Preliminary Investigation Of Antitumor Effects And Mechanisms Of The Melanoma Stem Cell Vaccine

Research Background:Malignant melanoma(MM)is an increasing incidence of invasive tumors in the world,accounting for 7%to 20%of skin malignancies,with a high degree of malignancy,easy to metastasize,relapse and secrete the transforming growth factor-beta(TGF-?)and other inhibitory factors,so once melanoma metastasis appeared,and it is difficult to be cured.In recent years,studies have indicated that there are cancer stem cells(CSCs),also known as tumor stem cells(TSCs),in ovarian cancer,breast cancer,colon cancer,myeloma and melanoma etc.It is known that CSCs have the characteristics that indicate the roots of an anti-radiotherapy and anti-chemotherapy,recurrence and metastasis.Theoretically,the elimination of CSCs can control tumor recurrence and metastasis,and decrease its resistant to radiotherapy and chemotherapy.Traditional treatments,such as radiotherapy and chemotherapy,these do not target CSCs that resulted in five-year survival rate being low in patients with tumors.Therefore,it is very important to find a new strategy for treatment of tumors.Tumor immunotherapy is the clinical treatment method to produce an effective treatment effects on inhibition of tumor growth by the activation of the body's immune system,because it can induce an effect immune responses by targeting tumor cells to play an anti-tumor effect.The effectiveness of a variety of immunotherapists has been demonstrated in preclinical and clinical treatments such as lymphokine-activated killer(LAK)cells,natural killer(NK)cells,T cells,dendritic cells,cytokine therapy and antibody treatment and so on,in which the therapeutic vaccines have made advancement.Objective:To investigate the effects and mechanisms of melanoma B16F10 CD133+CD44+CSC vaccine for the treatment of melanoma.Methods:B16F10 CD133+CD44+CSCs were isolated by immunomagnetic beads sorting technique.C57BL/6 mice were immunized with by 5×105 B16F10 CD133+CD44+CSC vaccine inactivated with MMC three times,an interval of 10 days between the immunizations.At 10 days after the last immunization,all mice were challenged with 1×105 B16F10 wild type cells.At the same time,the B16F10 non CSC vaccine group and the B16F10 wild type cell vaccine group were used as control vaccine groups.The levels of TGF-?,IFN-? and IL-4,cytotoxic activities of spleen cells and NK cells were respectively measured in the vaccine immunized mice.The tumor growth and survival time in each mouse was monitored.Meanwhile,the expression levels of NY-ESO-1,PD-1 and WISP-2 in tumor tissues of mice vaccinated with B16F10 CD133+CD44+CSC,B16F10 non CSC,and the B16F10 wild type cell vaccines were detected by QPCR and Western-Bolt,respectively.Results:The tumor volume and tumor growth rate of B16F10 CD133+CD44+CSC vaccine group were lower than those of control tumor vaccine groups,and the difference was statistically significant(**p<0.01).The levels of IFN-?/IL-4 ratio and cytotoxic activities of spleen cells and NK cells in B16F10 CD133+CD44+CSC vaccine group were higher than those in the control tumor vaccine groups,while the level of TGF-? was decreased in the CSC vaccine group.Analysis results of qPCR showed that the expression of NY-ESO-1and PD-1 in B16F10 CD133+ CD44+cells was higher than those in the control cells but the expression of WISP-2 was low.The results of Western-Bolt indicated that the expression of NY-ESO-1 and PD-1 in the tumor tissues immunized with B16F10 CD133+ CD44+ CSC vaccine group was lower than those in the control tumor vaccine groups,but the expression of WISP-2 was high.The difference was statistically significant between the CD133+CD44+ CSC vaccine group and the control vaccine groups(**p<0.01).Conclusion:The B16F10 CD133+CD44+ CSCs vaccine prepared in this study can effectively induce the specific cellular immune response and humoral immune response of B16F10,which shows an effective antitumor effects.The mechanisms may be involved in regulating expression of tumor-associated antigenic proteins by signal pathway proteins.This study provides experimental and theoretical basis for CD133+CD44+ CSC vaccine in the treatment of melanoma.