Study On The Role Of Human Epithelial Ovarian Cancer-associated Fibroblasts In Promoting Cancer

Epithelial ovarian carcinoma(EOC)accounts for more than 90%of all ovarian malignancies and is the second most frequent invasive malignancy of the female genital tract after cancer of the uterine corpus.The high mortality rate of ovarian carcinoma stems primarily from the lack of effective screening and early detection strategies.There has been little change to the 5-year cumulative survival rate for EOC since platinum-based treatment was universally introduced more than 30 years ago.Therefore,there is an urgent need for an in-depth understanding of the mechanisms involved in the progression of ovarian cancer in order to develop new diagnostic or therapeutic agents.The tumor microenvironment has been suggested to play an active role in cancer initiation and progression.Clinical and experimental evidence indicates that tumor development is intimately related to the complex tumor microenvironment.Neoplastic epithelia engage in reciprocal molecular crosstalk with surrounding cells,including pro-inflammatory cells,vascular cells,and fibroblast cells,resulting in the production of growth factors,proteases,chemokines,and cytokines.These cytokines promote angiogenesis and allow malignant tumor cells to escape host immunity and resist apoptosis.Carcinoma-associated fibroblasts(CAFs),a particular type of stromal fibroblast that exists within carcinomas,have been linked to multi-faceted tumor-promoting effects,including cytokine and protease secretion,regulation of tumor motility and metabolism,reorganization of the tumor extracellular matrix(ECM),and preparation of the metastatic niche.Therefore,identification of the specific factors derived from the stromal-epithelial biochemical interaction needs to be a primary focus of research.Detailed characterization of the tumor microenvironment and specific factors secreted by the synergistic interaction between CAFs and EOC cells has not been carried out.Thus,a thorough understanding of the biochemical interactions between stromal cells and epithelial cells is critical.Part I The relationship between carcinoma-associated fibroblasts and their clinical significance in epithelial ovarian carcinoma.Objective To investigate the relationship between the distribution of carcinoma-associated fibroblasts(CAFs)and their clinicopathological features as well as patient prognosis in epithelial ovarian cancer(EOC).Methods We used immunohistochemical technique to detect the expression of ?-SMA and MMP-9 in 145 cases of EOC,and analyzed its relationship with clinicopathological features.Resullts The expression of a-SMA in CAFs was upregulated as compared with normal tissue.Although the expression of a-SMA had no obvious relationship with age,tumor size and histological grade,it was associated with the clinical stage and lymph node metastases(P<0.05).The expression of MMP-9 in EOC cells was upregulated compared with normal tissue.The expression of MMP-9 was associated with the clinical stage and lymph node metastases(P<0.05),but had no obvious relationship with histological grade,tumor size and histological type(P>0.05).In addition,the expression of a-SMA in CAFs was correlated with the expression of MMP-9 in cancer cells.Conclusion The CAFs in EOC seem to play an important role in the ovarian cancer progression and lymph node metastases.It may become potential prognostic predictors of EOC.Part ? Purification,identification and cultivation of human epithelial ovarian carcinoma-associated fibroblasts and normal ovary fibroblasts.Purification,identification and cultivation of human epithelial ovarian carcinoma-associated fibroblasts.Objective To isolate,purify,culture and identify the carcinoma-associated fibroblasts(CAFs)from the human epithelial ovarian neoplasm.Methods:The primary ovarian CAFs were obtained from human epithelial ovarian neoplasm and purified by trypsin digestion and adherence repeatedly.The cells were verified according to morphological observation and immunocytochemical staining of certain proteins.Additionally,their proliferation indexes were assayed.Results:The purified ovarian CAFs were obtained successfully.The typical morphology of ovarian CAFs was simple fusiform fiber like cells.The ovarian CAFs exhibited lower proliferation than tumor cells and showed positive staining for fibroblast specific protein-1,vimentin,desmin and especially for a-smooth muscle actin,and negative staining for cytokeratin(broad-spectrum),cytokeratin 7,cytokeratin 20,CD31 and CD90.Conclusion:The primary cultured CAFs derived from the human ovarian carcinoma tissue can be obtained successfully with digestion method.This provides us an experimental foundation for further studies on the roles of CAFs in the initiation and progression of the cancer.Purification,identification and cultivation of human normal ovary fibroblasts.Objective To obtain the purified human ovarian normal fibroblasts(NFs)preliminarily.Methods The ovarian tissues were dissociated by 0.25%trypsin,after overgrowing on the bottom of the culture dish,the cells were adhered to the bottom of the dish repeatedly to depurate them.Then the cells were verified according to its morphology under the inverted-microscope and were further confined by a series of