Effect Of Ar-He Targeted Cryoablation On Immune Function And Its Clinical Significance

Cancer, a disease which has the highest mortality in the world, threatens human's life.70-80% patients are already in the advanced stage when diagnosed, losing the chance of surgery. However, the total curative ratio of surgery is less than 10% with a postoperative recurrence and metastasis rate of 50-70%. Cryotherapeutic ablation therapy, targeting killing cancer cells to eliminate tumor load, provides a new choice for patients with solid tumor who have lose the chance of radical surgery.Endocare CryocareTM Surgical System, invented by Endocare corporation in October 1980 based on clinical and basic research in ultralow temperature therapy, is a new therapeutic equipment for ultralow temperature intervened Ar-He targeted thermal ablation, which introduced a new concept in minimal surgery in curing cancer- Cryocare Targeted Cryoablation Therapy. It is brought in by Zhu-Jiang Hospital of southern medical university in 1999. Endocare Cryocare Surgical System is an advanced technique which is the only one that can undergo percutaneous thermal cryoablation therapy. Minimal invasive ablation, which can quickly relieve tumor load to elevate quality of life and guarantee the effect of comprehensive therapy, is the first choice for patients losing the chance of radical surgery and can't be taken placed by chemo and radioactive therapy. Endocare Cryocare Surgical System, which brings little wounds and better curative effect, is an important technique with an effective rate higher than 90%.Frozen tumor tissues are not removed in cryocare targeted cryoablation therapy so the operation is simple which brings less postoperative complications and has no effect on choosing other therapies. The curative effect of cryocare targeted cryoablation therapy can be evaluated when combined with radiotherapy, photodynamic therapy and traditional Chinese drug. It is also demonstrated by research that immune function can be enhanced to decrease recurrence and metastasis rate using cryotherapy. Cryocare targeted cryoablation therapy can enhance anti tumor immune function by inducing necrocytosis of cancer tissue to decrease immunosuppressive molecules secretion while necrotic in stiu cancer cells become tumor antigen.Immune response to cryotherapy is demonstrated by Yantorno in 1967 and Shulman in 1968 who settle the foundation of cryoimmunology. They inferred that antibodies targeting remaining tumor, including antibodies targeting tumor specificity membrane protein are produced after phagocyte present antigen when necrotic tumor tissues are cleared. These antibodies later bind to other membrane protein of alive tumor cells to induce complement fixation and chemotaxis of macrophagus and neutrophil to activate immune response and eliminate remaining tumor tissue and metastatic tumor. This procedure is called cryoimmnue response. Zhang Guo-Qiang et al detect the change of T-lymphocyte subpopulation in peripheral-blood and the serum levels of immunoglobulins in 10 patients before and after surgery, finding CD3+, CD4+, CD4+/CD8+ and immunoglobulins are significantly increased with CDT8+ decreasing significantly after cryosurgery. Peng Qiu-Ping et al found similar change of CD3+T, CD4+T and CD4+T/CD8+T in patients with hepatic cancer but no significant change in CD8+T cells level. Duan Yun-You et al reported that lung cancer cells treated with cryocare targeted cryoablation therapy could sensitize dendritic cells and anti tumor activity of NK cells and T lymphocytes when combined with IL-2. Researches of basic experiments on effect of cryotherapy on immune function are lacked so the mechanism of cryoimmnuology remains unclear needing more tumor cell lines experimented to be clarified. Anti tumor immune function of cryocare targeted cryoablation therapy and whether immunotherapy should be combined remain unclear in clinical practice. And thus, a summary of immune function of cryocare targeted cryoablation therapy from basic experiment to clinical practice is necessary.Subcutaneous xenotransplanted tumor model in SD rats were used in this study to undergo cryotherapy experiment to explore the change of local and overall immune function of SD rats. Principle of the cryoablation of local tissues is discussed and the presence of immune cytotoxicity in tissues around the cryotherapy ones is demonstrated. The effect of argon-helium cryoablation on immune function of SD rats is thoroughly explored through detecting the level of T-lymphocyte subpopulation and sIL-2R in peripheral-blood and cytotoxicity of single nucleus cells. The level of T-lymphocytes and NK cells in peripheral-blood of 76 patients before and after cryotherapy was detected to discover the clinical