Study Of Preparation Of The B16F10 Cancer Stem Cell Vaccine Expressing ESAT-6-gpi And IL-21 And Its Antitumor Effects

Melanoma (MM) is a malignant tumor and is of the leading cause of death related to skin cancers. The rapid progression of primary melanomas to locally invasive and metastatic other sites is a major obstacle for an early effective diagnosis and a therapeutic intervention for melanoma patients. But the molecular mechanism of metastasis in melanoma is little understood. More studies are needed to unfold the molecular mechanisms underlying melanoma progression and to explore new strategies for clinical therapy.Tumor immunotherapy has developed into a new method of tumor treatment. Immunotherapy generally includes:cytokine therapy, adoptive immunotherapy, tumor vaccine, antibody immune therapy, molecular targeted therapy, etc. The tumor therapeutic vaccines, especially tumor cell vaccine have made gratifying progress. Tumor vaccines change local microenvironment after inoculation, enhance the activities of antigen presenting cell (APC) and tumor specific cytotoxic T lymphocyte (CTL), natural killer cells (NK) cytotoxic, produce antitumor protective immune response. But due to weak immunogenicity of tumor antigens, complex tumor immune escape mechanism, how to improve the immunogenicity of tumor vaccine, stimulate the host immune system against tumor cell challenge has become one of research contents. In the early study, we used a novel strategy for developing tumor vaccine that expresses 6 kDa early secreted antigenic target (ESAT-6) in the glycosylphosphatidylinos-itol (GPI)-anchored form together with secreting IL-21. This novel vaccine can express artificial labeled heterogenetic antigen ESAT-6 on tumor cell membrane surface, secrete IL-21 into tumour inoculated site, and elicit anti-melanoma immune responses. However, how to evaluate this immune effect on tumor stem cells (TSCs) or cancer stem cells (CSCs), it is needed to further study.It has been increasingly evident that there is a small subpopulation of tumor cells possessing stem cell characteristic, and these cells are responsible for the tumorigenesis, chemoresistance, radioresistance, aggressiveness, metastasis and recurrence. These subpopulations are a subset of cells with stem/progenitorcell features known as CSCs. CSCs as the target cells of anti-tumor research, it have been deeply investigated, for example, the mice were immunized with dendritic cells together with CSCs, showing obvious anti-tumor effect.In this study, melanoma cell line B16F10 was selected as our investigation object. And the B16F10 CD133+CD44+cancer stem cell vaccine was developed, which express the GPI-anchored ESAT-6 together with secreting IL-21 (B16F10-ESAT-6- gpi/IL-21) and its anti-tumor efficacy and the mechanisms in murine tumor model was evaluated.Objective:To develop the melanoma B16F10 CSC vaccine expressing the gpi-anchored ESAT-6 and secreting IL-21 and to evaluate the antitumor efficacy of B16F10 CSC vaccine in murine tumor model.Methods:The constructed recombinant plasmid pIRES-ESAT-6-gpi/IL-21 were transfected into B16F10 cells and the stably transfected clones were isolated and identified. CD133+CD44+ CSCs were isolated from B16F10-ESAT-6-gpi/IL-21 cells with magnetic activated cell sorting system. The C57BL/6 mice were immunized with 5×105 B16F10-ESAT-6-gpi/IL-21 CD133+ CD44+CSCs inactivated with mitomycin C three times with interval of 14 days between the immunizations. All mice were challenged s.c. with 5×105 B16F10 cells 10 days after final immunization. An induced immune responses and antitumor efficacy as well as EMT associated molecules of tumor tissues were evaluated.Results:1. The results of immunofluorescence and Western blot demonstrated that the ESAT-6 was actually anchored on the surface of B16F10-ESAT-6-gpi/IL-21 cells and IL-21 was secreted.2. The tumour sizes in the mice immunized with inactivated B16F10-ESAT-6-gpi/IL-21 CD133+CD44+CSC vaccine were lower than that of control groups, but tumor-free mice were more than in the control groups, which was statistically significant (P< 0.01).3. The CSC vaccine could result in elongation of mice’s survival, increase of levels of anti-ESAT-6 and serum IFN-y, augmentation of the activity of CDC, NK, and CTL as well as decrease of Vimentin expression and increase of E-cadherin expression.Conclusion:These results suggest that our prepared B16F10-ESAT-6-gpi/IL-21 CD133+CD44+CSC vaccine induced powerful antitumor efficacy in mice, and CSC vaccine may serve as a new way for treatment of melanoma.