The Antitumor Effect And Mechanism Of A New Fusion Cell Vaccine Mediated By Bispecific Aptamer

Background: Malignant tumor has become a major disease that threatens the world,which is seriously endangering human health.Liver cancer is one of the most common primary malignant tumors,and its lethality rate ranks the third in the global malignant tumor mortality rate.At present,most of the patients are treated by chemotherapy,radiotherapy and operation,but the treatment is not adequately effective.In recent years,the fusion cell vaccine has a good application prospect in the tumor immunotherapy.How to improve the activity of fusion cell vaccine is an urgent problem to be solved.Due to the ability of aptamer combining with specific targets,aptamer has great potential in the targeted diagnosis and treatment of tumors.TLS11 a is a nucleic acid aptamer for mouse hepatoma cell BNL.1ME.A.7R.1.Our research group has found that it can bind with mouse hepatoma cell line H22.Previous studies showed FcR aptamers targeting FcγR III(CD16)molecule were selected successfully,which could bind with the DC FcγRIII on the surface of DC.Whether the antitumor effect of fusion cell vaccine could be enhanced by aptamers is worthy of further research.Objective: To explore a new fusion cell vaccine based on bispecific aptamers,provide a new strategy for tumor cell immunotherapy.Method:1.Bispecific aptamers TLS11a/FcR were synthesized.Flow cytometry was used to detect the binding ability of TLS11a/FcR with the mouse hepatoma cell line(H22)and BALB/c mice primary DC,mouse liver normal cell(BNL.CL2).Polyethylene glycol(PEG)was used to induce two kinds of cell fusion,and the TLS11a/FCR +PEG-DC/H22(TF@PEG-DC/H22)fusion cell vaccine which is mediated by bispecific aptamer was prepared.2.Flow cytometry was used to detect the expression level of costimulator CD80,CD86 and antigen presenting molecular MHC-II molecules on the surface of TF@PEG-DC/H22 fusion cell vaccine;ELISA was used to detect the level of cytokines(IL-6,IFN-β,IL-12p70,IL-10)in culture supernatant of TF@PEG-DC/H22 fusion cells.3.Flow cytometry was used to detect the expression level of activated molecules CD25 and CD69 on the surface of T cells after stimulating with TF@PEG-DC/H22 fusion cell vaccine.The proliferation of T cells after stimulating with TF@PEG-DC/H22 fusion cell vaccine was detected by flow cytometry;ELISA detected the level of cytokine(IL-2,TNF-α,IFN-γ)in culture supernatant of T cells after stimulating with TF@PEG-DC/H22 fusion cell vaccine.ELISPOT was used to detect the frequency of IFN-γ-secreted positive T cells stimulated by TF@PEG-DC/H22 fusion cell vaccine.Flow cytometry was used to detect the killing effect of T cells stimulated by TF@PEG-DC/H22 fusion cell vaccine on liver cancer cells(H22),Hepa1-6 and mice normal liver cells(BNL.CL2).4.HE staining was used to detect the cytotoxicity of fusion cell vaccine to all the important organs(heart,liver,spleen,lung,kidney and brain)of normal mice.BALB/c mice were used to construct liver cancer transplant tumor model using H22 cell line.After treating with TF@PEG-DC/H22 fusion cell vaccine through intratumoral injection for 3 times,the weight and size of the tumor in mice was measured and the survival rate of mice was observed to evaluate the antitumor activity of the fusion cell vaccine in vivo.5.One week later treatment for three times,blood of eyeball was taken out,and the level of serum IgG was detected by ELISA.The number of regulatory T cells(Treg)in the spleen cells of the tumor bearing mice after treating with TF@PEG-DC/H22 fusion cell vaccine were detected by flow cytometry,and the number of bone marrow derived suppressor cells(MDSC)in the spleen,bone marrow and tumor tissues of the mice after treating by the fusion cell vaccine was measured by flow cytometry.The killing effect of murine spleen T cells on H22 cells was detected by flow cytometry.6.When the size of tumor in tumor bearing mice was about 15 mm,the tumor was taken out for making paraffin slices.The local vascular density of tumor(CD34 antigen)and the expression level of the tumor cell nuclear antigen(PCNA)in tumor bearing mice were detected by immunohistochemistry(IHE).The number of tumor cells apoptosis in tumor bearing mice was examined by the TUNEL method.The antitumor mechanism of TF@PEG-DC/H22 fusion cell vaccine was investigated through the above methods.Result:1.The results of flow cytometry and fluorescence detection showed that the synthesized bispecific aptamer TLS11a/FcR could bind to mouse hepatoma cell(H22)and mouse primary dendritic cell(DC).2.A new DC/tumor fusion vaccine(TF@PEG-DC/H22)mediated by bispecific aptamer(TLS11a/FcR)was prepared,the fusion efficiency of cells reached 60%.3.The results of flow cytometry showed that the expression level of costimulator CD80,CD86 and antigen presenting molecules MHC-class II on the surface of TF@PEG-DC/H22 fusion cell vaccine were increased.The results of ELISA showed that the secreted level of cytokines(IL-6,IFN-beta,IL-12p70)in the culture supernatant of TF@PEG-DC/H22 fusion cells were increased,and the IL-10 level of each group was no difference.4.The results of flow cytometry showed that TF@PEG-DC/H22 fusion cells could activate the spleen T cells in mice,the expression level of activated molecules CD25 and CD69 on the surface of T cells were increased,and the proliferation index of T cells stimulated by TF@PEG-DC/H22 fusion cell vaccine were increased.The results of ELISA showed that the secreted levels of(IL-2,TNF-α,IFN-γ)in culture supernatant of T cells stimulated with TF@PEG-DC/H22 fusion cells were higher than those in other control groups.The results of ELISPOT showed that the frequency of IFN-γ-secreting T cells in TF@PEG-DC/H22 group were higher than other control groups.The results of flow cytometry showed that T cells activated by TF@PEG-DC/H22 fusion cell vaccine could specifically kill H22 cells,couldn’t kill liver cancer cells Hepa1-6 and mice normal liver cell BNL.CL2.5.HE staining showed that there were no obvious organic changes in the main organs of normal mice treated with TF@PEG-DC/H22 fusion vaccine,that is to say,no obvious side effects were observed.TF@PEG-DC/H22 fusion vaccine could inhibit tumor growth and prolong survival time of BALB/c hepatoma mice.The level of IgG in the serum of the tumor bearing mice was significantly increased after treating with TF@PEG-DC/H22 vaccine,and the number of Treg in the spleen of mice decreased significantly.The number of bone marrow derived suppressor cells(MDSC)in the spleen,bone marrow and tumor tissues of mice was lower than that of the other control groups.T cells isolated from spleen of tumor bearing mice can specifically kill H22 cells.6.The results of immunohistochemistry showed that the local blood vessel density in tumor bearing mice after treatment by TF@PEG-DC/H22 fusion cell vaccine could be reduced,and the expression levels of tumor cell nuclear antigen were decreased,the proliferations of tumor cells were inhibited and tumor cell apoptosis were increased.Conclusion:In this study,a new fusion cell vaccine mediated by bispecific aptamer(TF@PEG-DC/H22)was successfully prepared.It produced effective antitumor effect in vitro and in vivo.It opens up new ideas and directions for application of aptamers in the field of tumor cell immunotherapy.