The Anti-ovarian Cancer Effectiveness Of The Vaccine From Ovarian Cancer Stem Cells With High MUC-1 Expression

Background:Ovarian cancer(OC)is a common malignant tumor in the female reproductive system.Because of its insidious onset,diagnosis of this disease is usually at a later period.In addition,OC has the characteristics of relapse,metastasis,and drug resistance,resulting in its mortality being the first one in gynecologic malignant tumors.The pathological types of OC are complex,in which epithelial ovarian cancer(EOC)accounts for 85%to 90%of OC.At present,the preferred treatment for OC is a cytoreductive surgery regimen plus platinum-based chemotherapy,combined with bevacizumab or olaparib optionally based on patient’s condition is recommended,which gained slight therapeutic effect.However,the OC recurrence rate is more than 85%after treatment,and the 5-year survival rate remains less than 45%.The main reason is that current treatments are unable to effectively eliminate ovarian cancer stem cells(OCSC),which may be the main reason for OC drug resistance,recurrence,and difficult-to-cure cases.Cancer stem cells(CSC)or tumor stem cells(TSCs)possess unique features such as aberrant proliferation,self-regeneration,and multi-directional differentiation abilities,which contribute to tumorigenesis.These characteristics are closely correlated with drug resistance,recurrence,and metastasis observed in ovarian cancer.Therefore,targeting CSC through immunotherapeutic approaches has emerged as a novel treatment strategy for ovarian cancer.Epithelial cell molecule mucin1(MUC1)is a glycosylated transmembrane glycoprotein that is overexpressed in epithelial tumors including colorectal cancer,breast cancer,OC and lung cancer,etc.Especially in ovarian neoplasms,MUC1 expression level is a positively correlated with malignant degree of tumor.MUC1 has been demonstrated that it is involved in the biological functions of growth on tumor cells,differentiation,etc.MUC1 is one of tumor-associated antigens and has the epitopes of B and T cells.The previous researches have confirmed the CD44~+CD117~+molecules in human EOC cell lines HO8910 and SKOV3 may be considered the surface specific markers of OCSC.Thus,the CD44~+CD117~+cells were isolated from the human OC HO8910 and SKOV3 cell lines using the magnetic cell sorting(MACS)system,and the cells exhibited the CSC’s traits;the cancer stem-like cells were selected from the murine OC cell line ID8 by the serum-free medium(SFM)culture,and also exhibited the CSC’s traits.Since CSC have enriched more MUC-1 antigen than non-OCSC,CSC may elicit more powerful anti-tumor effectiveness in tumor immunotherapy,which has received more attention than non-OCSC.For this purpose,we used the MACS and SFM to isolate the OCSC from the human OC HO8910 and SKOV3 cell lines and the murine OC cell line ID8,and then developed the OCSC vaccines that were used for investigating immunological prophylaxis and treatment of OC.Research objective:To explore the anti-OC effectiveness and mechanisms of OCSC vaccines with high MUC1expression and to provide an experimental basis for future clinical use of OCSC vaccine in the treatment of OC.Research contents and methods:1.Verification of MUC-1 in EOC cell lines:MUC-1 expression in human HO8910,SKOV3,and ID8 cell lines was first demonstrated by Western blot assay.We designed the short hairpin RNA(sh MUC-1)plasmid by adopting Gateway site-specific recombination and amplification of MUC-1 gene to construct lentivirus recombinant plasmid.The lentivirus MUC-1(p LV-sh MUC-1 or p LV-sh NC MUC-1)was produced from the transient transfection of the HEK293T cells with p HAGE-CMV-sh MUC-1-IZs Green or p HBLV-U6-sh NC-Zs Green,ps PAX2,and p MD2.G plasmid DNAs plus Lipofectamine 2000.The stable expression colonies in HO8910,SKOV3,and ID8 cells were selected by limiting the dilution assay.The MUC-1 expression was identified by q RT-PCR and Western blot.2.Detection of MUC-1 expression