The Antitumor Effects Of Embryonic Stem Cells In Vitro And In Vivo

Lung cancer is the most dangerous threat to human health and life nowadays. Despite taking a comprehensive range of treatment, the survival rate of patients is still not significantly improved, the overall1-year survival rate is less than30%, while the5-year survival less than16%. The clinical treatment effects of widely used traditional therapies, such as surgical treatment, chemotherapy and radiotherapy is not ideal, even the effects the rapidly developed molecular targeted therapy and individualized treatment is not satisfied, the reason may be due to the distant metastasis of lung cancer and local recurrence after treatment. So if some treatment or drugs have not only the direct anti-tumor effect, but also improving immune function and eradicating the circulatory system micrometastasis and distant metastasis, are undoubtedly the gospel of patients with lung cancer, and these characteristics are precisely the advantages of tumor immunotherapy.From the point of view of immunology, tumor development is the result of the interaction between the tumor and the immune system, the body’s immune system is a powerful and complex regulatory body and performmance network, under normal circumstances, the tumor cells have clear antigenic markers can be immune recognition and clear away, however, the tumor cells undergo a series of changes in functional and structural aspects, to enable them to evade the immune system surveillance and killing effect. For example, by altering the expression of surface antigens, the immunogenicity of tumor cells weakened, resulting weak specific immune response produced in the body; by missing cell-surface MHC molecules, or by the downward or mutation of their expression, tumor cells could inhibit the antigen-presenting process in vivo, which can not be recognized by cytotoxic lymphoid T cells (CTL) and further affect tumor cells clearance in vivo; most tumor cells are abnormal in costimulatory molecule expression, which lead to lack of synergy in the cellular immune signaling, thereby evading the body’s immune system attacks; the body’s functional defects caused by tumor burden, lead to dendritic cells (DC) apoptosis, and thus the antigen-presenting ability becomes poor and unable to properly activate naive T cells which cause tumor immune tolerance in vivo; most tumor cells in the body can secrete a variety of immune suppression factors, such as transforming growth factor-β (TGF-β), interleukin-10(IL-10), vascular endothelial growth factor (VEGF), sialomucin and macrophages cell migration inhibitory factor (MIF), etc., which could greatly inhibit the differentiation and secretory function of T lymphocytes cells in the body, and destroy the balance state of Th1/Th2cytokines, thus contributing to the growth and proliferation of tumor cells in vivo.It was found that in clinical studies of tumor immunotherapy, because of the tumor inhibition on the body immune system and the altered expression of the tumor cell surface antigen molecules, cancer cells could escape from the body immune surveillance, and it was difficult to trigger an effective immune response in vivo. While ESCs and their derivative cells could effectively activate CD4+and CD8+T lymphocytes, and there were studies confirming that the MHC-I molecules and other protein antigens on the ESCs surface could induce T lymphocytes mediated cytotoxic lymphocytes as effecter cells in immune response in vivo. More importantly, the stable and durable immune response induced by embryonic stem cells, could cross-react with cancer cells because of the similar surface antigen, so the body had achieved effective anti-tumor immunity, which could find a new way of thinking for cancer immunotherapy. Looking back to the history of using ESCs antigen in tumor immunotherapy, we found that anti-tumor therapies involving embryonic tissues or ESCs had been carried out for many years. Although the mechanism was not clear, Schone had pointed out that animals vaccinated with embryonic tissues could significantly inhibit the growth of transplanted tumors many years ago. Since then, a series of studies had confirmed that inoculation by embryonic tissues could not only inhibit the growth of transplantable tumors of animals, but also could prevent tumor formation induced by cancer-causing factors, both tumor occurrence and development could be significantly inhibited. Researchers from Louisville University Cancer Center have found that the experimental mice inoculated with ESCs could prevent the occurrence of planting and induced tumors in2006, and this was the first time declaration of the antitumor effects of ESCs. The