The Mechanisms Of Host Anti-tumor Immune Response Triggered By Blocking STAT3Signal Pathway In Hepatocellular Carcinoma (HCC)

As a malignant disease, cancer poses a serious threat to human health with a high mortality rate. The investigation of WHO shows that cancer is the second largest cause of death after cardiovascular disease. Liver cancer is one of the most common malignant tumors worldwide, with the increase of its incidence and mortality. Due to dietary factors and cancer risk factors such as chronic hepatitis, liver cancer prevention and treatment are particularly serious in China. At present, the level of diagnosis and treatment of liver cancer has greatly improved. Treatments of liver cancer include surgery, radiation therapy, chemotherapy, targeted therapy, combined therapy, etc. but some disadvantages were found, such as great side effects, poor targeting, scarce resource of transplanted liver, which accounted for high cost, but poor efficacy. Due to the small side effects and the good position-relevant, Tumor gene therapy attracted much attention in recent years. To find the right target and carrier is the key to gene therapy.Signal Transducers and Activators of Transcription3(STAT3) is an importment cytoplasmic transcription factor with functions of transferring signal and initiating transcription. It can regulate the expression of many cancer genes enhancing cancer cells proliferation and survival, inhibiting apoptosis, inducing angiogenesis, such as antiapoptotic gene bcl-XL, mcl-1and cell cycle control gene cyclin D1, cyclin E, c-myc. Meanwhile, STAT3also regulate many cytokines associated with tumor immune tolerance, such as TGF-β,IL-10, IL-6and so on. Under normal physiological conditions, STAT3activation is transient and subject to strict regulation. Highly and sustained STAT3activation has been found in many tumors, can induce tumor cell proliferation. The STAT3signal path activation in tumor cells can induce expression of many cytokines associated with tumor immune tolerance, inhibit immune ability, and resist the immune system anti-tumor ability. Sustainly activated STAT3can increase the expression of vascular endothelial growth factor, induce tumor angiogenesis, thereby promot tumor cell metastasis. Therefore, STAT3has been considered as an important target for cancer diagnosis and therapy.As an important immune defense barrier, the immune system plays a vital role in the prevention of tumor formation and promoting the process of the tumor removal. As an independent lymph cells population, NK (natural killer) cells are an important component of the innate immune system, without pre-sensitized to recognize and kill target cells. It is the first line of defense in tumor defense. During the tumor development, NK cells can directly clean tumor cells by secreting perforin and granzyme, and secreting cytokines adjust the function of other immune cells. So NK cells play an important role in antitumor immunity. The NK cell cytoxity ability was regulated by many moleculars such as NK surface reprtors and cytokines. The immune system plays an important role in anti-tumor. Meantime the tumor cells can express immune suppressor or suface reportors to regulate immune cell function, such as inhibit NK cell funcation, and induce the immune inhibition cells to further negtively regulate anti-tunor immune response, resulting in immune escape environment.Our prevous study has found that the tumor cells STAT3signal pathway inhibition can suppress the tumor proliferation in vivo and in vitro. But there few research about the detail mechanism between the activated STAT3signal pathway in tumor cells and the immune system inhibition. Therefore, this study will use a series of molecular and cell biology techniques to discuss the mechanism between the liver cancer cells STAT3signaling pathway activation and anti-tumor immune response mechanism.At present, the level of diagnosis and treatment of liver cancer has greatly improved. Treatments of liver cancer include surgery, radiation therapy, chemotherapy, targeted therapy, combined therapy, etc. But some disadvantages were found, such as great side effects, poor targeting, scarce resource of transplanted liver, which accounted for high cost, but poor efficacy. Due to the small side effects and the good targeting, tumor gene therapy attracted much attention in recent years. To find the right target and carrier is the key to gene therapy. This study use mice liver cancer cells and mouse tumor model as research object, observe the influence of blocking tumor STAT3signal pathway by STAT3Decoy ODN in tumor cell proliferation, the anti-tumor immune response and the influence of NK cells function; study the detail mechanism. On this basis, our study use STAT3Decoy ODN targeted nanoparticles to treat tumor bearing mice, to study the feasibility of blocking STAT3signaling pathways for the treatment of liver cancer. The study provides technical support and theoretical basis for STAT3as a target in tumor gene therapy.Methods:1. In vitro assays:Western blotting technology was adopted to detect the expression and phosphorylation levels of STAT3in murine liver cancer H22and Hepal-6cell lines. Lipofectamine2000was used to transfect STAT3decoy ODN into liver cancer lines. MTT assay was used for detecting the influence of STAT3decoy ODN on cell proliferation. Cell apoptosis was assayed by Annexin V/P1.2. In vivo