Combination Therapy With PD-1 Blockade And GM-CSF Surface-modified Bladder Cancer Stem Cells Vaccine In Bladder Cancer

BackgroundBladder cancer(BCa)is a common malignancies.Recurrence of tumors may be due to the inability of traditional chemotherapy and radiotherapy to eliminate CSCs.As we all know,cancer stem cells(CSCs)may play an important role in tumor growth.Eliminating CSCs is a key issue in eradicating tumor.We have developed a technology that immobilizes streptavidin(SA)-tagged bioactive cytokines on the biotinylated tumor cell surface to prepare tumor cell vaccines.This type of therapeutic vaccine not only retains the biological activity of cytokines but also contains all of tumor antigen.Although the therapeutic CSCs vaccine could significantly enhance tumor-specific antitumor responses in vivo,this effect is usually restricted to the early stages of tumor growth.The tumor microenvironment exists tumor immune escape.PD-1/PD-L1 plays a very important role in the regulation of T lymphocyte-mediated cellular immunity,which has become a hot research topic in immunotherapy.We found in the study that the therapeutic CSCs vaccine enhanced the expression of PD-L1 and infiltration of PD-1+CD8+ T cells in tumor microenvironment.The tumor microenvironment after GM-CSF-vaccination exsit immune escape mediated by PD-1/PD-L1 signaling.Therefore,we speculated that:GM-CSF-vaccine can promote the growth and maturation of DCs,then activates the effector T cells,and induce specific immune response against tumor cells.On the basis of GM-CSF-vaccination,it is possible to induce a high-intensity,long-lasting,and specific immunity by blocking the PD-1/PD-L1 signaling pathway,which decrease the occurrence of T cell apoptosis and inhibit the associated immune escape.Methods and Results1.Establishment and identification of MCSCsMCSCs were extracted from MB49 cells by chemotherapy drug pressure screening combined with limited dilution and serum-free culture method.The results of FCM analysis showed that more CD133+CD44+ cells were detected in MCSCs than that in MB49 cells.The results of qPCR showed that the relative levels of CD133,OCT4 and NANOG mRNA were higher in MCSCs when compared to MB49 cells.MCSCs possessed strong tumorigenic ability.Compared to MB49 cells,MCSCs showed lower susceptibility to four traditional anticancer drugs(mitomycin,paclitaxel,cisplatin and doxorubicin).2.Immobilization and biological activity of SA-GM-CSFThrough our protein-anchor technology,the SA-tagged bioactive cytokines can be immobilized on the biotinylated tumor cell surface.FCM results showed that SA-GM-CSF could be efficiently anchored on the surface of ethanol-fixed MCSCs(98.7%).SA-GM-CSF fusion protein anchored on the biotinylated surface of MCSCs still retained their bioactivity.3.Immunotherapy with SA-GM-CSF-modified MCSCs vaccine and the associated immune escapeGM-CSF-vaccine could induce antitumor responses in vivo,but this effect is usually restricted to the early stages of tumor growth.The therapeutic CSCs vaccine enhanced the expression of PD-L1 and infiltration of PD-1+CD8+ T cells in tumor microenvironment.The tumor microenvironment after GM-CSF-vaccination exsit immune escape mediated by PD-1/PD-L1 signaling.4.Combination therapy with SA-GM-CSF-modified MCSCs vaccine and PD-1 blockade(1)The efficacy of combination therapy:The combination treatment of the MCSCs vaccine and PD-1 blockade produced the best antitumor response and significantly delayed tumor growth.(2)The related immunological mechanisms:GM-CSF-vaccine can promote the growth and maturation of DCs,then activates the effector T cells,and induce specific immune response against CSCs.The results of immunohistochemistry showed that more CD4+ and CD8+ T cells infiltrated the tumor tissues in the combined group than in the other groups.The combined group displayed higher concentration of IFN-g and IL-12 but lowest concentration of IL-10 than either the MCSCs vaccine or PD-1 blockade alone.The tumor-specific cytotoxic activities could be significantly enhanced by PD-1 blockade based on the presence of an effective immune response.Conclusions:1.The bladder cancer stem cells extracted by chemotherapy drug pressure screening combined with limited dilution and serum-free culture method have the characteristics of tumor stem cells.2.SA-GM-CSF could be efficiently anchored on the surface of ethanol-fixed MCSCs,and retained their bioactivity.3.GM-CSF-vaccine could induce antitumor responses,but the tumor microenvironment after GM-CSF-vaccination exsit immune escape mediated by PD-1/PD-L1 signaling.4.PD-1 blockade combined with MCSCS vaccine produced the best antitumor response in vivo,which significantly delayed tumor growth.GM-CSF-vaccine can promote the growth and maturation of DCs,then activates the effector T cells,and induce specific immune response against tumor cells.more CD4+ and CD8+ T cells infiltrated the tumor tissues in the combined group than in the other groups.The combined group displayed higher concentration of IFN-g and IL-12 but lowest concentration of IL-10 than either the MCSCs vaccine or PD-1 blockade alone.The tumor-specific cytotoxic activities could be significantly enhanced by PD-1 blockade based on the presence of an effective immune response.