biological markers with immunocytochemistry.And its growth cure was observed.Results The typical shape of ovarian NFS was fusiform,cords like,triangular,poly angular and irregular.Most of them contain Pseudopods-like structure.The NFs showed positive staining for vimentin,fibroblast specific protein-1,desmin and a-smooth muscle actin,negative staining for cytokeratin parn,cytokeratin 7,cytokeratin20,CD31,CD90,MMP-2,MMP-3,MMP-9,CA-125,CA-19.9,CEA,fibroblast activation proteina(FAP-a)and platelet-derived growth factor receptor a(PDGFR-a).Conclusions We've got human ovarian NFs successfully,thus will provide a tool for further studies on the cancer and its CAFs.Part III The effect of ovarian carcinoma-associated fibroblasts on the progression of epithelial ovarian carcinoma cells.Objective To elucidate the effects of CAFs on the progression of ovarian carcinoma cell lines including proliferation,migration,invasion and apoptosis.Methods Direct and indirect co-culture of EOC cells with fibroblasts were established.Cell proliferation was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Migration and invasion assays,transwell inserts and Matrigel-coated invasion chambers were used.Serum deprivation-induced apoptosis assays were identified using the conjugated human annexin V and propidium iodide double staining method.Results Proliferation assays showed that CAFs significantly increased the growth rate of OVCAR-3 and SKOV-3 cells(p<0.05).The migration of EOC cells was significantly increased after the co-culture with CAFs or NFs for 48h,moreover,there was an extremely significantly increase in CAFs group when contrast with the control group,The invasion of EOC cells was significantly increased,especially in CAFs group,when EOC cells co-cultured with CAFs or NFs for 48h.Culture in serum-free conditions strongly induced the apoptosis.The percentage of apoptotic EOC cells was reduced by co-cultured for 48h with CAFs compared to control media.Conclusions CAFs and the CM from CAFs/EOC co-cultures stimulated ovarian tumor cell proliferation,migration and invasion in vitro.Moreover,CAFs and the CM from CAFs/EOC co-cultures also inhibited serum-deprivation-induced EOC apoptosis.Part IV Cytokine,chemokine,and growth factor levels were screened using a biotin label-based human antibody array system in the co-cultured conditioned mediumObjective The aforementioned observations indicate that CAFs supply locally acting paracrine cues that induce EOC cells to progress in vitro.To understand this crosstalk better,the cytokines shall be screened for the further investigation.Methods The CM was harvested from CAFs,OVCAR-3 cells,or CAF+OVCAR-3 cell co-cultures.The levels of various factors in the cell-free culture supernatants were measured with the RayBio(?)Biotin Label-based Human Antibody Array system.In this system,biological samples are labeled with biotin.The biotinylated proteins are then incubated with antibody chips.The presence of proteins captured by the antibody chip is detected using streptavidin-conjugated fluorescent dye(Cy3 equivalent)as a reporter.The signals,which are visualized by laser scanning,are normalized using positive,negative,and internal controls.Results Using this biotin label-based antibody array technology,the expression levels of 507 human target proteins can be simultaneously detected,including of cytok:ines,chemokines,growth factors,angiogenic factors.We defined an arbitrary cutoff signal ratio of>2.0 or?-2.0-fold changes as significant expression.Of the 507 cytokines,there was significant expression of 57 in the CC-CM from CAF+OVCAR-3 cells,Of these 57 cytokines,44 were upregulated and 13 were downregulated.Conclusions Our results suggest that biotin labelbased antibody arrays have great potential in applications for cytokines discovery.With this information,targeting the stroma in ovarian cancer may not only be effective in suppressing tumor development,but may also prevent tumor metastasis.Further investigation of the role of these factors may be a high priority for future work.Part V The effects of carcinoma-associated fibroblasts in the mouse model of human ovarian carcinoma.Establishment subcutaneous and abdominal xenografted tumor mouse model with stable expression of green fluorescent protein Objective In order to provide a useful and visualizing human ovarian cancer mouse model,we establish a subcutaneous and abdominal xenografted tumor mouse model with stable expression of green fluorescent protein(GFP).Methods Green fluorescent protein(GFP)-labeled EOC cell lines were transfected with 2 ml of pLenti-GFP lentivirus combined with 12 mM polybrene,cells with stable expression of GFP was collected.The dynamic growth of the xenografted tumors was observed and analyzed through tumor bioluminescent imaging weekly using a whole-body imaging system.Results The established human ovarian cancer cells could stably express high level of GFP in vitro.After subcutaneous and abdominal inoculation into the nude mice,the visualizing human ovarian cancer models were established.Conclusions The xenografted tumor mass with stable expression of GFP can be observed and measured conveniently,useful and visualizing human ovarian cancer mice models were provided.The study of carcinoma-associated fibroblasts contribution to the initiation