| BackgroundGlioma is one of the most common malignant tumors, accounting for 40% to 50% of brain tumors. According to the latest statistics, the occurence rate of glioma is 3-10 in every 100,000 people. The current dominant treatment of glioma is microsurgical excision, combined with postoperative chemotherapy and radiotherapy. But glioma, especially glioblastoma, show infiltrative growth, invading into normal brain tissues, and are not sensitive to radiotherapy and chemotherapy. Surgical trauma and chemotherapy also produce immunity suppression and destruction. Accordingly, recurrence and mortality rates of glioma generally remain high. Therefore, it is essential to eradicate tumor cells while minimizing surgical trauma and improving immunity levels after operation. It is also currently the most promising strategic direction of glioma therapy. With the rapid development of molecular biology and tumor immunology, immunotherapy has become the fourth treatment of cancer, the other three being surgery, radiotherapy and chemotherapy. Our previous study showed, C6 glioma bearing rats treated by argon-helium cryoablation not only destroyed the tumor cells, but also induced apoptosis around the lesion, while peripheral blood CD3+, CD4+, CD8+, CD14+, CD16+ 56+ lymphocytes and Thl /Th2 ratio were significantly increased. The survival time of tumor-bearing animals was significantly prolonged (P<0.01) than the control group. We confirmed that cryoablation can not only directly kill tumor cells, but also enhance the cellular immune function, improve the anti-tumor ability. It has achieved a perfect combination of a minimal invasion and immunity boosting, while laying the foundation for further study of its mechanism.It has been internationally confirmed that cryoablating tumor-bearing animals can effectively activate the body’s anti-tumor immune response, but its mechanism is still subject to further study. The immune mechanism of cryoablation is mainly based on the fact that the environment created by cryoablation is conducive to antigen-presenting cells in this microenvironment of tumor-specific antigen response, which further stimulates T cells immunity, killing any residual tumor. Cryoablation is an effective means to produce tissue necrosis. Cell necrosis includes damage of cell membrane and the release of its internal cytoplasmic organelles. This process is a strong stimulus signal to cellular immunity. Meanwhile, necrosis also stimulates the release of additional cytokines, such that the possibility of these antigen presenting cells being activated greatly increases. Unlike hyperthermia, cryoablation does not severly damage the internal tumor antigen peptide. With necrotic tumor cells, these relatively intact tumor-specific antigen peptides are released into the surrounding environment, making it easy to be absorbed by antigen-presenting cells and presented to T cells, thus producing immune response. Some scholars used cryoablation on mice with breast cancer, and found that mice were immune to secondary challenge after treatment. They also discovered the existence of T cell response in the local lymph nodes near the tumor. There are arguments that, necrosis caused by cryoablation can cause the release of tumor antigens. These antigens may enter the circulatory system of the body and reach the peripheral immune organs, being ingested by B lymphocytes, and activating the production of antibodies against these antigens.The normal immune system can recognize and kill mutant cells, thereby removing or inhibiting the growth of tumor cells. But tumor cells through its high mutation characteristics, can evade immune surveillance and establish immunosuppressive microenvironment, resisting and inhibiting the body’s anti-tumor immune response. In tumor tissues, in addition to a large number of tumor cells, there are also fibroblasts, immune cells, myeloid-derived suppressor cells and other cytokines and extracellular matrix containing a variety of functions, which constitute the support for the growth of the tumor microenvironment. With the development of tumors, the tumor microenvironment, especially tumor immune microenvironment, shifts towards immune suppressive microenvironment, including the eradication or de-function of anti-tumor immune effector cells, the increase of cellular immune suppression, the reduction of immune effector molecules and the increase of immunosuppressive molecules, thereby helping the tumor further escape its own immune surveillance, and exacerbating tumor progression. Some inhibitory cytokines in the tumor microenvironment affect the function of dendritic cells. Tregs can also affect DCs’ function through their own immunosuppressive molecules. These dendritic cells can further induce the differentiation and proliferation of Tregs. These tolerogenic dendritic cells with low expression of the stimulus signal coordination and MHC2 molecules, and induce T cell disability through IDO. However, the effect of cryoablation on the