in sorted OCSC:The HO8910 CSC,SKOV3 CSC,HO8910-sh MUC-1 CSC,and SKOV3-sh MUC-1 CSC were isolated from the human EOC SKOV3 and HO8910 cell lines,the HO8910-sh MUC-1 and SKOV3-sh MUC-1 cells using a MACS,respectively.The SFM was used to select ID8 CSC and ID8-sh MUC-1 CSC.The MUC-1expression in sorted OCSC was identified by Western blot.We will also verify whether the target antigen MUC-1 was higher expression in OCSC than non-OCSC.3.The anti-OC effectiveness and mechanisms induced by the HO8910 and SKOV3 CSC vaccines:The 1×10~5HO8910 CSC,SKOV3 CSC,HO8910-sh MUC-1 CSC,and SKOV3-sh MUC-1 CSC were respectively developed into various vaccines by the freezing and thawing three times,which were subcutaneous(s.c.)injected into BALB/c nude mice thrice,between the vaccinations with 10-day interval.Mice were s.c.challenged with 1×10~6 HO8910 or SKOV3 cells 10 days after the final vaccination.The tumor growth was observed diebus tertius by measuring the diameter of tumors using calipers.The tumor-free mice,survival mice,and tumor sizes were severally evaluated.Tumor growth was observed until the end of the experiments.The ratio of CD117~+CD44~+CSC and ALDH~+cells in the tumor tissues were analyzed by FCM.The levels of perforin and granzyme B secreted by the immunocytes in the tumor tissues were detected by q RT-PCR.The murine splenocytes were used for killing experiments,including NK cytotoxicity,splenocyte cytotoxicity,and antibody-dependent cell-mediated cytotoxicity(ADCC).The complement-dependent cytotoxicity(CDC)was performed,and the enzyme-linked immunosorbent assay(ELISA)was also carried out to analyze the levels of anti-MUC-1 antibody,IFN-γ,and TGF-β.The hematoxylin-eosin(H&E)stain assay was used to evaluate whether the different vaccines induced toxic and side effects on the heart,lungs,liver,kidneys,and intestine.4.The prevent OC effectiveness and mechanisms elicited by ID8 CSC vaccine:the 1×10~5ID8,ID8 CSC and ID8-sh MUC-1 CSC were respectively prepared for the different vaccines by the freezing and thawing thrice,which were s.c.injected into C57BL/6 mice three times,between the immunizations with 10-day interval.Mice were s.c.challenged with 1×?10~6 ID8 cells 10days after the final vaccination.The tumor growth was observed every three days by measuring the diameter of tumors using calipers.The tumor-free mice,survival mice,and tumor sizes were separately assessed.The anti-OC effectiveness and mechanisms were evaluated until the end of the experiments.The ratio of ALDH~+cells and the infiltrated CD45~+CD3~+CD8~+lymphocytes in the OC tissues were analyzed by FCM.q RT-PCR was used to detect the levels of perforin and granzyme B secreted by the immunocytes in the OC tissues.The murine splenocytes were used for killing experiments including NK cytotoxicity,splenocyte cytotoxicity,CD4~+T cytotoxicity,and CD8~+T cytotoxicity,and the ADCC.The CDC was also carried out.ELISA was used to analyze the levels of anti-MUC-1 antibody,IFN-γand TGF-β.The H&E stain assay was used to evaluate whether the different vaccines caused toxic and side effects on the heart,lungs,liver,kidneys,and intestine.5.The treatment of OC effectiveness and mechanisms by ID8 CSC vaccine:(1)Immunotherapy of OC with ID8 CSC vaccine:The 1×10~6ID8 cells were s.c.implanted into C57BL/6 mice for three days and then the OC bearing mice were respectively treated with PBS,ID8 vaccine,ID8-sh MUC-1 CSC vaccine,and ID8 CSC vaccine thrice,between the treatments with a 7-day interval.The tumor growth was observed every three days until the end of the experiments by measuring the diameter of tumors using calipers.The ratio of ALDH~+cells in the tumor tissues was analyzed by FCM.The levels of perforin and granzyme B secreted by the immunocytes in the tumor tissues were detected by q RT-PCR.The NK cytotoxicity,splenocyte cytotoxicity,the ADCC activity and the CDC activity were analyzed respectively.The levels of anti-MUC-1 antibody,IFN-γand TGF-βwere detected by ELISA.