assays:STAT3Decoy ODN was used to block murine liver tumor lines STAT3signal pathway. The BALB/c and C57b1/6tumor model were established to investigate tumor growth and mice survival. The percentage and activation of NK cells or T cells were analyzed by flow cytometry. NK cells from tumor bearing mice liver and spleen was isolated, and used as effector to lysis tumor cells by MTT assay in vitro.3. Cell depletion and transfer:NK or T cells were depleted by injecting anti-ASGM1or anti-CD3antibody to study the roles in tumor STAT3blockage induced anti-tumor immune response. NK cells were collected by MACs and the purity was detected by Flow cytometry. Different source of NK cells were injected i.p. into NOD-SCID mice, and tumor growth and mice survival were observed.4. Molecules assays:q-PCR were performed to confirm the mRNA levels of cytokines IL-10, TGF-β,IFN-α, IFN-β.Flow cytometry was used to confirm ligands Rael, H60and Multl expressed on tumor cells. ELISA was used to detect the cytokines IL-10, TGF-(3and cytokine IFN-y. The antibodies neutralization and lymphocytes deletion were performed to analyze the influence of IL-10, TGF-β and Treg cells to NK cell functions.5. Evaluation for targeting nanoparticle delivery of STAT3Decoy ODN: Using targeting nanoparticle carrier prepared by cooperation team, STAT3Decoy ODN was deliverd into HCC cells. Flow cytometry and fluorescence microscope was used to detect the transfection efficiency. FA receptor blocking assay was used to investigate the carrier targeting ability. STAT3Decoy ODN targeting therapy was evaluated by i.v. and intratumor injection in tumor bearing mice.Results1. Total STAT3and p-STAT3were constitutively expressed in two murine HCC cell lines Hepal-6and H22. Blocking STAT3by decoy ODN promoted murine HCC cell apoptosis in vitro.2. Blocking STAT3signal pathway by Decoy ODN inhibited HCC growth in vivo, prolonged the mice survival. Meantime, we found the NK frequency and activation were increased, but T cells didn’t show significant change. These results indicated that blocking STAT3signal pathway can enhance NK anti-tumor immune response.3. In the T cells immunodeficiency nude mice model, the STAT3Decoy ODN treated H22proliferation was inhibited, and the NK frequency and activation were increased. NK or T cells depletion was performed by injecting anti-ASGM1antibody and anti-CD3antibodies i.v., separetly, and liver tumor growth was increased in NK deletion mice, more than that in T deletion mice. These findings showed that bearing tumor mice anti-tumor immune response induced by blocking STAT3in HCC depended on the NK cells function.4. The purified nude mice NK was adoptively transfered into the NOD-SCID mice, and the tumor growth was observed. The survival rate of NOD-SCID mice transferred with NK cells from donor mice-bearing Decoy-treated-H22cells prolonged significantly.5. The sensitivity of HCC cell lines Hepal-6and H22to NK cytotoxity was increased by blocking STAT3signal pathway. Immune inhibitory cytokines IL-10and TGF-β were decreased, whereas the immune stimulatory cytokine IFN-a expression and NKG2D ligands Rael, H60and Multl were increased.6. STAT3blockade in HCC downregulated immune inhibitory cytokines and Tregs frequency decreased in mice bearing STAT3-blocked HCC cells, and HCC-induced immunosuppressive status was broken. 7. Using FA receptor targeting nanoparticle as carrier, STAT3Decoy ODN can be efficiently dilivered into tumor cells and inhibited the tumor growth efficiently.ConclusionIn our research, we use STAT3Decoy ODN to block liver cancer STAT3signal pathway in vivo and in vitro to study its influence in tumor cell proliferation and host anti-tumor immune response, especially NK function. We found the tumor proliferation was inhibited, and mice survival was prolonged by blocking STAT3signal pathway using STAT3Decoy ODN. Futhermore, NK frequency and activation were increased, but T cells didn’t show obvious change. Additionally, blocking HCC STAT3signal pathway can increase NKG2D ligand expression on HCC cells, which increased the sensibility of HCC cells to NK cells cytotoxity; meanwhile, the immune inhibition cytokines IL-10, TGF-β expression in the tumor cells decreased, as well as immune regulatory T cells numbers decreased. So, blocking tumor STAT3signal pathway can break HCC-induced host immune inhibitory status, and NK cell fuctions recovery played a key role in the process. Targeting STAT3might be a potential strategy for HCC therapy.Significants1. The study discovered for the first time that blocking STAT3signaling pathway in mouse hepatoma cells can reverse mouse anti-tumor immune response, especially the members of the innate immune system, NK cells function.2. By a variety of murine tumor models, lymphocyte clearance experiment, and adoptive transfer experiment, NK cells have been proved to play an extremely important role in anti-tumor immune responses induced by the STAT3blocked hepatoma cells.3. By antibodies neutralization and cells clearance experiments, it was demonstrated the immune inhibitory cytokines and immunoregulatory T cells regulated by STAT3play an important role in immune suppression induced by mouse hepatoma cells.4. The feasibility analysis showed that STAT3Decoy ODN delivered by FA receptor nanoparticle displayed anti-HCC effects. This study provided detail theoretical basis and technical supports in application of STAT3in tumor targeted therapy.