study from a Chinese scholar Yi Li has shown that, mice inoculated with human embryonic stem cells could also produce anti-tumor immunity against colon cancer cells, they believed that this kind of anti-tumor effect was a broad-spectrum one, and common anti-cancer vaccines could be developed on this basis. Recent researches from Kavitha Yaddanapudi confirmed that the induced anti-tumor immunity by embryonic stem cells was not foreign body reaction, which may be specific immune responses against ESCs constituted by a variety of effecter cells and cytokines.On the other hand, there have been a lot of studies on the anti-tumor effects of embryonic stem cells in vitro, these studies also has shown us a series of satisfactory results. Raof and Postovit et al declared that the micro-environment of embryonic stem cells could induce tumor cell re-programming and positive transformation, and be able to inhibit the occurrence and metastasis of local tumor in vivo to a certain extent; Giuffrida and other researchers found that embryonic stem cells in vitro environment are capable of secreting a soluble anti-tumor factor, having the role of inhibition of growth of various tumor cells, and can block the cell cycle of the tumor cells; Lee et al confirmed that embryonic stem cell microenvironment was able to change the mode of growth of tumor cells, inducing malignancies obtained certain benign phenotype, which has anti-tumor effects; Kulesa et al found that the development of embryonic neural crest microenvironment enables highly metastatic melanoma to normal phenotypic transformation, reduce the degree of malignancy and the performance of their cells of origin cytology shape; recent research results show that embryonic stem cells can be induced to differentiate into and to stimulate the body’s anti-tumor immunity antigen presenting cells-dendritic cells, or directly kill tumor cells and differentiate into immune effector cells, such as natural killer cells (Natural killer cell, NK), monocytes and granulocytes in vitro environment.This study was built on the summary of previous researches, and took a deep look into anti-tumor effects and mechanisms of ESCs from two aspects in vitro and in vivo: observing and detecting the ESCs direct inhibitory effect on cancer cells and the interaction between ESCs and cancer cells by the establishment of ESCs-Lewis lung carcinoma cells co-culturing system in vitro; building tumor mouse model to explore the mechanism of induced anti-tumor immunity by ESCs from the aspects of effecter cells, cytokines, and local pathological changes of tumor tissue. This study will open up new research areas of immunotherapy for lung cancer and the anti-tumor effect of ESCs, and bring new hopes for the prevention and treatment of lung cancer.CHAPTER I The anti-Lewis Lung Carcinoma effects of ESCs in vitroSECTION I The inhibition effects of embryonic stem cells on Lewis lung carcinoma proliferationOBJECTIVE:To study the inhibition effects of ESCs on Lewis lung carcinoma cells under co-cultured condition in vitro, and to explore the possible mechanisms of ESCs against cancer.METHODS:Building the C57/BL6mouse ESCs-Lewis lung carcinoma cells non-contact Transwell co-culture system in vitro, studying the important changes of Lewis lung carcinoma cells in biological behavior, including cell cycle, apoptosis, and tumor cell invasion ability under co-culturing condition.RESULTS:After the non-contact co-culture with ESCs, the cell proliferation of Lewis lung carcinoma cells was significantly inhibit, cell cycle was in stagnation of G2/M phase and the cell apoptosis increased significantly, and the cell invasion ability was significantly decreased, all results showed substantially statistical significance (P<0.05).CONCLUSION:The embryonic stem cells have significant anti-tumor effects in vitro, including the ability to inhibit tumor cell proliferation and to promote apoptosis of tumor cells, and significantly change the invasion ability of the tumor cells. SECTION II The anti-tumor effects of embryonic stem cells in the treatment of Lewis lung developmentOBJECTIVE:To study the inhibition effects of ESCs on the tumorigenic ability of Lewis lung carcinoma cells under co-cultured condition in vitro, and to explore the possible mechanisms of this kind of effects.METHODS:Through the establishment of C57/BL6mice with Lewis lung cancer animal model to study tumorigenicity change of Lewis lung carcinoma cells after co-cultured with embryonic stem cells in vivo. Tumor growth of mice in experimental group and the control group were detected, and the tumor cell apoptosis is detected by the TUNEL method.RESULTS:The tumor formation ability of Lewis