The Safety Of Apoptotic Glioma Stem Cell Antigens Loaded Dendritic Cells Vaccine

BackgroundGlioma is the most common primary malignant tumor of the central nervous system(CNS),it has a high recurrence rate and poor prognosis.The operation resection is the primary treatment at present,combined with chemotherapy and radiotherapy.However,new approaches to reduce recurrence is required to be investigated.Glioma stem cells(GSC)is a group of glioma cells that have capacitis of self-renewal,unlimited proliferation and multi-directional differentiation.GSC possess a variety of phenotypes even in one glioma cell lineage,which may cause the progression and recurrence of gliomaTumor immunotherapy mainly includes immunity cell vaccine,peptide vaccine,radioimmunotherapy,cytokine therapy,and immune checkpoint blocker therapy,etcDendritic cell(DC)is the most powerful professional antigen-presenting cell.Many studies indicated that the DC vaccine loaded with glioma cell antigens had satisfying effect on reducing tumor.Our early research on autologous DC vaccine targeted GSC also proved that the GSC-DC vaccine is effective and safe.To further investigate the safety of the GSC-DC vaccine loaded with apoptotic glioma stem cell antigens,this study mainly focuses on the following aspects:firstly,whether the GSC is contaminating the DC vaccine and able to induce tumor;secondly,whether the DC vaccine would cause autoimmunity since the common antigens expressed in GSC and normal cells such as neural stem cell and astrocyte.MethodsThis study have three major parts:(1)Sorting of GSC from glioma cell line and preparation of apoptotic GSC antigen:CD133+glioma cells was sorted from glioma cell line GL261 as GSC,and then the purity of GSC was determined by flow cytometry(FCM);Then GSC was induced apoptosis and tested the portion of apoptotic cells by FCM.(2)Culture of DC and T cell and T cell killing assay:The bone marrow was extracted for primary culture of DC from C57BL/6 mouse,which organism GL261 cell line extracted from;at the 5th day after culture,the DC was loaded with GSC apoptotic antigens,and then the maturation and purity of DCwas measured by FCM;CD3+T cell was derived from splenocyte of C57BL/6 mouse by sorting with magnetic activated cell sorting(MACS),also the purity of T cell was determined by FCM.The DC vaccine was co-culture with T cell for 24 hours to activate it.Then T cell killing assay was performed by lactate dehydrogenase(LDH)release method after co-culturing T cell with NSC and astrocyte respectively for 6 hours(3)Nude mice tumor formation assay and tests of immune function disorder:After flow cytometry activated cells sorting(FACS),the GSC-DC vaccine prepared in the second parts was subcutaneously injected in nude mice,with GSC injection as positive control and untreated DC injection as negative control.Observation lasted for a month after this assay was performed;on the other hand,the GSC-DC vaccine was intravenously injected in the caudal vein of C57BL/6 mice,with lipopolysaccharide(LPS)stimulated DC injection as positive control and untreated DC injection as negative control.Two weeks later,histology exam and blood exams was demonstrated to find out whether there is immune inflammation cell infiltrating in mice's brain,and whether abnomalitis of immune hemocytes and adverse effects on liver and kidney exist.Results(1)The purity and function of GSC reached 89.5%and the portion of apoptotic GSC came up to 98.6%after the inducement(2)The maturation of DC was facilitated after apoptotic GSC antigens loaded and the purity of DC came up to 96.7%after FACS;The purity of T cell reached 93.6%after sorted;NSC and astrocyte both had good vitality;In T cell killing assay,damage was not observed on NSC and astrocyte;(3)As nude mice tumor formation assay,the GSC-DC vaccine group are healthy,while the positive control group had 4 in 6 nude mice carried tumor subcutaneously;Two weeks after the intravenous applying of GSC-DC vaccine in C57BL/6 mice,abnormal inflammation cell infiltration was not occured in the brain of mice,and blood exams were normal as well.Conclusion(1)Well-prepared GSC-DC vaccine is unlikely tumorigenic;(2)GSC-DC vaccine did not cause obvious immune abnormalities and adverse reactions.