(2)After 1×10~6ID8 cells challenged mice three days the mice were respectively treated with PBS,ID8 CSC vaccine,Cisplatin(50μg/100μl/each)intraperitoneal injection(i.p.),ID8 CSC vaccine plus Cisplatin(50μg/100μl/each)i.p.three times,between the treatments with 7-day interval.The tumor growth was observed and the effects and mechanisms of immunotherapy of OC were investigated.6.The effectiveness and mechanisms of immunological prevention and treatment of OC by the naked MUC-1 mRNA vaccine and ID8 CSC vaccine:(1)The naked MUC-1 mRNA was transfected into ID8 cells with Lipofectamine 2000 and MUC-1 expression was identified by q RT-PCR and Western blot assays.(2)In the MUC-1 mRNA vaccine prevention of OC experiment,the mice were respectively vaccinated with PBS,ID8 CSC vaccine,Lipofectamine 2000+10μg naked MUC-1 mRNA vaccine(s.c.),ID8 CSC vaccine plus Lipofectamine 2000+10μg naked MUC-1 mRNA vaccine(s.c.)three times,between the treatments with 10-day interval.Mice were s.c.challenged with1×?10~6 ID8 cells 10 days after the final vaccination.The tumor growth was observed and the effects and mechanisms of the combinated vaccines prevention of OC were investigated.(3)In the MUC-1 mRNA vaccine treatment of OC experiment,after 1×10~6ID8 cells transplanted mice three days the mice were respectively treated with PBS,ID8 CSC vaccine,Lipofectamine 2000+10μg naked MUC-1 mRNA(s.c.),ID8 CSC vaccine plus Lipofectamine2000+10μg naked MUC-1 mRNA vaccine(s.c.)three times,between the treatments with 7-day interval.The tumor growth was observed and the effects and mechanisms of the various vaccines treatment of OC were investigated.Research results1.The MUC-1 expression was found in the HO8910,SKOV3,and ID8 cell lines.After the recombinant lentiviral p HBLV-U6-sh MUC-1-Zs Green was respectively infected into HO8910,SKOV3,and ID8 cells,the results of q RT-PCR and Western blot indicated that the MUC-1expression was significantly reduced compared with the controls.The CD117~+CD44~+CSC were respectively isolated from human EOC SKOV3 and HO8910 cell lines using MACS,and the MUC-1 expression was significantly increased compared with the controls;while ID8 CSC were obtained from SFM and the MUC-1 expression was also significantly increased compared with the ID8 cells and ID8-sh MUC-1 CSC,and the differences were a statistically significant(p<0.05).2.The anti-OC experiment results indicated that,in comparison with the mice immunized with the other control vaccines,the mice immunized with the HO8910 or SKOV3 CD44~+CD117~+CSC vaccine developed tumor for long time and tumor grew slow,so much as no tumor generation in one mouse,and tumor sizes were small.The results of detecting tumor tissues showed that the ratio of CD117~+CD44~+CSC and ALDH~+cells were the lowest but the levels of perforin and granzyme B secreted by the immunocytes were the highest in CSC vaccinated mice than those of mice immunized with the other control vaccines.In addition,the effective mechanisms by CSC vaccines indicated that the NK cell cytotoxicity,splenocyte cytotoxicity,and the ADCC and CDC activities,as well as the levels of serum anti-MUC-1 antibody and IFN-γwere the highest but TGF-βlevel was lowest than those of the other control vaccines;the differences between the CSC vaccines and the other control vaccines were statistically significant(p<0.05).The result of H&E staining showed that the HO8910 and SKOV3 CSC vaccines had no any pathological damages of toxic and side effects on the heart,lungs,liver,kidneys and intestine in the immunized mice.3.The preventing OC experiment results indicated that the ID8 CSC vaccinated mice generated tumor for long time,tumor grew slow and tumor volumes were small compared to the mice immunized with the other control vaccines.It was also found that the ratio of ALDH~+cells were the lowest but the infiltrated T cell rate in the tumor tissues and the levels of perforin and granzyme B secreted by the immunocytes were the highest