lung cancer cells after co-cultured with embryonic stem cells was significantly decreased, the increase of subcutaneous tumor volume and weight of the experimental group mice was subject to significant inhibition, and the apoptosis of the experimental group mice tumor tissue was significantly increased, all the results have statistical significance (P<0.05).CONCLUSION:In vitro, the tumor formation ability of Lewis lung carcinoma cells was significantly inhibit due to co-culture with embryonic stem cells, the tumor growth was significantly inhibit in vivo entities and the cell apoptosis was increased. The tumorigenic ability was a typical ability of malignant cells; the possible mechanism may be due to the inhibition effect of embryonic stem cells on cancer stem cells. CHAPTER II The anti-Lewis Lung Carcinoma effects of ESCs in vivoSECTION I The vaccination effects of embryonic stem cells in Lewis lung carcinoma occurrencesOBJECTIVE:To study after embryonic stem cells inoculation, the mechanism of induced anti-tumor immune response in mice, and its role in the prevention of Lewis lung carcinoma occurrences in vivo.METHODS:Embryonic stem cells (y-rayed) were injected subcutaneously into the C57/BL6mice as immunization (three times at7days interval), and then the number and proportion of peripheral blood lymphocytes (CD3+, CD4+, CD8+T cells and CD19+B cells) were detected by flow cytometry, serum-free cell factor (IL-2, IL4and of IFN-y) level was detected by ELISA. After Lewis lung carcinoma cells inoculation, subcutaneous tumor growth was closely monitored and the tumor growth curve was draw. When the animal experiments were finished, spleen lymphocyte proliferation was detected, the tumor tissues were isolated and weighed, tissue sections were embedded and CTL lymphocyte infiltration within the tumor tissue was detected by IHC, cancer cell apoptosis in tumor tissue was detected by TUNEL.RESULTS:The absolute value and proportion of peripheral CD3+/CD8+T lymphocytes in experimental mice was significantly higher compared with the control group, serum level of anti-tumor cytokine (IL-2and IFN-y) was significantly elevated and splenic T lymphocytes proliferation was obviously increased, while the tumor incidence rate was significantly lower (P<0.05); tumor growth in experimental mice and the tumor growth rate was significantly inhibited, pathological tumor CTL lymphocytes infiltration within the tumor tissues were significantly increased and cell apoptosis was significantly different (P<0.05).CONCLUSION:The vaccination by embryonic stem cells can induce significant anti-tumor effect in mice; the anti-tumor immune levels were significantly increased, which can effectively prevent the occurrence of transplanted tumors in mice. SECTION II The anti-tumor effects of embryonic stem cells in the treatment of Lewis lung developmentOBJECTIVE:To explore the possible mechanism of embryonic stem cells induced anti-tumor immune response in mouse tumor models, and its therapeutic effects on tumor development.METHODS:The C57/BL6mice were inoculated subcutaneously with Lewis lung carcinoma cells to establish tumor animal models, and the tumor inoculation time and growth of tumors were used to simulate different tumor development period. The y-ray inactivated embryonic stem cells were injected subcutaneously into mice as immunotherapy (three times at7days interval), the changes in number and proportion of peripheral blood lymphocytes were detected by flow cytometry and mice serum cytokine levels was checked by ELISA. The subcutaneous tumor growth was closely monitored and tumor growth curve was made, the tumors were isolated and weighted and the CTL lymphocytes infiltration was checked by IHC, spleen lymphocytes proliferation was detected tumor cell apoptosis was checked by TUNEL up to the end of the animal experiments.RESULTS:Inoculation of embryonic stem cells in tumor mice model was able to stimulate the CD3+/CD8+T lymphocyte proliferation, significantly increase the serum level of Thl-type anti-tumor cytokines (IL-2and of IFN-y)(P<0.05). The immunosuppression status in bearing mice was significantly improved and the growth of solid tumors was obviously inhibited. Pathology experiments showed that spleen T lymphocyte proliferation in experimental mice was significantly increased, more CTL lymphocytes infiltration was detected in tumor tissues, and there was a significant difference in the degree of tumor cell apoptosis (P<0.05).CONCLUSION:Inoculation of embryonic stem cells in a tumor mice model could significantly improve the body immunosuppression status and induce anti-tumor immunity which could significantly inhibit the solid tumor growth and progress.