Establishment Of Human Ovarian Cancer Omentum Metastasis Models And The Activation Of Omental Microvascular Endothelial Cells By Ovarian Cancer Cells

PurposeEstablishment of ovarian cancer orthotopic xenograft(OCOX)mouse models for in vivo imaging and comparison of two OCOX models,i e.cellular orthotopic injection(COI)and surgical orthotopic implantation(SOI),regarding xenograft formation rate,in vivo imaging,tumor growth and metastasis,and tumor microenvironment.Establishment of an omentum-derived highly metastatic ovarian cancer cell subline and an orthotopic-omental metastasis mouse model via in vivo selection using OCOX mouse models.To investigate whether the omental angiogenesis were initiated and activated before omentum metastasis formation.Methods1.Ovarian cancer cell lines that stably expressing GFP and firefly luciferase were established by lentivirus transfection;2.The tumor formation and progression of COI and SOI tumors were monitored by bioluminescent in vivo imaging.At the endpoint,the primary tumor volume and peritoneal metastases were compared between the two groups;3.Proliferation and migration abilities of tumor cells that generate COI and SOI tumors were detected by EdU and scratch assays,respectively;4.Expression of a-SMA,CD34,MMP2,MMP9,Vimentin,E-cadherin and Ki67 in tumor samples were detected by immunohistochemistry;5.A firefly luciferase-labeled ovarian cancer cell line ES2-luc was used to establish the SOI-OCOX mouse model.After three cycles of in vivo primary tumor-omentum metastasis selection,the omentum tissues of the passage 3 mice were obtained;6.The omentum-derived highly metastatic ovarian cancer cell subline was isolated and cultured in vitro;7.The cell phenotype was identified by in vitro assays:EdU assays,transwell migration and invasion assays,matrigel adhesion assays,colony formation assays and anoikis;8.The ability to metastasize of tumor cells was assessed in vivo:the highly metastatic cells and parental cells were used for orthotopic transplantation,and the tumor formation,growth and metastasis were compared;9.HE staining and immunohistochemical staining for CD34 and CD 105 were performed to detect the metastases and angiogenesis in mice omentum.10.CD34 and CD 105 marked microvessel endothelial cells(MVECs)in omental specimens obtained from ovarian cancer patients were assessed by immunohistochemistry;11.Human omental MVECs were primarily isolated and cultured in vitro;immunocytochemistry(ICC)and flow cytometry for CD31 and CD34 were performed to identify the cells;12.After co-culture with ovarian cancer cell conditioned medium(CM),the EdU,scratch assays,transwell migration and invasion assays,tube formation assays and endothelial cell permeability assays were performed to detect functional changes of MVECs;the expression of MVEC activation marker CD 105 was detected by western blot.Results1.Through lentivirus transfection,two luciferase labeled ovarian cancer cell lines,ES2-luc and SKOV3-luc were obtained.Then we successfully established COI-and SOI-OCOX mouse models using the cell lines.The tumor formation rate in the COI and SOI models were 87.5%and 100%,respectively.Suspected tumor cell leakage occurred in 37.5%of the COI models;2.The SOI xenografts grew faster,held larger primary tumors,and were more metastatic than the COI xenografts;3.The migration and proliferation properties of the cells that generated SOI xenografts were significantly starker than those deriving COI xenografts in vitro.The tumor cells in SOI xenografts exhibited a mesenchymal phenotype and proliferated more actively than those in the COI xenografts;4.Compared with the COI tumors,the tumor microenvironments in SOI tumors were more pro-metastatic.5.During the in vivo selection,from the first in vivo passage to the third,the time interval between inoculation and the formation of intraperitoneal metastases and the survival of the mice was becoming shorter.The target cell subpopulation was successfully isolated form the omentum tissues of the passage 3 mice and named ES2-luc-OMP3;6.The highly metastatic cells were generally refractible and exibited mesenchymal morphology.And they showed enhanced capabilities of migration,invasion and anoikis resistance,while their proliferation,colony formation and adhesion abilities were reduced;7.Tumors derived from ES2-luc-OMP3 exhibited a more metastatic and aggressive phenotype with significantly increased ability to form omental metastases,compared with parent ES2-luc.Histological assays confirmed the increased omental metastases and angiogenesis in the mice harboring ES2-luc-OMP3 cells.8.There is abundant CD34-and CD 105-positive microvessels in the omentum specimens with metastasis,whereas only few CD34-positive vessels and no CD 105-positive microvessels were observed in specimens derived from early stage(FIGO I)ovarian cancer patients.Most metastasis-free omental specimens presented with CD 105-positive microvessels with varied density;9.Isolated MVECs were CD34-positive in ICC,and the purity of MVECs was more than 90%;10.After co-culture with ovarian cancer cell CM,the MVECs changed to a fibroblast-like morphology,their CD 105 expression was increased,and their proliferation,migration,invasion and tube formation abilities were enhanced.The permeability of endothelial cell monolayer was decreased.Conclusions1.SOI is a feasible and reliable technique to establish OCOX mouse models mimicking the clinical process of ovarian cancer growth and metastasis,although SOI is more technically difficult and time-consuming than COI.2.We have successfully established an omentum derived highly metastatic ovarian cancer cell subline and an orthotopic-omental metastasis mouse model.We believe this cell line and mouse model can serve as a useful tool for in vitro and in vivo studies on ovarian cancer omental metastasis.3.The omental angiogenesis is activated before metastatic colonization by the secretion from ovarian cancer cells,which facilitates omental metastasis.