tumor immunosuppressive environment is still unknown and requires further study.CTLA4 is an expression of advanced metastatic melanoma treatment of patients in the activation of T cells and inhibition of Treg cell costimulatory molecules on the receptor. In 2011, anti-CTLA4 antibody (ipilimumab) was approved by the US FDA to be used on melanoma patients. CTLA4 blockade can inhibit CTLA4 signaling in T cells, thus reversing CTLA4’s suppression of effector T cells, as well as blocking Treg activation. Studies have shown that in a mice model, systemic injection of anti-CTLA4 can increase tumor infiltrating effector T cells, reduce the number of Treg cells, and increase the Teff/Treg ratio. Anti-CTLA4 antibody combined with GM-CSF vaccine in micee glioma also achieved significant results. Although some studies have confirmed that anti-CTLA4 antibody combined with argon helium cryoablation for the treatment of melanoma has a good effect, whether it also applies to mice glioma needs further study. Besides, compared with the serious side effects of systemic administration, whether application of local tumor monoclonal antibody is more advantageous also requires further study.Based on the above background, we first used cryoablated subcutaneous glioma in mice model. We observed changes of immunosuppression in tumor-draining lymph nodes before and after the cryoablation, especially tumor-associated immune suppression on dendritic cell function. As a result, we further confirmed cryoablation’s effect on the recovery of tumor immune function. Secondly, we established a mice model of glioma brain in situ, in situ injecting anti-CTLA4 antibody and cryoablated tumor lysates. The anti-tumor effect was observed in each group to study the anti-tumor mechanisms. We hope to explore a new treatment of glioma and provide experimental evidence for further clinical applications.Chapter I:The influence of cryoablating tumor on the function of immunosuppressive dendritic cellsObjective:To study the effect of cryoablation on the function of immunosuppressive dendritic cells in tumor-draining lymph nodes.Methods:By establishing subcutaneous GL261 mouse glioma model, using argon-helium cryoablation or general surgery treatment, the mice were divided into control group, tumor group, cryoablation group and surgery group. Two weeks after treatment, we isolated tumor-draining lymph nodes, tested flow cytometry the number and phenotype of dendritic cells in tumor-draining lymph nodes, and the expression of interleukin-10 (IL-10). We also used CFSE to test flow cytometry the ability of dendritic cells in TDLN to stimulate T cell proliferation. We used T cell cytotoxicity assay to detect T cells’ ability to kill tumor cells. Treated mice were inoculated again with GL261 glioma cells. We detected their anti-tumor immunity against the rechallenge, measured the volume of the second tumor, and drew a secondary tumor growth curve. We also recorded the survival time of the secondary tumors in mice, and drew survival curves. Immunofluorescence detection of the expression of infiltrating dendritic cells of secondary tumors. After each group of mice was treated again, we depleted regulatory T cells (Treg), recorded the survival time of mice in each group, and drew survival curves.Results: We successfully constructed a mouse model of subcutaneous GL261 gliomas and divided mice into groups. We successfully isolated tumor-draining lymph nodes in each group, and tested with flow cytometry the dendritic cells in tumor-draining lymph node:the expression of CD80 and CD86 in tumor group was significantly lower than in the control group (P=0.001, P= 0.001), while that in cryoablation group was significantly higher than that of surgical group(P=<0.001, P=0.001). MHC II dendritic cells in cryoablation group was significantly higher than the control group, tumor group, and surgical group, which has with statistical significance (P= 0.012, P= 0.022, P= 0.004). The absolute number of dendritic cells in TDLN of cryoablation group was significantly higher than the tumor group (P= 0.003), in which the expression of DC IL-10 in cryoablation group was significantly lower than the control group (P=<0.001) and surgical group (P=<0.001), while the tumor group and surgical group was significantly higher than the control group (P= O.001, P= 0.001). The amount of secreted IL-10 in control group, tumor group, cryoablation group, and surgical group, the amount of secreted IL-10 was:78 ± 37.24pg/ml,766.6 ± 115.9pg/ml,177.7 ± 46.7pg/ml,596.6 ± 310.9 pg/ml. The secreted IL-10 in tumor group and surgical group was higher than the cryoablation group, which is statistically significant (P=<0.001, P= 0.001). But there was no significant difference between cryoablation group and control group (P= 0.272). In lymph mixed culture experiments, with the increase of DC:T cell ratio (1:3,1:1,3:1), the T cell proliferation rate in control group and cryoablation group rose significantly, while that of tumor group and surgical