Investigation On Specific Aanti-glioma Immunity In Mice Induced By In Situ Administration Of Dendritic Cells Following Argon-helium Cryosurgery

Gliomas, characterized by high morbidity and high mortality, compose up to 70% of adult malignant primary brain tumors. Although we have made progress in the managements of glioblastoma, patient’s survival time is still unsatisfactory. The main treatment of malignant glioma is surgical excision and postoperative radiotherapy or chemotherapy, but the invasive growth of glioma is not sensitive to radiotherapy and chemotherapy, resulting in no big improvement in an average survival time of patients. With the development of science and technology, molecular biology and immunology, the immunotherapy of tumor has become a promising treatment modalities, and has become the fourth cancer treatment after surgery, radiotherapy and chemotherapy.Dendritic cells (DC) are the most powerful immune system antigen presenting cells (APC), who can initiate a specific immune response. Dendritic cells are key to antitumor immune response, which can effectively present antigen and activate naive T lymphocytes. Under normal conditions, most of the dendritic cells remain in an immature state in peripheral tissues, with low expressions of costimulatory molecules, adhesion molecules, however, more expressions of FcR and pathogen receptors, with a strong ability of uptake and processing antigen. The antigen peptide from antigen after uptaked and processed by immature dendritic cell, are loaded in MHC-I and MHC-II molecules to form an antigen peptide-MHC molecules complexes. Then the antigen peptide-MHC molecules complexes express on the surface of dendritic cells. Meanwhile the dendritic cells up-regulate the expression of costimulatory molecules on cell surface, adhesion molecules, and differentiate into mature dendritic cells. Mature DC can identify and activate tumor antigen-specific T cells to proliferate, which secrete a large amount of cytokines such as IL-12, leading to tumor degression.Currently, tumor antigen pulsed DC as vaccine has been widely used in clinical and achieved good results. The main method for preparing dendritic cell vaccines include tumor antigen peptide sensitized dendritic cells, DNA or mRNA extracted from tumor cell stimulated DC, DC fused with tumor cells, tumor-derived cell DNA/ mRNA sensitized DC, cytokines and chemokines genetically modified DC and so on.Argon-helium cryoablation is a new kind of frozen surgical treatment in recent years, and has advantages of high efficiency, targeting, minimally invasive, low cost, and less pain for patients. Compared with liquid nitrogen refrigeration system, the use of argon-helium cryoablation system has the advantages of high freezing rate, less time of thermal and freeze circulation. And the helium rewarming can quickly stop the further formation of ice hockey, reducing damage to the surrounding tissue. After tumors cryoablated by argon-helium, a large number of local tissue necrosis, then the necrotic tissue as a tumor antigen uptaken by immature dendritic cells. Mature dendritic cells differentiated from immature dendritic cells induced the activation and proliferation of T lymphocytes. Research shows that, cell necrosis including cell membrane rupture, cytoplasm and organelles release, and the release of cytokines, which caused by argon-helium cryoablation is an effective way to release antigen. Internal tumor antigen peptides catch no major damage, which is different from the traditional hyperthermia. These relatively complete tumor specific antigens which are easily uptaken by antigen presenting cell and presented to T lymphocytes, result in cellular immune.Therefore, based on argon-helium cryoablation could provide a large number of tumor antigens in situ and dendritic cells’ strong ability of uptake and presenting antigen, In this study, we investigated whether administration of immature DCs could make full use of in situ tumor antigen after cryosurgery to enhance anti-glioma immunity in vivo. In this study, the C57BL/6 mice and GL261 glioma cell lines were used for the study. First of all, immature bone marrow denreitice cells were induced by GM-CSF and IL-4.The expression of DC surface molecules were detected by flow cytometry. C57BL/6-GL261 glioma models were established.106 immature DCs were injected intratumoral 24 hours after argon-helium cryoablation. The change of CD3+, CD4+, CD8+T cells in the peripheral blood, serum IL-12, the percentage of Thl/Th2 in spleen lymphocytes after treatment, spleen CTL toxicity after cryotherapy were detected. Tumor volume