significance of argon-helium cryoablation combined with traditional Chinese drugs.This study includes three parts.Part 1:Establishment of subcutaneous xenotransplanted tumor model in SD ratsObjectiveTo establish subcutaneous xenotransplanted tumor model of W256 cell in SD rats to offer a animal model for argon-helium cryoablation.MethodsW256 cells were inoculated to enterocoelia of SD rats with proper concentration after being unfrozen to passage culture to establish mouse model of ascites. Concentrated ascites was inoculated in right rear of rats subcutaneously to establish subcutaneous xenotransplanted tumor model. Two tumors were removed two weeks after inoculation and all tumors removed at the end of experiments to undergo HE staining and be observed under light microscope.ResultsInoculation in 66 SD rats was successful with all rats growing well and no infected focus around tumors. Subcutaneous nodules could be percussed 4 days after inoculation. The average size of tumor was 3.5cm3 about 3 weeks after inoculation with the longest major axis of 5.5cm. Tumor nodules were removed 2 weeks after inoculation to undergo gross inspection and HE staining. Tumors were made up of multiple soft grey white nodules. Tumor cells, whose nuclei were large with clear nucleolus and multiple mitotic figures under light microscope, were tightly packed in a mass or compact sheet with no intercellular matrix between and abundant small vessels. Patchy necrosis were observed in tumors at the end of experiment.ConclusionEstablishment of subcutaneous xenotransplanted tumor model of W256 cell in SD rats using the method of injecting concentrated ascites is simple and economical with precise inoculation and high tumor proliferating rate. It's easier to undergo minimal invasive targeting ablation with the size of tumor can be 3.5 cm3 2-3 weeks after inoculation. In a word, this animal model is perfect for experiments using argon-helium cryoablation to treat cancer. Part 2 Change of immune function in subcutaneous xenotransplanted tumors of SD rats after Argon-Helium CryoablationObjectiveTo observe the effect of argon-helium cryoablation on the tissue necrosis and cell apoptosis as well as the immune function in subcutaneous xenotransplanted tumor of SD rats to further discuss the immune function effect of argon-helium cryoablation in treating cancer and supply new theory for clinical practice.Methods1, Rats were divided randomly into 4 groups:cancer control group(A), cryoablation group(B), surgery group(C), normal control group(D) (n=10). They were treated after anesthesia as follow. Cryoablation group:subcutaneous xenotransplanted tumors underwent argon-helium cryoablation for 3-5 minutes and then rewarmed for 30 seconds. Surgery group:tumors were removed with wounds cleared and seamed. Cancer control group:Cryocare surgical system was put into tumor for 3 minutes with no cryotherapy. Normal control group:dermotomy were operated with wounds cleared and seamed.2, Rats were killed with tumor tissues removed 3,12,24 hours and 3,7,14 days after cryoablation.3 rats from each group were killed with tumor tissue removed thoroughly at each time point. Tumor tissues were stored in 10% formaldehyde solution for histopathology observation and detection of immunohistochemistry indexes.1.5 ml blood of each SD rats from each group were collected from venous sinus of eye-orbit 1 day before treatment and 1,3,5 weeks after treatment to detect T-lymphocyte subgroup (CD3+T,CD4+T,CD8+T) via flow cytometry and level of serum sIL-2R by double antibody sandwich method. Result1,Necrosis of tumors with argon-helium cryoablation Three obvious zones of focus could be seen after subcutaneous xenotransplanted tumors underwent argon-helium cryoablation. It could be seen after cryotherapy at the center of focus (area around the place where probe were inserted) that cells were completely destructed with broken nucleic and coagulative necrosis. Obvious damaging area around necrosis could be seen with some tumor cells which had typical characteristics of apoptosis cells with cell shrinkage and condensation and margination of nuclear chromatin. Vessels at the area were congested, embolized and effusing. Tumor cells at the peripheral area were not damaged and had normal morphology.2, Detection of Cell Apoptosis by TUNEL Subcutaneous xenotransplanted tumors tissue was observed with TUNEL staining after argon-helium cryoablation. Apoptosis cells with typical apoptosis characteristic, whose nucleic were brown and pyknotic with concentrated chromatin and some broken cells mainly gathered in damaging areas around ice balls. There were few apoptosis cells in control group. Compared to cancer control group, positive rate of apoptosis cells in cryoablation group at each time poin