Study On The Anti-angiogenesis Mechanism Induced By A DNA Vaccine Encoding Extracellular Domain Of Porcine Endoglin

Background Cancer is one of the most common causes leading human death. However, to date, despite aggressive approaches have made in the therapy of human cancer in the past decades, the prognosis of cancer remains poor, five year mortality exceeds 90% worldwide. It is clearly need to seek more rational alternative strategy for treatment of cancer. Nowadays, cancer active immunotherapy is an attractive strategy for cancer treatment, but some limitations remain to be solved. Thus, active immunotherapies, which target the blood vessels that nourish the growing tumor cells rather than the tumor cells themselves, are possible alternatives to overcome these obstacles of conventional immunotherapy. Angiogenesis is important for normal embryonic development, menstruation, inflammation, and for the development of pathologic conditions such as tumor, rheumtoid arthritis, retinopathies, etc. Several lines of direct and indirect evidence indicate that the growth and metastasises of tumor are angiogenesis-dependent. As a strategy for cancer therapy, antiangiogenic therapy attempts to stop new vessels from forming around a tumor and break up the existing network ofabnormal cappillaries that feeds the cancerous mass The breaking of immune tolerance against important molecules such as some receptor associated with angiogenesis on autologous angiogenic endothelial cell should be a useful approach for cancer therapy by active immunity. However, the immunity to the self-molecules on angiogenic vessels is presumably difficult to elicit by autologous or syngeneic molecules as vaccine due to the immune tolerance acquired during the development of the immune system In addition, endoglin has been shown to be one of the powerful markers for tumor angiogenesis. Endoglin may be used as an ideal model molecule to explore the feasibility for tumor active immunotherapy. Objective The current studies explore the feasibility of immunotherapy of tumors with the plasmid DNA encoding the extracellular domain of porcine endoglin as a vaccine by the breaking of the immune tolerance against mouse endoglin in a cross-reaction between the porcine and murine xenogeneic homologous endoglin molecules. Methods The full-length cDNAs of extracellular domains of porcine and murine endoglins were amplified by RT-PCR from the total RNA isolated from the porcine and murine embryo livers. Then the cDNAs was inserted respectively into the eukaryotic-expression vector pcDNA 3.1(+), the resultant recombinant plasmids (named as ppEDG and pmEDG respectively) were confirmed to be correctly expressed in eukaryotic COS-1 cells in vitro. Thereafter, these DNA plasmids were used as vaccine to observe whether or not they hadanti-tumor activities in murine tumor models. Autoantibodies against self-endoglin were detected by ELISA, Western blot analysis and ELISPOT assay. The anti-tumor activities of the autoantibodies were certified by the immunoglobulins purified from the serum of the immunized mice. Anti-angiogenesis capability was detected by immunohistochemistry and Alginate encapsulated tumor cell assays. Results We found that tumor therapy with plasmid DNA encoding extracellular domain of porcine endoglin (ppEDG) was effective at both protective and therapeutic antitumor immunity in three murine tumor models. Murine endoglin specific autoantibodies in sera of mice immunized with ppEDG could be found in Western blotting analysis and enzyme-linked immunospot assay. The major antibody subclasses induced by the ppEDG immunization were IgGl and IgG2b. The purified immunoglobulins were effective at the inhibition of tumor growth similar to that by active immunization with ppEDG Histological examination revealed that IgG autoantibody was deposited on the micro-vessels within the tumor tissue from mice immunized with ppEDQ and intra-tumor angiogenesis was significantly suppressed. Furthermore, balls from alginate encapsulatd tumor cell assay in mice immunized with ppEDG indicated significantly vessels suppression happened. Conclusion Immunization with ppEDG vaccine inhibits tumor growth and angiogenesis, these observations may provide a vaccine strategy for cancer immunotherapy through the induction of autoimmunitiesagainst the molecule-associated with angiogenesis on autologousangiogenic endothelial cell in a cross-reaction with single xenogeneichomologous gene.