group was relatively slow. With the DC:T ratio being 3:1, the T cell proliferation rate in cryoablation group was significantly higher than that of the tumor group (P=<0.001) and surgical group (P= 0.001), which is statistically significant. In tumor cell killing experiments, effector T cells and target cells GL261 mouse glioma cells were co-cultured for 4 hours with the respective E:T ratio of 50 and 100, detecting the killing ability of its target cells by CCK-8. The killing rate of target cells GL261 mouse glioma cell in cryoablation group was higher than the other groups (E:T= 10,50,100, P= 0.001; E:T= 10,50,100, P=<0.001; E:T= 10,50,100, P=<0.001). The size of the second tumor in cryoablation group was 514.8 ± 310.8mm3, significantly less than that of control group 995.6 ± 225.7mm3 (P= 0.049) and surgical group 1032.7 ± 277.4mm3 (P= 0.037), but no significant difference in survival time (P= 0.109, P= 0.091). After immunofluorescence staining of secondary tumors, the infiltrating dendritic cells of secondary tumor tissues in surgical group express more IL-10, while that of cryoablation group express less IL-10. After removing the Treg cells, the average survival period of the control group was 36.6 ± 3.37 days, tumor group 35.2 ± 4.8 days, cryoablation group 66.6 ± 7.5 days, and surgical group 44.6 ± 5.4 days. The survival time of cryoablation group was significantly better than the control group, which difference is statistically significant (P= 0.011).Conclusion: cryoablating GL261 mouse glioma can alter the number and phenotype of dendritic cells in tumor-draining lymph nodes, reduce the expression of immunosuppressive factor IL-10, enhance the ability to stimulate T cells, and enhance the killing ability of effector T cells towards mouse GL261 glioma cells. It can also delay the growth of secondary tumor, reduce secondary tumor infiltrating dendritic cell expression of IL-10, remove immunoregulatory cells Tregs, enhance the resistance to secondary tumors, and prolong the survival time of secondary tumors.Chapter Ⅱ:Analysis of the treatment of cryoablation lysates combined with anti-CTLA4 antibody in situ on mice intracranial gliomaObjective:To explore cryoablation lysates combined with anti-CTLA4 antibody in situ treatment of mice intracranial glioma, analyze its anti-tumor effect, and study its anti-tumor mechanism.Methods:Established intracranial glioma mice model. In situ intracranially injected cryoablation lysates and/or anti-CTLA4 antibody. Divided mice into tumor group (right brain caudate nucleus in mice inoculated with 1× 105/5μl GL261 mouse glioma cells, no treatment.), anti-CTLA4 group (right brain caudate nucleus in mice inoculated with 1 × 105/5μl GL261 mouse glioma cells, in situ injected anti-CTLA4 antibody once each day on the 3rd,6th,9th, and 12th day,10μg each time.), cryoablation group (right brain caudate nucleus in mice inoculated with 1 x 105/5μl GL261 mouse glioma cells, in situ injected cryoablation lysates each day on the 3rd and 6th day,10μg each time.), combined treatment group (right brain caudate nucleus in mice inoculated with 1 x 105/5μl GL261 mouse glioma cells, in situ injected anti-CTLA4 antibody once each day on the 3rd,6th,9th, and 12th day, lOμg each time; in situ injected cryoablation lysates each day on the 3rd and 6th day, lOμg each time). Calculated each group’s survival. Assessed the neurological toxicity of each group. Isolated brain infiltrating lymphocytes. Tested with flow cytometry changes in CD4+, CD8+ T lymphocytes. Calculated CD4+/Foxp3+Treg ratio and CD4+/Foxp3+Treg ratio. Tested brain infiltrating lymphocyte cells’ killing capacity. Immunofluorescence confirmed brain infiltration of lymphocytes. Extracted brain tumor tissues. Tested the expression of IL-10, IL-12, IL-4 and IFN-gama in tumor microenvironment. Analyzed the survival of each group and its mechanism after depleting CD4+, CD8+ T lymphocytes.Results:The mouse glioma model was successfully established and treatment in each group was implemented accordingly. The treatments were reliable. No mice died due to surgery operations. The average survival in tumor group, anti-CTLA4 group, cryoablation group, and combination treatment group were 28.8+1.31 days,39.9 ± 3.8 days,32.7 ± 1.7 days, and 51.9 ± 3.4 days. The survival in all treatment groups were better than the control group, (P= 0.01, P= 0.028, P= 0.00). There was no significant difference between the survival of anti-CTLA4 group and the survival of cryoablation group (P= 0.123). Survival in the combination group was better than survival of anti-CTLA4 group and cryoablation group (P= 0.046, P= 0.001). In the neurological score system, on the 7th day of tumor inoculation, tumor group mean rank score of 12.05, the combined treatment group mean rank score of 8.95, Z=-1.33, P= 0.181, the difference was not statistically significant. On the 13th day of tumor inoculation, tumor group was 14.2 scoring average rank, the combined treatment group mean rank score of 6.8, Z=-2.87, P= 