change curve and the survival analysis were also measured.Objective:1. To establish a method for amplification and purification immature* dendritic cells (imDC) from murine bone marrow in vitro, which could provide enough dendritice cells for glioma immunotherapy.2. To investigate specific anti-glioma immunity in mice induced by in situ administration of dendritic cells following argon-helium cryosurgery.Methods:Bone marrow cells obtained from mice were cultured with 10ng/mL GM-CSF and 10ng/mL IL-4. Cell morphology was observed under a microscope. The cell surface molecules (CD11c+, CD80+, CD86+, and MHC-II+) detection of the loosely adherent cells on five days. C57BL/6 mice glioma models were established by subcutaneously inoculating GL261 cells into the left flank. All mice were randomly divided into four groups:control group (n=14), immature DC group (n=14), cryosurgery+PBS group (n=15), cryosurgery+imDC group (n=15). The percentage of CD3+、CD3+CD4+、CD3+CD8+T cell subsets in blood were tested with flow cytometry before and 10 days post treatment. IL-12 levels in the serum samples were determined by ELISA 10 days post treatment. The frequencies of Thl/Th2 cells in spleens of C57BL/6 mice were measured with flow cytometry 10 days post treatment. A CCK-8 assay was performed to measure the cytotoxic reactivity of splenocytes from different groups.10 days after treatments; Tumor sizes were measured with a vernier caliper every 3 days, and calculated by using the formula:A × B, in which A was the largest dimension and B was the shortest. The survival analysis of the mice was also measured.Results:The loosely adherent cells on five days show the typical characteristics of imDC in the morphology. Moreover, they expressed significantly high level of DC surface markers CD11c+(80.28±4.94%), low levels of CD80+(31.58±3.94%) and CD86+ (28.31±6.08%), and moderate level of MHC-II+(64.78±7.63%). These suggested that most of the DCs were considered as immature DCs. In comparison with other treatments, additional DCs injections accompanying with cryosurgery significantly reduced tumor sizes (P=0.000) and prolonged survival (P=0.000). The mean survival times for no treatment group, DC group, cryosurgery group and cryosurgery+DC group were:39.11 ± 4.86 days,42.11 ± 6.47 days,61.30 ± 11.63 days, and 70 days. Ten mice in cryosurgery+DC group survived more than 70 days. There were no significant differences in the percentages of CD3+, CD3+CD4+, CD3+CD8+ cells and the ratio of CD3+CD4+/CD3+CD8+before treatments among groups (P=0.782, P=0.642, P=0.813, P=0.959). However, the percentages of CD3+(50.65±4.42%), CD3+CD4+(32.90±3.76%) cells and the ratio of CD3+CD4+/CD3+CD8+ (1.99±0.26) greatly increased 10 days after treatments in cryosurgery+DC group and were significantly different from those in the other groups (P=0.000;.P=0.000; P=0.000). Apart from that, the percentages of CD3+, CD3+CD4+cells and the ratio of CD3+CD4+/CD3+CD8+ in cryosurgery group 10 days after treatments were significantly higher than those in no treatment group (40.39±2.17% vs.35.24±1.55%, P=0.014; 23.68±1.31% vs.18.48±1.15%, P=0.002; 1.52±0.11 vs.1.19±0.17, P=0.034). The percentages of CD3+, CD3+CD4+ cells in DCs group increased too, but compared with those in no treatment group, the differences were not statistically significant (38.57±2.77% vs.35.24±1.55%, P=0.092; 21.36±1.79% vs.18.48±1.15%, P=0.060). In addition, as compared with no treatment group, the percentages of CD3+CD8+in other groups showed no difference (P=0.547). IL-12 levels in no treatment group, DC group, cryosurgery+ PBS group and cryosurgery+DC group were 37.22±9.16 pg/mL,68.82±6.23 pg/mL,72.95±9.93 pg/mL and 132.50±7.16 pg/mL, respectively. Statistically, IL-12 levels in the cryosurgery+DC group were highest among groups (P=0.000). Notably, the frequency of Thl in cryosurgery+DC group (13.98±2.52%) was considerably higher than other groups (P=0.000, P=0.000, P=0.000)。 As compared with other groups, the cytotoxic effects in cryosurgery+ DC group were higher at all E/T ratios (P=0.000).Conclusions:1. Preliminary purification to obtain high purity of murine bone marrow-derived immature dendritic cells by manual screening culture method is simple and economical, which could provide enough dendritice cells for glioma immunotherapy.2. On the basis of the reduction of tumor load by the argon-helium cryoablation, in situ administration of DCs effectively completed antigens loading by uptake the tumor antigen, and enhanced systemic antitumor immunity which provides the experimental basis for argon-helium cryoablation immunotherapy of glioma.