Experimental Research Of Mesenchymal Stem Cell Derived From Endometrial Adenocarcinoma On Development Of Endometrial Cancer

Objective:To explore the effect and mechanism of endometrial adenocarcinoma-derived mesenchymal stem cell on the development of endometrial cancer.Content:1.Isolation and identification of endometrial adenocarcinoma-derived mesenchymal stem cell?ECMSC?and endometrium-derived mesenchymal stem cell?EMSC?.2.ECMSC and EMSC mediated the biological function of human endometrial adenocarcinoma cell line HEC-1A in vitro.3.The effect of ECMSC and EMSC on the endometrial tumor progression was evaluated by nude mouse tumor model.Method:1.MSC was isolated from human endometrial adenocarcinoma and endometrium by tissue digestion.The morphology of MSC was observed by inverted microscope.The immunophenotype of the MSC derived from endometrial cancer and endometrium was analyzed by flow cytometry and the induction ability was determined by different induction reagents including oil-red-O and alkaline phosphatase?ALP?staining.The key transcription factor PPAR?expression of adipogenic differentiation and osteogenic differentiation marker ALP for ECMSC and EMSC was detected by quantitative real-time polymerase chain reaction?RT-PCR?.2.To assess the effect of ECMSC and EMSC on the biological function of human endometrial adenocarcinoma cell line HEC-1A,the cultural supernatant of ECMSC and EMSC was harvested and concentrated by centrifugal filter method.The evaporated supernatant was co-cultured with HEC-1A,and then the morphology of co-cultured HEC-1A was observed by inverted microscope and the cell growth curve was assessed by CCK8 kit.The migration ability of HEC-1A cell was measured by scratch test and the cell cycle was analyzed by flow cytometry.Furthermore,the apoptosis of HEC-1A was determined by flow cytometry after TNF?treated the evaporated supernatant of MSC.The expression of anti-apoptotic gene DcR3 and epidermal growth factor?EGF?in ECMSC and EMSC was analyzed by RT-PCR.3.To address the function of ECMSC on the development of endometrial adenocarcinoma in vivo,the nude mice were subcutaneously injected with HEC-1A cells and then ECMSC or EMSC were injected intravenously into mice at day 1 and day6.Tumor volume was measured every two weeks and tumor weight was measured at the endpoint of the experiment.Animals were sacrificed by cervical dislocation under anesthetic status after 28 days.To evaluate the histopathology changes of tumor,the tumor tissue was embedded in paraffin and stained with hematoxylin and eosin?H&E?.The expression of CD31 and DcR3 in tumor tissue was detected by immunohistochemistry staining.Western blot assay was used to analyze the expression of CD31,VEGF and Dc R3 in tumor tissue.Results:1.MSCs were isolated from human endometrial adenocarcinoma and endometrium.These cells were morphologically similar to fibroblast-like cells.Flow cytometry was used to detect the phenotype of these cells,and the results showed that the cell was positive for mesenchymal markers?CD73,CD90,CD105,CD166?and human leukocyte antigen?HLA-ABC?,and negative for marker of hematopoietic stem cells?CD34,CD45?and costimulatory molecules?CD80,CD86?and human leukocyte antigen?HLA-DR?.These cells are multipotent,as showed by their ability to differentiate into adipocytes and osteoblasts by oil-red-O staining lipid-rich vesicles and alkaline phosphatase staining.RT-PCR analysis also demonstrated that MSCs displayed corresponding transcriptional expression of paroxysm proliferation activated receptor gamma 2?PPAR?2?and ALP under specific adipogenic and osteogenic inductive cultures,respectively.These results strongly indicated that the MSC was successful isolated from human endometrial adenocarcinoma and endometrium.2.To address the effect of MSC on the biological function of human endometrial adenocarcinoma cell line HEC-1A,the serum-free supernatant of ECMSC and EMSC was collected and concentrated by centrifugal filter method.The evaporated supernatant was cocultured with HEC-1A,the results showed that ECMSC and EMSC supernatant treated HEC-1A were morphologically similar to fibroblast-like cells.In CCK8 cell viability assay,a significant increase proliferation was observed in the ECMSCs supernatant-treated HEC-1A cells compared to cells treated with EMSC supernatant?P<0.05?.Furthermore,we observed that ECMSC significantly up-regulated the cancer cells in G₂/S phase after being treated with ECMSC supernatant for 48 hours by using flow cytometry?P<0.05?.Moreover,TNF-?treated ECMSC supernatant could down-regulated the percentage of apoptotic cell in comparison to the EMSC plus TNF-?group.To further assess the mechanism of ECMSC on regulating the apoptotic pathway,RT-PCR assay was used to analyze the expressions of anti-apoptotic gene DcR3 and epidermal growth factor?EGF?,we found that ECMSC could significantly increase DcR3 and EGF expression at the transcriptional level,suggesting that ECMSC promote HEC-1A proliferation maybe in anti-apoptosic pathway and facilitate tumor growth.3.To evaluate the effect of ECMSC on development of endometrial adenocarcinoma in vivo,nude mice tumor model expreiment was performed.The results showed that ECMSC could significantly increase the volume and weight of xenografts in vivo,suggesting the oncogenic effects of ECMSC in tumor microenvironment.By using hematoxylin and eosin?H&E?staining,we observed that the tumor tissues obtained from the ECMSC group presented decreasing of inflammatory infiltrating cell and increasing angiopoiesis in comparison to the EMSC group.Immunohistochemistry?IHC?assay was used to test the level of CD31 and DcR3 in the tumor tissues,which is the key executor of apoptosis in the intrinsic apoptosis pathway and tumor proliferative factor.We observed that the percentage of CD31 and DcR3?+?cells were significantly increased in tumor tissues obtained from ECMSC group compared with that obtained from the EMSC control group?P<0.01?.To further assess their oncogenetic properties,western blot was performed to detect the expressions of CD31,DcR3 and VEGF in ECMSC group or EMSC group.Results showed that the expression of CD31,DcR3 and VEGF in ECMSC group was significantly higher than that of the EMSC group,indicating an acceleration of ECMSC in endometrial adenocarcinoma.Conclusion:We sucessfully isolated MSC from the endometrial adenocarcinoma.ECMSC exert promoting neoplastic properties in vitro and in vivo.This research provides an alternative way of ECMSC-based tumorgenesis process,such as stimulating the proliferation of HEC-1A,accelerating migration and inhibiting apopotosis.