in CSC vaccinated mice than those of mice immunized with the other control vaccines.Moreover,the NK cytotoxicity,splenocyte cytotoxicity,CD4~+T and CD8~+T cytotoxicities,the ADCC and the CDC cytotoxicities,as well as the levels of serum anti-MUC-1 antibody and IFN-γwere the highest but TGF-βlevel was lowest in ID8 CSC vaccinated mice than the other control vaccines,which were a statistically significant(p<0.05).4.The treating OC experiment results indicated that the ID8 CSC vaccine treated mice developed small tumor,and the ratio of ALDH~+cells was the lowest compared with the ID8WT and the sh MUC-1 CSC vaccine.In addition,ID8 CSC vaccine treated mice exhibited that the NK cytotoxicity,splenocyte cytotoxicity,the ADCC and the CDC cytotoxicities,as well as the levels of serum anti-MUC-1 antibody and IFN-γwere the highest and the TGF-βlevel was lowest than the control vaccine treated mice,and the differences were a statistically significant(p<0.05).5.The treating OC experiment results of ID8 CSC vaccine combined with Cisplatin showed that the mean tumor volumes were smaller in all vaccines treated mice than PBS treated mice,and the tumor volume was smaller in ID8 CSC vaccine combined with Cisplatin treated mice than ID8 CSC vaccine treated mice;the NK cytotoxicity,splenocyte cytotoxicity,the ADCC and the CDC cytotoxicities in ID8 CSC vaccine combined with cisplatin treated mice were the higher than the other control vaccines treated mice,and the differences were a statistically significant(p<0.05).6.The treating OC experiment results of ID8 CSC vaccine combined with the naked MUC-1mRNA vaccine showed that the MUC-1 expression was increased in ID8 cells transfected with the naked MUC-1 mRNA.The effects of immunoprevention and treatment of OC in the naked MUC-1 mRNA vaccine was effect but inferior to ID8 CSC vaccine,however,the tumor volume of immunoprevention and treatment of OC in ID8 CSC vaccine combined with the naked MUC-1 mRNA vaccine treated mice was smaller than ID8 CSC vaccine alone;the NK cytotoxicity,splenocyte cytotoxicity,the ADCC and the CDC cytotoxicities,as well as the levels of serum anti-MUC-1 antibody and IFN-γwere the higher and the TGF-βlevel was lower in ID8 CSC vaccine combined with the naked MUC-1 mRNA vaccine treated mice than the other control vaccines treated mice,and the differences were a statistically significant(p<0.05).Conclusions1.The human SKOV3 and HO8910 CSC,and murine ID8 CSC vaccines could induce the immunized mice to generate powerful immunity for antagonism OC growth,and reduced the CSC numbers in tumor tissues.The effectiveness and mechanisms may be that,compared with the non-CSC vaccine,the CSC vaccine enriched high level of MUC-1 and related TAAs,and elicited the stronger immune response to CSC,which was benefited to improve killing more CSC for inhibiting OC growth.While the MUC-1 expression was down-regulated in CSC and developed vaccines,the anti-OC effectiveness was reduced,suggesting the MUC-1 serves as a TAA that was a key one of dominant targe antigens in CSC vaccine against OC.2.The ID8 CSC therapeutic vaccine could induce the immunized mice to paly anti-OC effectiveness;the MUC-1 expression was down-regulated in ID8 CSC led to vaccine reducing anti-OC effects.The ID8 CSC vaccine combined with Cisplatin have a synergistic treatment effect on the therapeutic OC,and Cisplatin may enhance the effectiveness of ID8 CSC vaccine against OC.3.The MUC-1 mRNA vaccine alone has an immunological prevention and treatment effects on the mouse OC,but the naked MUC-1 mRNA vaccine combined with the ID8 CSC vaccine has a stronger anti-OC effect than ID8 CSC vaccine alone,suggesting the MUC-1 mRNA vaccine is expected to play an important role in prevention and treatment of OC.4.The OCSC vaccines performed a pivotal effect on anti-OC in mouse animal experiment,and provided the experiment evidence that possesses the reference value for immunological prevention and treatment of OC.