and growth of human ovarian carcinoma in vivo.Objective To evaluate whether the presence of stromal fibroblasts contribute to the initiation and growth of ovarian cancer in in the mice model.Methods we injected G-Ovcar-3 cells,CAFs,or both CAFs and G-Ovcar-3 cells in a tumor-to-stroma ratio of 1:1 to null mice.All mice were subcutaneously inoculated in the left mid-dorsal flank with(a)G-Ovcar-3 cells alone(0.1 × 106 cells per mouse),(b)G-Ovcar-3 cells with CAFs in a tumor-to-stroma ratio of 1:1(0.1 × 106 cells per mouse),(c)CAFs alone(0.1 × 106 cells per mouse),(d)G-Ovcar-3 cells alone(1 × 106 cells per mouse),(e)G-Ovcar-3 cells with CAFs in a tumor-to-stroma ratio of 1:1(1 ×106 cells per mouse)and(f)CAFs alone(1 × 106 cells per mouse).Results All formed tumors were GFP-labeled.We found that in the group injected with G-Ovcar-3 cells at a concentration of 0.1 × 106 cells per mouse,none of the mice developed tumors,but in the group injected with both tumor and stromal cells at the same cell concentration(both at 0.1 × 106 cells/mouse),48.1%of the mice formed tumors.The difference between the two groups was significant(P<0.01),When the number of G-Ovcar-3 cells was increased to 1 × 106,92.5%of the mice formed tumors,and in the group injected with both tumor and stromal cells(1 × 106 cells/mouse),100%of the mice formed tumors as anticipated(P>0.05).No tumors developed when the mice received CAFs either at 0.1 × 106 or 1 × 106 cells per mouse.Besides tumor formation,we also evaluated the tumor size in the groups injected with 1 × 106 cells per mouse.All tumors were measured using the whole-body imaging system.Throughout the 10-week study period,the mean tumor volume was greater in the group injected with both tumor and stromal cells than in the group injected with G-Ovcar-3 cells alone,but the difference was not statistically significant(P>0.05).Interestingly,no distant tumor metastases were observed in this model.Conclusions The presence of CAFs contributed to the initiation of tumor formation,particularly,when tumor cells were injected at a low concentration,and also contributed to the growth of ovarian cancer in vivo.A novel humanized ovarian microenvironment mouse model of ovarian carcinomaObjective To establish an ideal model of ovarian cancer,which can not only mimic the processes of tumor progress and metastasis,but also provide a normal human ovarian microenvironment for the ovarian cancer cells to proliferate.Methods In this study,normal human ovarian tissues were subcutaneously implanted into severe combined immunodeficient(SCID)mice to create a normal human ovarian microenvironment,after which the human ovarian cancer cells were inoculated into the implants.Results The implants became vascularized and retained their original morphology for about 4 weeks following implantation.Immunohistochemical staining for cytokeratin-7 confirmed the ovarian origin of the epithelial cells.CD34 staining demonstrated human-derived vessels.Positive estrogen receptor and partially positive progestin receptor staining indicated the estrogen and progestin dependence of the implants.Only vascular pericytes expressed a-smooth muscle actin,indicating the normal ovarian origin of the xenografts.The new model not only mimick the processes of tumor progress,but also allowed the orthotopic human ovarian cancer cells to proliferate in the normal human ovarian microenvironment.Conclusions It is a better mouse model for the research of ovarian cancer in mimicking all the interactions between ovarian cancer cells and human ovarian microenvironment.Part VI The study of carcinoma-associated fibroblasts prevetion cisplatin-induced apoptosis in human epithelial ovarian cancer cells through upregulation of PI3K/Akt/Xiap cell signaling pathway.Objective To investigate the effects of CAFs prevetion cisplatin-induced apoptosis through upregulation of PI3K/Akt/Xiap cell signaling Pathway in epithelial ovarian cancer Cells.Methods Ovarian cancer cell Ovcar3 were exposed to DDP with various concentrations(O?g/ml,2.5?g/ml,5.0?g/ml and 10?g/ml)and different duration(0,24h,48h and 96h),then CAFs cultured with Ovcar3 cell.The apoptosis of Ovcar3 cell was assessed by the conjugated human annexin V and propidium iodide double staining method.The mRNA level of PI3K/Akt/Xiap was detected by real-time PCR.The protein level of PI3k,Xiap,Akt and Akt phosphorylation was detected by Western blot.Signaling transduction inhibitors,LY294002 was used to block PI3K/Akt signaling pathways.Results Cisplatin induce ovarian cancer cell apoptosis in a dose and time-dependent manner.Cisplatin decreases PI3K/XIAP mRNA level and protein level so that induction apoptosis in ovarian cancer cell chemotherapy.After co-culture,CAFs prevent Cisplatin-induced apoptosis by upregulate the mRNA level and protein levels of PI3K/XIAP.In addition,phosphorylation of Akt was enhanced by CAFs.Remarkably,inhibition of Akt phosphorylation by LY294002 can block the effects on DDP-induced Xiap up-regulation.Conclusions The results indicate that CAFs prevents apoptosis through a PI3K-dependent inhibition,of the caspase cascade.These results demonstrate a novel mechanism by which CAFs regulates apoptosis and the possible involvement of the PI3K/Akt survival pathway in chemoresistance of ovarian cancer cells.