Study Of Anti-ovarian Cancer Effects And Mechanisms Induced By Ovarian Cancer Stem Cell Vaccines

Ovarian cancer is one of the three main malignant tumors in female reproductive system,in which epithelial ovarian cancer?EOC?accounts for 85%to 90%of ovarian cancer.Although chemotherapy regimens are constantly optimized,EOC therapeutic effects are not so satisfied,and the 5-year survival rate remains about 30%-40%.The recurrence rate is more than 60%and its mortality ranks the first one in gynecologic malignant tumors.So far,the EOC has become a serious threat to women's lives and health.Cancer stem cell?CSC?are a small subset in tumor tissues and is capable of self-renewal,differentiation,unlimited proliferation,and posses the features of stem cells.CSC are responsible for the origin of tumorigenesis,tumor development,chemoresistant,metastasis,the invasion and recurrence.Therefore,it is necessary that exploring the characteristics of EOC CSC and finding the efficient methods to target CSC and to improve the therapeutic effects on EOC are the key to control and eradicate ovarian cancer.Tumor immunotherapy has been highlighted in recent years since some encouraging progress has been made both in basic research and clinical application research.Due to CSC's characteristics,researchers have focused on targeted immunotherapies towards CSC,which includes following aspects:targeted immunotherapies directed toward CSC by surface specific markers,survival diverticulum,blocking critical signal pathways,inhibiting efficient DNA repair,manipulation of ncRNAs,screening drug-resistance of CSC in three dimensional culture as well as DC vaccine targeting the CSC,etc,in especial vaccine research,which is outstanding for its ability of activating the body's natural killer cells?NK?and specific cytotoxic T cells?CTL?,producing protective antibodies from antitumor immune responses,thus,tumor vaccine is a promising method for tumor immunotherapy.Because of the weak immunogenicity of tumor antigens,the complicated mechanisms of tumor immune escape and the heterogeneity of tumor cells,the actual efficacy of tumor vaccines still remain a big gap with the expected effect as a therapeutic cancer vaccines in clinic in spite of many encouraging researches reported.There are some obstacles that need to overcome and the key of them is to find a way to improve the immunogenicity of tumor vaccines and stimulate the host immune responses for attacking tumor cells.Recent studies have shown that CSC vaccines,due to its high expression of the dominant antigens,can trigger an effective targeting CSC immune responses to inhibit tumor growth and metastasis.Previous work of our research group has found that the CD44⁺CD117⁺EOC SKOV3/HO8910 cells own the characteristics of CSC.Therefore,we isolated EOC CSCs from human SKOV3/HO8910 cell lines or ovarian cancer stem-like cells?OCSC?from mouse ID8 cell line,and developed the deliquescent and intact CSC vaccines with a simple and economical way to explore its anti-anti-ovarian effects and mechanisms in this study.Objective:To explore the anti-tumor effects and mechanisms induced by EOC CSC vaccines in animals and to provide experimental and theoretical basis for future clinical use of EOC CSC vaccines in the treatment of ovarian cancerMethods:1.The CD117⁺CD44⁺CSC were isolated from human EOC SKOV3 and HO8910 cell lines by using a magnetic-activated cell sorting system?MACS?,respectively.Each nude mouse was subcutaneously inoculated with the 4×10⁵inactivated SKOV3 or HO8910 CD117⁺CD44⁺CSC three times,which were repeated freezing and thawing three times before inoculated,an interval of 10 days between the immunizations,and then all immunized mice were challenged subcutaneously with 5×10⁶SKOV3 or HO8910 cells after final inoculation.The anti-cancer effects of SKOV3 and HO8910 CSC vaccines were evaluated by tumorigenicity and survival conditions of various vaccine immunized mice.Subpopulation analysis of CD117⁺CD44⁺and ALDH-1^highigh tumor cells via flow cytometry?FCM?;in addition,detection of mice serum IFN-?and TGF-?using enzyme-linked immunosorbent assay?ELISA?;NK cell cytotoxicity assay and both qRT-PCR and western blotting were carried out to measure the expression level of NKG2D and MICA for the preliminary analysis of anti-tumor effects and its molecular mechanisms.Meanwhile,the NOD/SCID mice served as a mouse model to proof an immunological effects CSC vaccines from the opposite direction.2.The receptor