Study On The Treatment Of Lung Adenocarcinoma With Specific Immune Response Induced By HiPSCs Combined With CpG

Objective:Lung cancer holds the world's highest morbidity and mortality rate among all kind of tumors.Previous studies have proved that cancer stem cells(CSCs)play a key role in tumorigenesis,metastasis and recurrence.CSCs-based vaccine has specific potential to clear the dormant cancer cells in vivo,which prevent tumor metastasis and recurrence so as to improve the recovery rate.However,the low proportion of CSCs in tumor tissues makes it difficult to isolate and cultivate,leading to the difficulties of CSCs-based specific antigen acquisition,therefore limit its application.The similarities between embryonic stem cells,tumor cells and induced pluripotent stem cells(iPSCs)remind similar possibilities between CSCs and iPSCs.Therefore,in this study,a scientific hypothesis that iPSCs replace tumor-specific antigens to CSCs and may induce anti-tumor immunity in vivo was proposed.Tumor vaccine based on human iPSC combined with CpG was prepared and the effect of inducing anti-tumor immune response in the mouse model of human lung cancer was investigated.The ability of tumor growth inhibition and the mechanism of the vaccine enhancing in vivo antigen presentation was also studied,which expected to provide theoretical basis for the development of therapeutic human pluripotent stem cell vaccine for lung adenocarcinoma.Methods:The ALDEFLUOR reagent staining was used to A549-ALDH+cells from the isolated lung tumor cells according to FCM,and their characteristics of CSCs were verified from differentiation ability,Value-added fast,typical stem characteristics and tumor formation ability.The A549-CSCs RNA sequencing was performed using Illumina Hi Seq X10 platform compared with iPSC for analysis.Human iPSC-based tumor vaccine combining with CpG was irradiated by6000rads.Animals were performed by subcutaneous injection each week for four times and tumor-bearing on 28 days.Serum samples were collected at 2 and 4 weeks after first immunization were added to A549 and H1975 cells and FCM was used to detect specific antibody Ig G.To evaluating the ability of iPSC+CpG combined vaccine to induce an effective cellular immune response,FCM was used to detect spleen antigen presenting cells(CD11c+HLA+CD80+and CD11b+CD80+),the cytotoxic T cell(CD8+T cells)and Treg ratio(CD4+CD25+Foxp3+)after 2 weeks of tumor bearing.Four weeks after tumor introduction,T cells were detected in spleen and the peripheral blood and CD8+T cell infiltration in tumor tissues.Simultaneously,the tumor growth was monitored after bearing.After that,the spleen CD3+T cells were isolated to evaluate whether the raised cytotoxic T cells in preimmunized groups could terminate tumor growth in tumor-bearing model.Each of the lung tumor-bearing mouse received an intravenous injection and the tumor growth was monitored.The percentage of CD8+CD44+T cells and spleen T cell group(Th1,Th2,Th17)was calculated to evaluate whether adoptive transplantation of cytotoxic T cells in the pre-immunized group can prevent growth and elicit anti-tumor specific responses in tumor-bearing mice and study its mechanism of action.Results:Flow cytometry(FCM)successfully selected A549-CSCs.The isolated A549-CSCs showed typical stem characteristics and verified the similar gene expression profile between CSCs and iPSCs.After human iPSCs immunization,the tumor binding Ig G was detected and gradually increased.Two weeks after tumor bearing,higher percentage of iPSCs+CpG spleen antigen presenting cells(1.55%±0.18%?1.11%±0.26%)and the cytotoxic T cells(3.79%±0.31%)compared with PBS group(0.81%±0.26%?0.78%±0.13%?0.74%±0.15%)were observed(P<0.05),while those of Treg ratio(1.86%±0.12%)compared with PBS group(3.04%±0.29%)were decreased(P<0.01).Four weeks after tumor introduction,the tumor volume of iPSCs+CpG group was 69.57mm~3,which was significantly lower than other groups(P<0.01).The iPSCs+CpG group CD8+T cell subsets in the spleen(11%±3.48%iPSCs+CpG vs.2.98%±0.43%PBS)(P<0.05)and effector/memory cytotoxic T cells in the peripheral blood(2.89%±0.93%CD8+T cells,7.5%±3.45%CD4+T cells)were higher than PBS group(0.78%±0.23%CD8+T cells,3.64%±1.09%CD4+T cells)(P<0.05).Higher CD8+T cell infiltration in tumor tissues of iPSCs+CpG group was observed according to immunohistochemistry.Higher percentage of spleen Th1 cells was also raised in iPSCs+CpG group(19%±1.14%iPSCs+CpG vs.11%±0.57%PBS)(P<0.05)and Treg and Th17 ratio(3.11%±0.55%)was reduced compared with PBS group(5.78%±0.82%)(P<0.05).There was no significant change in Th2 ratio during the experiment.Tumor volume in iPSCs+CpG group was significantly reduced after T cell transfer.Furthermore,the cytotoxic T cell subsets(12.5%±3.92%)in iPSCs+CpG group was higher than PBS group(2.48%±0.78%)(P<0.01)and Th1 subset(26.67%±3.83%)in the spleen in iPSCs+CpG group was also higher than PBS groups(16.2%±1.28%)(P<0.05),while Th17 ratio(3.53%±1.31%)was reduced compared with PBS group(5.65%±0.89%)(P<0.05),and there was no significant difference in Th2 subset in all groups(P>0.05).Conclusion:This study suggests that iPSCs derived from human fibroblasts exhibited the similar antigen characteristics to CSCs and demonstrated that human iPSC-based tumor vaccine combine with CpG could induce anti-tumor specific immune responses in a humanized mouse model.The preimmunization of iPSCs+CpG could elicit stronger antigen presentation cells(DC)to present antigen-sensitized cytotoxic T cells,which specific killing tumor cells and suppress tumor growth after introduction.Adoptive transplantation of T cells from the spleen in immunized mice not only can produce specific immune response,killing tumor inhibited tumor growth so as to reduce burden,but also can trigger anti-tumor responses in vivo.