Ovarian Epithelium Carcinoma Cells Influence The Differentiation And Function Of Dendritic Cell Precursors Through Notch1 Pathway

IntroductionOvarian epithelial carcinoma is one of the most common gynecological malignancies encountered in the clinic.It has a biological behavior of metastasis initiated at an early stage and rapidly spread over the peritoneal cavity.Published literatures revealed that the immune deficiency in tumor microenvironment plays an important role in the pathogenesis of ovarian cancer,and also influences the disease progression,and overall survival rate.One evidence to support the above statement is that the matured myloid DCs(mDCs,HLA-DR+CD11C+CD123-),which are known as the most effective antigen-presentation cells,infiltrate rarely into the tumor site in the patients with ovarian carcinoma.Of note,mDCs play an important role in promoting immune response,while the plasmcytoid DCs(pDC,HLA-DR+CD11C-CD123+)are oppositely to promote immune escape.It is known that mDC precursors would probably present as Lin-CD45RA-cells because no specific surface markers such as CD45RA were expressed and lineage negative.However,each DC subtypes have the potential ability to differentiate through either myeloid or lymphoid-lineages and the DC development is quite flexible rather than their lineage restriction.In our previous study,we cultured such precursors that generated from CD34+ human progenitor cells(HPC)with GM-CSF and TNF-a in the ovarian carcinoma microenvironment.Then,we observed that the Lin-CD45RA-DC precursors differentiated into more pDCs than mDCs,which indicated that it may favor tumor progression.However,the mechanism is still unclear.Notch proteins(Notch 1-4)constitute a group of highly conserved transmembrane receptors that regulate cell fate decision during the development of many mammalian cell lineages.A lot of studies have provided much data on the critically important role of the Notch pathway in the differentiation of myeloid cells.Recent studies also confirmed the expression of Notch protein in antigen presentation cells and matured DC.More and more papers concluded that Notch pathway is associated with DC differentiation.But the conclusions were controversial.Cheng et al.described that the differentiation of DC in Notch-1 anti-sense mice was significantly down-regulated that only half of the normal level of Notch-1 were expressed in HPC.Yet,Radtke et al.found that in Notch-1 conditional knockout mice,the thymic DCs,conventional DCs,and Langerhans cells were in normal level.Based on our previous research works,the current study is aimed to address the expression of Notch1 in Lin-CD45RA-DC precursors,and then to illustrate the relationship between ovarian epithelial carcinoma cells and dendritic cell precursors whether or not through Notch 1 pathway.PART I Detection of Notchl receptors and ligands in dendritic cell precursorsObjective:The aim of this study was to confirm the expression of Notchl receptors and ligands in Lin-CD45RA-DC precursor cells.Materials and Methods:1.The CD34+ hematopoietic stem cells were extracted from umbilical cord blood in term parturition,and Lin-CD45RA-DC precusors were separated;2.Expression of Notchl receptor and ligands in Lin-CD45RA-DC precusors wasdetected by Real-time RT-PCR.Results:In the Lin-CD45RA-DC precursors developed from CD34+ cells,we can detect the expression of Notchl receptors and their ligands,Jagged 1,Jagged 2,and Delta 1 ligands.Among them,Notchl receptor and Jagged1 ligand had high expressions,while expressions of Jagged2 and Delta1 were much lower.Conclusion:The expression of Notchl and Jagged1 were relatively high in Lin-CD45RA-DC precursor cells,while the experession of Jagged2 and Delta1 were relatively low,suggesting that Notchl and Jagged1 may play a major role in the function of Lin-CD45RA-DC precursor cells.PART ? Ovarian epithelial carcinoma cells influence the differentiation of dendritic cell precursors through Notchl pathwayObjective:The aim of the study was to confirm the effect of SKOV3 culture supernatant on the differentiation of dendritic cell through Notch1 pathway.Materials and Methods:1.Flow cytometry was used to analyze the subset of DCs and the effect of SKOV3 culture supernatant on the differentiation of Lin-CD45RA-DC precursor cells was compared;2.Notchl was down-regulated by shRNA or DAPT and flow cytometry was used to analyze the subset of DCs,the effect of Notchl inhibition on the differentiation of Lin-CD45RA-DC precursor cells was compared;3.Based on step 2,the effect of SKOV3 culture supernatant on the differentiation of Lin-CD45RA-DC precursor cells before and after Notchl receptor inhibition was compared.Results:1.SKOV3 culture supernatant can influence the differentiation of Lin-CD45RA-DC precursors,resulting less mDCs(p=0.011)and more pDCs(p=0.006);2.Notchl receptor inhibition can influence the differentiation of Lin-CD45RA-DC precursors,generating more mDCs(DAPT:p>0.05;shRNA:p=0.007),but less pDCs(p=0.015,p=0.007).3.Notchl receptor inhibition can partly reverse the effect of SKOV3 on the differentiation to pDC(p=0.02,p=0.02),but had no effect on mDCs(p=0.25,p=0.07).Conclusion:SKOV3 supernatant might affect the differentiation of dendritic precursor cells to pDC through Notch 1 pathway.PART III Ovarian epithelial carcinoma cells influence the function of dendritic cell through Notchl pathwayObjective:The aim of the study was to confirm the effect of SKOV3 culture supernatant on the function of dendritic cell trough Notch1 pathway.Materials and methods:1.The effect of SKOV3 culture supernatant on the function of DC cells was compared by the IL-12 level,detected by ELISA;2.The effect of Notchl inhibition on the function of DC cells was compared by the IL-12 level,detected by ELISA;3.Based on step 2,the reversal effect by Notch1 inhibition to SKOV3 culture supernatant was compared by the IL-12 level,detected by ELISA;Results:1.SKOV3 supernatant can promote the decrease of IL-12 secretion by DC cells(p=0.001);2.The inhibition of Notch1 receptor expression can increase the secretion of IL-12 by DC cells(p=0.0002,p=0.0002);3.Inhibition of Notchl receptor expression can partially reverse the effect of SKOV3(p=0.02,p=0.01);Conclusion:SKOV3 supernatant might affect the secretion of IL-12 by dendritic cells through Notchl pathway.