tyrosine kinase like orphan receptor 1?ROR1?may be one of dominant antigens of EOC CSC,which plays an important role in the activation of immune responses triggered by CSC vaccines.We designed the short hairpin RNA?shROR1?plasmid by adopting Gateway site-specific recombination and amplification of ROR1 gene to construct lentivirus recombinant plasmid.The lentivirus ROR1?pLV-shROR1 or pLV-ovROR1?was produced from the transient transfection of the HEK293T cells with pHAGE-CMV-shROR1-IZsGreen or pLV-ovROR1,psPAX2,and pMD2.G plasmid DNAs plus Lipofectamine2000.Forty-eight hours after the co-transfection,the lentivirus-bearing supernatants were collected and passed through a 0.45-mm filter.HO8910 cells were infected with the pLV-shROR1 or pLV-ovROR1.The stable expression colonies were selected by limiting the dilution assay.The HO8910-pLV-shROR1CD117⁺CD44⁺CSC or HO8910-pLV-ovROR1 CD117⁺CD44⁺CSC were isolated for making the vaccine and then the various vaccines were inoculated into mice,respectively,as above mentioned method.The anti-cancer effects of HO8910 CSC vaccine with down-regulated or up-regulated ROR1 expression were evaluated by tumorigenic experiments.In addition,the immune effects of HO8910-pLV-shROR1 CSC and HO8910-pLV-ovROR1 CSC vaccines were respectively analyzed by FCM,ELISA,NK cytotoxicity,qRT-PCR and western blotting assay.According to these results,the effect of dominant antigen ROR1 on anti-tumor efficacy of HO8910 CSC vaccine was further assessed.3.OCSC were obtained from serum-free culture medium?SFM?cultivation.2×10⁵ OCSC after repeated freezing and thawing three times were inoculated into each C57BL/6 mouse,a total of three times with an intervals of 10 days,and the mice were challenged subcutaneously with 2×10⁶ ID8 cells 10days after last immunization.All vaccinated mice's tumors growth and survival were respectively observed.The ROR1 expression of down-regulation in mouse EOC ID8 cell was established as the same way of HO8910 cells.Moreover,the anti-cancer effect of ID8 OCSC-shROR1 vaccine was evaluated in C57BL/6mice respectively using the same method as above.FCM,ELISA,NK cytotoxicity,CD4⁺T,and CD8⁺T cytotoxicities,complement dependent cytotoxicity?CDC?assays,as well as qPCR and Western blotting assays were respectively used for further analysis of the vaccine's immune responses and anti-tumor effect mechanisms.Besides,the vaccine developed by the dendritic cell?DC?loaded with OCSC lysate were used in vaccinal experiment to compare an immune effects between the developed vaccine from the OCSC lysate and the developed vaccine from the DC loaded with OCSC lysate in this study.Results:1.The vivo experiment showed that the tumor growth were slower,tumor sizes were smaller,and the tumor-free life was longer in SKOV3/HO8910 CD117⁺CD44⁺CSC vaccinated mice than those of SKOV3/HO8910 cell,non-CD117⁺CD44⁺CSC,and PBS vaccinated mice.Moreover,the mice vaccinated with SKOV3/HO8910 CD117⁺CD44⁺CSC vaccines could significantly increased the serum IFN-?but decreased the TGF-?levels as well as enhanced the NK cytotoxicity compared with the control groups?p?27?0.05?.The results of qRT-PCR and western blotting showed that higher levels of perforin and granzyme,and higher levels of NKG2D and MIC were found in the mouse tumor tissues from SKOV3/HO8910 CD117⁺CD44⁺CSC vaccination groups compared with SKOV3/HO8910 cell,non-CD117⁺CD44⁺CSC,and PBS vaccination groups?p<0.05?.Besides,SKOV3/HO8910 CD117⁺CD44⁺CSC vaccinated mice could induce strongly immune response to target CD117⁺CD44⁺cells or ALDH-1^highigh tumor cells so as to shrink the subsets of both CD117⁺CD44⁺tumor cells and ALDH-1^hightumor cells in tumor tissues.On the contrary,there was no significant effect in NOD/SCID mouse model.All above data give evidence that CSC vaccination probably enhanced the immune responses in nude mice to generate adequate efficacy of against tumors.2.HO8910-pLV-shROR1 cells?HO8910-shROR1?infected with the lentiviral plasmid pLV-shROR1 and HO8910-pLV-ovROR1 cells?HO8910-ovROR1?infected with the lentiviral plasmid pLV-ovROR1 were successfully selected,respectively.The down-regulation or up-regulation of ROR1 expressions in both mRNA and protein levels in HO8910-pLV-shROR1 cells and HO8910-pLV-ovROR1 cells were verified by qRT-PCR and western blotting assays.After expanded cultivation,HO8910-shROR1 CD117⁺CD44⁺cells and HO8910-ovROR1 CD117⁺CD44⁺cells were isolated via MACS for developing vaccines through repeated freezing and thawing three