M-sPD1 virus activity and in vitro targeting ability by QPCR,cell infection and bioluminescence.(4)To examine the ability of EC1-AAV2M-sPD1 virus to target 4T-1 cells in vivo,the expression of Luciferase in B16 and 4T-1 double-transplanted tumor BALB/c mice was analyzed by small animal live imaging.(5)To analyze the effect of EC1-AAV2M-sPD1 virus on tumor proliferation,EC1-AAV2M-sPD1 virus was injected into the tail vein of BALB/c female tumor-bearing 4T1-Luc mice,and mice injected with EC 1-AAV2M-GFP virus or PBS solution were used as controls.Tumor volume and body weight were measured in tumor-bearing mice and Ki67 expression in the tumor was analyzed by IHC staining.(6)The expression of E-cadherin in the tumor was analyzed by Western Blot.Analysis of tumor metastases in lung tissue and pathological changes in heart,liver,kidney and spleen tissue by H&E staining.Results(1)pAAV-sPD1 plasmid was constructed successfully,which could express sPD1.(2)EC1-AAV2M-sPD1 virus with specific infectivity was successfully packaged.(3)In vitro and in vivo experiments demonstrated that EC1-AAV2M-sPD1 virus can selectively target 4T-1 triple negative breast cancer cells but not B16 melanoma cells.(4)Administration of EC1-AAV2M-sPD1 virus effectively alleviated the proliferation and lung metastasis of 4T-1 cell transplanted tumors in tumor-bearing BALB/c mice compared to control animals.(5)Gene therapy has no obvious toxic and side effects on mice.Conclusion In this study,EC1-AAV2M-sPD1 virus was successfully packaged,which was able to target 4T-1 tumor cells in vivo and ex vivo,and the viral signal was detected for a longer period of time in tumor-bearing mice after receiving a single viral treatment.Most importantly,this gene therapy strategy significantly reduced the growth and lung metastasis of 4T-1 triple-negative breast cancer cells in mice with transplanted tumors.In summary,Adeno-associated viral vectors carrying immune-blocking checkpoints successfully inhibited tumor growth and metastasis,and it is possible that this novel therapeutic strategy could overcome tolerance caused by repeated administration.Therefore,treatment with EC1-AAV2M-sPD1 virus is a potential new strategy for the treatment of TNBC.These studies have established a novel gene therapy strategy for delivering immunosuppressive molecules,which provides a new method for further treatment of TNBC and has potential clinical application prospect in clinic.