Anti-Tumor Effects Of SiRNA-PD-1 And CD28 Combined Therapies,Delivered By Attenuated Salmonella,on Mice Burdened Melanoma

BackgroundPD-1 blockade has been used in clinical practice for the treatment of melanoma,but some patients still have no significant effect.As reported,rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent,however,CD28 molecules is lost on the surface of infiltrative T cells in many patients,which is an important cause of PD-1therapy tolerance.We previously showed that siRNA-PD-1 delivered by attenuated Salmonella was able to effectively inhibit melanoma growth.However,the response of tumor-bearing mice to this therapy was also inconsistent.Compared with the effective mice,the expression of CD28 was significantly lower in mice that failed to respond to treatment.Therefore,we designed and synthesized a pCD28-siRNA-PD-1 co-expression plasmid to study its therapeutic effect on melanoma-bearing mice in the hope of improving the efficacy of siRNA-PD-1 by increasing CD28 expression.The study will provide some theoretical and experimental basis for the clinical application of PD-1intervention therapy in melanoma patients with low expression of CD28.ObjectiveTo investigate the effect and mechanism of attenuated Salmonella vectored pCD28-siRNA-PD-1 co-expression plasmid on melanoma.Methods1.Cell experimentEL4 cells in good condition were plated in six-well cell plates(concentration of 3×10~5/well)and incubated for 16–24 h.pCD28-siRNA-PD-1 plasmids of different clones were transfected into six-well plates and cultured for another 24 h.Western blot was used to detect the expression of PD-1 and CD28 and other related proteins.2.Animal experiment On the 7th day after the establishment of the melanoma tumor-bearing mouse model,the successfully inoculated mice were randomly divided into five groups:PBS group,Scramble group,CD28 group and siRNA-PD-1 group.On days 7 and 14,mice were treated with PBS,Scramble,pcDNA3.1-CD28,siRNA-PD-1 and pCD28-siRNA-PD-1twice.To observe the incidence of tumor and measure the weight of tumor in tumor-bearing mice;The expression changes of PD-1,CD28,CD4,CD8 and other related proteins in tumor tissues were detected by Western blot;the expression levels of immune cells and apoptotic cells in tumor tissues of mice were detected by immunofluorescence and TUNEL assay;the proportions of CD4 and CD8 T cells,CD4CD25 Foxp3 regulatory T cells(Treg)and NK cells in spleen,as well as the proportions of peripheral blood associated immune cells of mice were detected by FACS;the proportions of tumor-specific cytotoxic T cells in different treatment groups were detected by CTL in vitro killing assay by culturing splenocytes in vitro.Results1.The co-expression plasmid of siRNA-PD-1 and CD28 significantly inhibited PD-1 expression and increased CD28 expression in EL4 cells.2.In melanoma tumor-bearing mice,attenuated Salmonella carrying siRNA-PD-1 and CD28 co-expression plasmids more significantly inhibited the growth of mouse tumors and improved the survival rate of tumor-bearing mice.3.Attenuated Salmonella carrying siRNA-PD-1 co-expression plasmid with CD28 increased apoptosis of cells in tumor tissues.4.Attenuated Salmonella carrying siRNA-PD-1 and CD28 coexpression plasmids increased CD4~+and CD8~+T cell infiltration in tumor tissues.5.Attenuated Salmonella carrying siRNA-PD-1 co-expression plasmid with CD28 inhibited the expression of cell surface molecule PD-1 and increased the expression of CD28 molecule in tumor tissues.6.Attenuated Salmonella carrying siRNA-PD-1 co-expression plasmid with CD28 increased the expression of related proteins CD4,CD8 and CD28 in tumor tissue cells and inhibited the expression of PD-1.7.Attenuated Salmonella carrying siRNA-PD-1 and CD28 co-expression plasmids increased the ratio of CD4~+,CD8~+T and NK cell lymphocytes and decreased the ratio of Treg cells in the spleens of tumor-bearing mice.8.Attenuated Salmonella carrying a co-expression plasmid of siRNA-PD-1 with CD28 inhibited the expression of PD-1 on the surface of PBMCs and increased the expression of CD28 on the cell surface in the blood of tumor-bearing mice.9.Attenuated Salmonella carrying siRNA-PD-1 with a CD28 co-expression plasmid enhanced CTL function in splenocytes.ConclusionsThe delivery of pCD28-siRNA-PD-1 by attenuated Salmonella can effectively inhibit the tumor growth of melanoma-bearing mice,solve the problem of ineffective PD1 treatment to a certain extent,improve the survival rate and enhance the anti-tumor immune response of mice.This topic provides a potential new therapeutic strategy for improving the ineffectiveness of PD-1 monotherapy in tumors in clinical practice.