was significantly higher(P=0.000). Apoptosis cells increased 3 hours after cryoablation, reaching the peak at 12 hours after(67.25±5.51)%.3, Cytotoxicity of Mononuclear cell after cryoablation Cytotoxicity of mononuclear cell was significantly enhanced after cryoablation, which is significantly higher in cryoablation group(P=0.05). Cytotoxicity of cells in normal control group was higher than that in tumor control group and surgery group but had no statistically difference.4, Detection of T-lymphocyte Subgroup and Serum sIL-2R levelCompared to normal control group, there is significant difference in percentage of CD3+T, CD4+T cells and CD4+T/CD8+T ratio which eventually decreased over time in cancer control group(P=0.000). Percentage of CD3+T, CD4+T cells and CD4+T/CD8+T ratio in surgery group which eventually increased over time was significantly different compared to normal control group(P=0.000). Percentage of CD3+T, CD4+T cells and CD4+T/CD8+T ratio in cryoablation group which also increased over time had significantly increased compared to surgery group(P=0.000).②Percentage of CD8+T cells in cancer control group which increased over time was significantly different compared to normal control group at each time point. Percentage of CD8+T cells in surgery group and cryoablation group which decreased over time was significantly different compared to cancer control group 3 and 5 weeks after treatment.③sIL-2R level of peripheral blood of rats in cancer control group which increased over time was significantly different at each time point compared to normal control group(P=0.000). sIL-2R level of peripheral blood of rats in surgery group which decreased over time was significantly different 3 and 5 weeks after treatment compared to cancer control group(P=0.000). sIL-2R level of peripheral blood of rats in cryoablation group which decreased more rapidly than that in surgery group over time was significantly different 3 and 5 weeks after treatment compared to surgery group(P=0.000).Conclusion1, That cryoablation area as well as peripheral immune cytotoxicity together indicated that argon-helium cryoablation took effect through necrosis and apoptosis. Tumor tissue should be completely packed in the ice ball when using argon-helium cryoablation. Some tumors with irregular shape may reoccur with incomplete cryoablation and thus argon-helium cryoablation should be combined with chemotherapy, radiotherapy, immunotherapy or other anti tumor methods. 2, Argon-helium cryoablation could effectively activate immune response. Secretion of tumor associated antigen was released continuously after argon-helium cryoablation, leading to the activation and proliferation of T-lymphocyte with tumor associated antigen specificity cytotoxicity which activated and enhanced anti tumor immune fuction. Cytotoxicity of mononuclear cells in peripheral blood was also increased, which might be higher when combined with cytokine.3, There is limitation in the enhancement of anti-tumor immune fuction by argon-helium cryoablation. So further exploration should be made in how to promote and maintain immunoregulation of argon-helium cryoablation.Part 3 Effect of Ar-He Targeted Cryoablation Combined with Traditional Chinese Drugs on Immune function and its clinical significanceObjectiveTo observe the effect of Ar-He targeted cryoablation combined with traditional Chinese Drugs on immune function and clinical outcome and its clinical significance.Methods1,76 patients pathologically diagnosed to have III or IV stage malignant advanced cancer were divided into two groups:argon-helium cryoablation group and argon-helium cryoablation combined with traditional Chinese drugs group, each 38 patients.2, Fasting venous blood from the patients'elbow were drawn 2 and 4 weeks before or after treatment to detect the subgroup of T-lymphocytes (CD3+T,CD4+T,CD8+T), CD4+T/CD8+T ratio and NK cell level via flow cytometry.ResultsLevel of CD3+T,CD4+T,CD4+T/CD8+T,NK cells of 76 patients was significantly higher after argon-helium cryoablation (P=0.000). Level of CD3+T,CD4+T,CD4+T/CD8+T,NK cells of patients in argon-helium cryoablation combined with traditional Chinese drugs group was slightly higher 2 week after therapy than that in argon-helium cryoablation group but had no statistics significance but 4 weeks after was significantly higher with statistics significance(P=0.000).ConclusionArgon-helium cryoablation can release immnosuppression in patients with advanced cancer while enhancing immune function. But there is limitation in the effect of argon-helium cryoablation on immune function and thus traditional Chinese medicine should be combined. Combination of these two therapy will decrease the recurrence of tumor, relieve patients'suffering and improve quality of life...