Preparation Of Tumor Vaccine By Targeting Blockage Of STAT3 In Hepatoma Cells And Its Anti-tumor Mechanisms

ObjectLiver cancer is the most common malignancy in the world, whether the incidence or mortality rates top out. Our country is disaster area of liver cancer, accounting for about 55% of the world. Liver cancer has become a major killer which seriously threatens Chinese health and lives. At present, the main means for the treatment of liver cancer include surgery, radiotherapy and chemotherapy, but the effect is not ideal, because of obvious side effects, easy to relapse and metastasis, as well as the poor prognosis. In recent years, along with the rapid development of biomolecular technology and immunology, tumor biological therapy plays a more and more important role in the comprehensive cancer treatment. As an safe and effective treatment, and biological therapy has became the fourth mode following the surgery, chemotherapy and radiotherapy.Cancer biotherapy is to regulate host immune system or tumor cell proliferation by using cellular biology and molecular biology methods to inhibit tumor growth Its benefits include the specificity, exact effect, low toxicity, anti-cancer recurrence after surgery and good long-term anti-cancer effect. Recent years, the breakthrough in cancer biotherapy is to find efficient and specific targets. STAT3 is an important member in the family of signal transduction and transcription activating factor. As a transcription factor, STAT3 sustained activated and overexpressed in many primary tumors. And, the activated STAT3 can not only promote the proliferation of tumor cells, inhibit apoptosis by regulating its target genes, but also promote the tumor angiogenesis and immune escape by regulating the secretion of cytokines. Therefore, the development of cancer is closely related with the abnormal expression of STAT3.Our previous findings confirm that blocking STAT3 signaling pathway in hepatoma cells can inhibit the proliferation and promote the apoptosis of tumor cells. Most importntly, the sensitivity of the hepatoma cell to NK cell cytolysis was significantly enhanced. Mice innoculated with HCC cells with blocked-STAT3 could effectively activate the the immune system and break the immune tolerance, resulting in an effective anti-tumor effect. The results suggest that the tumor antigen component of hepatoma cell may have been changed by blocking STAT3, so that the immunogenicity of hepatoma cell was enhanced. Therefore, based on these, we prepared a kind of tumor vaccine by using HCC cells treated with STAT3 decoy-ODN. The results showed that mice immunized with the vaccine displayed an effective anti-rumor immune response. And the tumor growth was slow down with the prolonged survival. At last, we depth studied the anti-tumor mechanisms of this vaccine. Our findings provide a theoretical basis and reasonable proposals for clinical prevention and treatment of liver cancer.Methods 1. Different concentrations of FITC-labeled Decoy-ODN were transfected into hepatoma cells Hepal-6 and H22, the transfection efficiency was analyzed by using flow cytometry, and the optimal condition for transfection was determined. Under this condition, the apoptosis rate of HCC cells transfected with Decoy-ODN was detected by Annexin-V/PI double staining method.2. After HCC cells transfected with Decoy-ODN, these cells were prepared to the tumor vaccine by freez-thawing method with liquid nitrogen. H22 cells and Hepal-6 cells vaccines were used to immunize BALB/c and C57BL/6 mice 3 times, separately, and then these mice were inoculated with HCC cells. Tumor growth and survival rates of mice bearing HCC cells were observed.3. After immunized three times with the vaccine, the lymphocytes from lympho organs were separated, and the proportions and activation of lymphocyte subpopulation were detected, as well as the secretion of cytokines associated with anti-tumor immunity. The antigen-presenting cells of DC proportion and expression of costimulatory molecules were analyze.4. After immunized three times with the vaccine the lymphocytes from lympho organs were separated, and the proportion of immune memory lymphocytes in mice were detected, investigating the immune memory function of vaccine. And through the induction of proliferation in vitro to verify the effect of immune memory.5. To discuss the mechanism of anti-tumor immunity, the lymphocytes were separated from tumor-bearing mice immunized with the vaccine, and then the activation state of lymphocyte subsets, the proportion of Treg cells and the secretion of immunosuppressive cytokines were detected by using flow cytometry.6. To investigate whether NK cells display key function in immune memory, athymic nude mice were immunized with this vaccine, and the tumor growth and the activation of the immune system were analyzed.7. To explore the role of humoral immunity in cancer clearing process, HCC cells incubated with serum from tumor-bearing mice, then tumor-specific IgG levels were detected by flow cytometry.Results1. Decoy-ODN could be effectively transfected into HCC cells at the concentration of 100nM for 12h. And the apoptosis rate of HCC cells was significantly increased by Decoy-ODN, indicating that Decoy-ODN could block STAT3 effectively under this condition.2. Compared to control groups, the tumor formation rate of tumor-bearing mice immunized with tumor vaccine decreased obviously, and the tumor growth was significantly slowed, accompanied by prolonged survival.3. The proportion of T cells and NK cells in PBMC from immunized mice increased obviously, as well as the expression of CD69. Further more, the intracellular cytokines IFN-γ and TNF-α elevated significantly. Further analysis of antigen-presenting cells DC indicated that the proportion of DC and the expression of costimulatory molecules CD80 and CD86 were higher than the control group.4. The proportion of memory T cells (CD3+CD44+CD62L+) in PBMC increased obviously in mice immunized with tumor vaccine. Further more, the spleen lymphocyte from immunized mice displayed stronger proliferative ability under the stimulation of tumor cells for 5d.5. The proportions of T cells and CD69+T cells in peripheral blood and tumor-infiltrating lymphocytes from tumor-bearing mice preimmunized with vaccine were significantly higher than control. Moreover, CD 107a a marker of granule exocytosis expressed on T cell surface was also significantly elevated. Meanwhile, although the proportion of NK cells did not show significant change, CD69+NK cells were improved accompanied with high expression of CD 107a. In contrast, the proportion of the immune negative regulatory cells Treg and secretion of inhibitory cytokines IL-10 and TGF-β in tumor-bearing mice were markedly decreased.6. The tumor growth rate and lymphocyte activation did not show significant change between tumor-bearing athymic nude mice pre-immunized with vaccine and control mice.7. After HCC cells incubated by serum from vaccine-immunized mice, the proportion of IgG+ HCC cells was significantly increased, indicating that tumor-specific IgG in serum were significantly elevated.ConclusionsIn this object, STAT3 in HCC cells was blocked by decoy-ODN, and then the whole tumor cells were prepared as tumor vaccine via freeze-thawing. Subsequently, mice were immunized with this tumor vaccine, and vaccine-induced preventive efficiency was evaluated. Furthermore, we analyzed vaccine-induced immune responses. Our results showed that the immune system was activated obviously by immunized with this tumor vaccine, and immune memory was generated. Importantly, we found the tumor formation ability and tumor growth rate were significantly lower in mice preimmunized with this tumor vaccine than control group. Finally, we found this vaccine-induced anti-tumor effect was T cell-dependented. And, the humoral immunity also played an importantrole in this process. Our research provides a new strategy and a theoretical basis for the clinical cancer prevention and treatment.