Study Of Isolation And Identification Of Tumorstem-like Cell From ID8 Cell Line And Its Vaccine Effects

Ovarian cancer(OC)is one of the most common gynecologic malignant tumors.Epithelial ovarian cancer(EOC)occupies 85%in OC and is the most important pathological type.Because of its failure of early diagnosis,higher malignancy,metastasis and resistance to chemotherapy,OC mortality rate ranks top of all the gynecologic cancers.It is indicated that there are a small amount of cancer stem cells(CSC)in tumor tissue,which have the abilities of infinite proliferation,self-renewal and radiation resistance and which are believed to be responsible for the tumorigenesis,chemoresistance,metastasis and recurrence.Therefore it is necessary to find new strategies for treatment of OC.In recent years,with the rapid development of tumor immunology,cell biology and molecular technology,tumor immunotherapy has developed into a new era.Tumor immunotherapy is a therapeutic approach which can induce anti-tumor specific immune response and plays full functions in inhibition and elimination of tumor.Tumor immunotherapy includes blocking of immune check point,adoptive immunotherapy and tumor vaccine and so on.Moreover,It is confirmed that the immunotherapy can obviously improve the disease prognosis.However,because CSC and its progeny exhibit different gene expression lineages and different antigens,conventional immunotherapy can't effectively wipe out CSC,which leads to tumor recurrence.Therefore,effectively targeting of CSC is the key to eliminate tumor and prevent recurrence.It is believed that CSC has a variety of tumor specific antigens and tumor related antigens.The vaccine which consists of whole CSC lysates could stimulate the host immune system against CSC.In this study,murine ovarian cell line ID8 is selected as the investigation object.Serum free culture method is adopted to enrich and identify ID8 cancer stem-like cell(OCSC).Then ID8 OCSC vaccine is developed and its anti-tumor efficacy and the mechanisms in murine tumor model are evaluated.Objective:Isolation and identification of OCSC in murine ovarian cell line ID8.On the basis,we are to evaluate the antitumor efficacy of ID8 OCSC vaccine in murine tumor model.Method:1.ID8 cells were cultured in the serum-free medium(SFM)to derive tumor sphere cells.The CCK8,the plate colony formation,transwell assays and chemosensitivity testing were adopted to analyze the biological differences between suspending sphere cells and conventional cultured ID8 cells.Real-time PCR and Western blot assays were carried to examine the expression level of tumor stem cell marker(ALDH1)and the mice tumor model was used to observe the tumorigenesis in vivo.2.C57BL/6 mice were immunized with 2×10~5 ID~8 OCSC vaccine three times an interval of 10 days between adjacent immunizations,and the OCSC vaccine was inactivated by repeated freeze-thawing method.Then all mice were subcutaneously challenged with 2×10~6 ID8 cells 10 days after the final immunization.At the same time,ID8 cell vaccine group was used as control vaccine group,the tumor growth and mice survival time were monitored.Meanwhile,the levels of cytokines,CTL,CDC and NK activities were examined.3.The expression of tumor dominant antigen,type I receptor tyrosine kinase-like orphan receptor(ROR1)in ID8 cell and ID8 suspending sphere cell were examined by Real-time PCR and Western blot.The target gene sequences of musROR1-shRNA sequence were designed,then constructing the recombinant plasmid and packaging lentivirus.And then lentivirus was cotransfected into 293T cell and virus titre was tested.Finally,ID8 cell was infected with lentvirus.In order to detect the inhibition of ROR1 expression,the assays of Real-time PCR and Western blot were adpoted.4.C57BL/6 mice were immunized with 2×10~5 shROR1-ID8 OCSC vaccine three times an interval of 10 days between adjacent immunizations,and the OCSC were inactivated by repeated freeze-thawing method.Then all mice were subcutaneously challenged with 2×10~6 ID8 cells 10 days after the final immunization.At the same time,ID8 cell vaccine group and ID8 OCSC vaccine group were used as control vaccine groups.The tumor growth and mice survival time were monitored.The tumor formation time and tumor volume were observed in mice in order to explore the molecular mechanism of CSC anti-tumor effect.Result:1.Ovarian ID8 cells grew into stable suspending tumor sphere cells(OCSC)in SFM.Compared with ID8 cell,OCSC significantly exhibited their abilities of proliferation,self-renew,invasion,drug resistance and tumorigenesis.Moreover,the expression level of ALDH1 was higher in OCSC group than that of conventional cultured ID8group.2.The tumor grew more slowly and tumor sizes were smaller in ID8 OCSC vaccine group than that of ID8 vaccine group.Moreover,ID8 OCSC vaccine can increase the level of serum IFN-?,strengthen the activities of CDC,NK and CTL as well as decrease the level of serum TGF-?1.3.The expression of ROR1 in OCSC was higher than that of conventional cultured ID8cell,and the difference was statistically significant.The result of recombinant plasmid DNA sequencing is the same as the expected DNA sequence,which demonstrates the recombinant plasmid was successfully reconstructed.The titre of lentivirus showed the lentivirus was successfully constructed.Silencing ROR1 can inhibit the expression of ROR1 protein in ID8 cell.4.The shROR1-ID8 OCSC vaccine was developed by application of repeated freezing-thawing method,and the vaccine was immunized into C57BL/6 mice.The result shows that the tumor grew more quickly and tumor sizes were bigger in ID8shROR1-ID8 OCSC vaccine group than that of ID8 OCSC vaccine group,which was statistically significant.Conclusion:SFM can effectively enrich ID8 suspending sphere cell which has the properties of OCSC.The vaccine of ID8 OCSC with high ROR1 expression that has stronger immunogennicity can induce powerful antitumor efficacy in mice.The findings indicate that OCSC vaccine may serve as a new way for treatmen of ovarian cancer.