times,and then were immunized into mice.The in vivo vaccine experiment results showed that anti-tumor effects of HO8910-shROR1 CSC or HO8910-ovROR1 CSC or HO8910 CSC vaccination in athymic mouse model revealed the tumors grew most slowly,tumor sizes were smallest and the tumor-free life was longest in mice of HO8910-ovROR1 CSC vaccination than those of other groups,in which the HO8910 CSC vaccine group ranked two and HO8910-shROR1 CSC vaccine group ranked three.Moreover,the mice vaccinated with HO8910-ovROR1 CSC vaccine markedly enhanced the serum IFN-?and decreased the TGF-?levels as well as enhanced the NK cytotoxicity compared with the control groups?p?27?0.05?.In addition,the highest levels of the granzyme,perforin,NKG2D and MICA analyzed by both qRT-PCR and western blotting assays were found in tumor tissues of mice vaccinated with HO8910-ovROR1 CSC vaccine.Furthermore,HO8910-ovROR1 CSC vaccination could significantly reduce the proportion of CD117⁺CD44⁺CSC cells and ALDH^highcells in xenograft tumor tissues detected by FCM.The above results demonstrated that the ROR1 molecule may be the dominant antigen of EOC CSC vaccines and play a key immunogenicity role in HO8910 CSC vaccine.3.Based on tumor growth rate,tumor sizes and tumor-free survival in C57BL/6 mouse experiments,the anticancer effects showed that ID8-OCSC vaccine was better than non-OCSC vaccine,which is statistical significance?p?27?0.05?.When the ROR1 molecule was down-regulated,the anticancer effects of ID8-OCSC-shROR1 vaccine was lower than that of ID8-OCSC vaccine,and the differences were statistically significant between two groups?p<0.05?.Moreover,the mice vaccinated with ID8-OCSC-shROR1 vaccine group remarkably reduced the serum IFN-?,decreased the NK cytotoxicity,splenocyte cytotoxicity,antibody level,and CDC activity,respectively but strengthened the TGF-?level compared with ID8-OCSC vaccine group,and the differences were statistically significant between two groups?p<0.05 or p<0.01?.Meanwhile,CD4⁺T,and CD8⁺T cytotoxicities from the mice immunized with ID8-OCSC-shROR1 vaccine were also decreased,in especial CD8⁺T cytotoxicity,which were statistically significant compared with the ID8-OCSC vaccine group?p<0.05 or p<0.01?.In addition,the lower levels of granzyme and perforin,NKG2D and MIC A analyzed by both qRT-PCR and Western blotting assays were lower in tumor tissues of mice vaccinated with ID8-OCSC-shROR1 vaccine group than those of ID8-OCSC vaccine group,which is statistical significance?p?27?0.05?.Besides,ID8-OCSC-shROR1 vaccination could significantly increase the proportion of ALDH^highcells in mouse ovarian cancer tumor tissues compared with ID8-OCSC vaccination,in spite of a little bit of lower than that non-OCSC vaccination,detected by FCM,showing that ID8-OCSC-shROR1vaccination induced the weaker killing activity of targeting OCSC than that of ID8-OCSC vaccination.These results not only manifested that ID8-OCSC vaccination has a better against OCSC effects but also demonstrated ROR1 may be the dominant antigen of EOC ID8-OCSC vaccine and played a key immunogenicity role in ID8-OCSC vaccine.While the immune response against OCSC effects of ID8-OCSC-shROR1 vaccine became attenuated in C57BL/6 mouse model.Additionally,the anti-tumor effects of developed vaccine by DC loaded with ID8-OCSC lysate was a slight better than that of ID8-OCSC vaccine developed by the OCSC that were repeated freezing and thawing three times,however,there was no statistically significant between two vaccines?p?29?0.05?.Conclusions:1.Human EOC SKOV3/HO8910 CD117⁺CD44⁺CSC vaccination could induce nude mice to produce antitumor immune against ovarian tumor growth and the mechanisms were associated with the CSC vaccine with high expression of the dominant antigen ROR1 that induce a strong immune responses;down-regulation or up-regulation the expression of ROR1 can affect the CSC vaccine immune effects and anti-ovarian cancer effects.2.Mouse ID8 OCSC vaccination could induce C57BL/6 mice to generate immune effects against murine ovarian cancer growth,and its effect mechanism was also related to ID8 OCSCwith high expression ROR1 antigen;down-regulation of ROR1 expression can influence ID8 OCSC vaccine immune effects and its anti-ovarian cancer effects.3.In this study,our developed the EOC CSC vaccines could effectively enhance the antitumor effects in the immunized mice,and the data may provide the experiment basis for treatment of EOC in clinic by using CSC vaccine.