0.004. The difference was statistically significant. Tumor group scored higher than combined treatment group. Brain tumors in the control group were HE stained, showing tremendous brain glioma infiltrating normal brain tissues, while the combined treatment group were also HE stained showing normal brain tissues, with infiltration of lymphocytes, but not tumor cells. After immunofluorescence staining, the tumor group had little brain CD4+ T lymphocytes and CD8+ T lymphocytes infiltration. Combined treatment group showed more brain infiltration of CD4+ T lymphocytes and CD8+ T lymphocytes. We tested with flow cytometry brain infiltrating lymphocytes. With respect to the percentage of CD4+ T lymphocytes in brain infiltrating lymphocytes, the combined treatment group was significantly higher than that of tumor group (P=<0.001), anti-CTLA4 therapy group (P= 0.002) and cryoablation group (P=<0.001); In terms of the percentage of CD8+ T lymphocytes in brain infiltrating lymphocytes, the combination treatment group was significantly higher than that of the tumor group (P =<0.001), anti-CTLA4 therapy group (P=<0.001) and cryoablation group; the relative numbers of CD4+ and CD8+ lymphocytes in the combination treatment group were 0.46 ± 0.048/Million, and 0.71 ± 0.019/Million, significantly higher than the other three groups (all P=<0.001). Similarly, there was no significant statistic difference between the CD4+Fox3/Foxp3+ ratio of spleen lymphocytes in tumor group and the combined treatment group (P= 0.713). But the CD4+Fox3/Foxp3+ ratio of brain infiltrating lymphocytes in the combination treatment group was significantly higher than that of the tumor group (P= 0.005); There was no significant difference (P= 0.102) between the tumor group and the combination treatment group in the CD8+Foxp3-/Foxp3+ ratio of spleen lymphocytes. But the CD8+Foxp3-/Foxp3+ ratio of brain infiltrating lymphocytes in the combination treatment was seven times higher than that of the tumor group (P= 0.003). In tumor cell killing experiments, when effector cells: target cells were 10,20, 40, the killing efficiency of brain infiltrating lymphocytes in the combined treatment group was higher than the spleen lymphocytes group (P= 0.005, P=<0.001, P= 0.001) and tumor group (P= 0.007, P= 0.001, P=0.001). In the combination treatment group, the killing efficiency of brain infiltrating lymphocytes towards the GL261 mouse glioma cells was higher than towards B16 murine melanoma cells (T ratio= 10, P-0.002, T ratio= 20, P=<0.001, T ratio= 40, P= 0.001) and Hepal-6 mouse hepatocellular carcinoma cells (effector to target ratio= 10, P= 0.002, T ratio = 20, P= 0.001, T ratio= 40, P=0.001). Meanwhile, the combined treatment group brain infiltrating lymphocytes dose of anti-IFN-gama GL261 mouse glioma cells release significantly greater than normal mouse spleen lymphocytes (P=<0.001) and in brain tumor infiltrating lymphocytes (P= 0.001) of release. In real-time PCR detection of brain tumor tissue factor experiment, the expression of IL-10 in the combined treatment group was only one third of the tumor group (P= 0.02), while the IL-10 expression in the cryoablation group was 1.6 times higher than the tumor group (P= 0.036). Expression of IL-12 in the combination treatment group was 7.17 ± 1.51 times higher than the tumor group, showing a significant difference (P= 0.001). Expression of IL-4 in the combination treatment group was 0.53 ± 0.24 times higher than the tumor group, showing a significant difference (P= 0.024). Expression of IFN-gama in the combined treatment group was 3.76 ± 0.25 higher than the tumor group, showing a significant difference (P= 0.001). After depleting CD4+ or CD8 + lymphocytes, the survival of mice in the combination treatment group was not significantly different than that of combination treatment+ CD4-depletion group (P= 0.134). The survival of mice in the combination treatment+ CD8-depletion group was significantly shorter than the combined treatment group, showing significant difference(p= 0.001).Conclusion:In situ injection of anti-CTLA4 antibody with tumor cryoablation lysates can treat brain gliomas in mice, prolong the survival time and improve survival. It has less severe nervous system toxicity. Combination treatment can increase the percentage and number of CD4+, CD8+ T lymphocytes in brain infiltrating lymphocytes. It can also increase the ratio of effector T cells and regulatory T cells, especially the ratio of CD8+ effector T cells and regulatory T cells. The combination treatment altered glioma tumor microenvironment, increased the expression of tumor local IL-12, IFN-gama, and reduced the expression of IL-10, IL-4. In the in vitro experiments, brain infiltrating lymphocytes in the combination treatment group have GL261 mouse glioma cell specific killing capacity, with strong anti-tumor capabilities. Specifically, CD8+T lymphocytes in the combination treatment of glioma plays a key role. |
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