| Background:Ovarian cancer has the lowest incidence and the highest mortality rate among the three major gynecological tumors.Ovarian cancer has the characteristics of insidious onset,rapid progression,early metastasis and poor prognosis.Ovarian cancer with pelvic and abdominal cavity extensive implantation and metastasis is very common,often accompanied by a large amount of ascites,which brings great challenges to clinical treatment.Although in recent years,in addition to traditional tumor cytoreductive surgery and chemotherapy,many new treatment methods have appeared,but the actual clinical application has not significantly improved the prognosis.Finding new treatment methods is particularly critical.Immunotherapy is developed based on the basic principles of cellular immunity and humoral immunity,including cytokines,monoclonal antibodies,tumor vaccines,immune cell infusion,and immune checkpoint blocking.Among them,in the field of immune cell therapy,chimeric antigen receptor T cells(CAR-T)have gradually attracted attention,whose basic principle is to specifically recognize the target antigen on the surface of tumor cells and form specific cytotoxicity against tumor cells through the activation of intracellular signals.The efficacy of CAR-T cells in hematological tumors is very significant.Many anti-CD19 CAR-T cell methods have been approved by the FDA,and some patients can be cured totally.However,CAR-T cells have not made any breakthrough in the treatment of ovarian cancer.The main problems are closely related to the characteristics of solid tumors.For example,the microenvironment of solid tumors is complex,and the function of immune cells is often inhibited.The tumors of solid tumors are essentially highly heterogeneous,there are very limited choices of immune targets;solid tumors are dense masses of tissues,and it is difficult for conventional immune cells to break through the physical barriers of solid tumors.In short,it is necessary to explore new treatments for ovarian cancer.At the same time,fundamentally,immunotherapy methods must also be based on the disease characteristics of ovarian cancer.Urokinase-type plasminogen activator receptor(uPAR)is a GPI-anchored membrane protein,which belongs to one of the members of the plasmin system.In a variety of malignant tumors,significant high expression of uPAR has been found,which can bind urokinase-type plasminogen activator(uPA)to initiate plasminogen activation and participate in the degradation of extracellular matrix,affecting pathological processes such as tumor proliferation,metastasis,invasion,drug resistance and recurrence,and is one of the tumor markers that have gradually received attention.Existing studies show that uPAR is specifically and highly expressed in ovarian cancer tissues,not only on ovarian cancer parenchymal cells but also on tumor stromal cells.Then,unlike traditional tumor treatment targets,uPAR-targeted therapies are expected to simultaneously capture the stromal components of ovarian cancer and the substantial components that inhibit ovarian cancer.In this study,uPAR was used as a therapeutic target for ovarian cancer.Firstly,the effect of uPAR on the metastatic ability of ovarian cancer cells was verified,and the animal model was used to verify the ability of uPAR to promote the peritoneal implantation and ascites formation of ovarian cancer.Considering characteristics of progression function and specific expression of uPAR in ovarian cancer,CAR-T cells targeting uPAR were designed to kill ovarian cancer cells,and the efficiency of cytotoxicity and cytokine secretion were verified.Objective:Construct stable transfection ovarian cancer cell lines with uPAR gene overexpression and gene knockout,respectively;study the effect of uPAR on ovarian cancer cell metastasis ability;study uPAR influence on the changes of various parameters of epithelial-mesenchymal transition during ovarian cancer metastasis;Mouse ovarian cancer intraperitoneal implant tumor model was used to investigate the effects of uPAR on ovarian cancer intraperitoneal implantation and ascites formation.CAR-T cells targeting uPAR were constructed.Validate the cytotoxicity ability of uPAR-targeted CAR-T cells to ovarian cancer cells,and provide new ideas and strategies for clinical cell therapy of ovarian cancer.Methods: Part 1: In vitro experiments with uPAR to enhance metastatic capacity of ovarian cancer1.Detection of uPAR protein expression in ovarian cancer cell lines cultured in vitro by flow cytometry;2.Design and construct a uPAR gene overexpression plasmid vector and transfect the uPAR negative cell line A2780;design and construct a shRNA lentiviral vector targeting the uPAR gene to transduce the uPAR strong positive cell line ES-2;construct and identify the uPAR gene stably transfected cell lines at the overexpression and knockout sites;3.To explore the effect of uPAR on the metastatic ability of ovarian cancer cells through cell scratch experiments and Transwell chamber experiments;4.The changes of various indexes in epithelial-mesenchymal transition and the expression of uPA and PAI-1 were detected by Western blotting.Part 2: In vivo experiments uPAR promotes ovarian cancer metastasis and ascites formation1.Establish a mouse abdominal cavity implant tumor model using ES-2 cell line and its uPAR gene knockout cell line to verify the effect of uPAR on abdominal cavity metastasis of ovarian cancer;2.Measure the effect of uPAR on the formation of ascites in mice by measuring the weight and abdominal circumference of mice;3.To investigate the effects of uPAR gene knockout on tumor lesions,liver and spleen organs of ovarian cancer mice by HE staining and immunohistochemical staining.Part 3: Construction and preparation of targeted uPAR-CAR-T cells1.Utilize the basic framework of the third generation CAR-T cells to design X CAR-T cells that target uPAR,and creatively select natural binding fragments that target uPAR as extracellular recognition regions;2.Construct a CAR lentivirus using a three-plasmid lentivirus packaging system;3.T lymphocytes in peripheral blood were extracted by density gradient centrifugation,CAR-T cells were constructed by lentiviral transduction method,and the expression of CD3,CD4,CD8 of T cells was detected by flow cytometry.Part 4: Targeting uPAR-CAR-T cells to kill ovarian cancer cells1.Using anti-uPAR-CAR-T cells,uPAR positive ovarian cancer cell lines,and uPAR negative ovarian cancer cell lines for effector cell and target cell incubation experiments;2.Use the LDH kit method to detect the tumor lysis rates of the efficacy target experiment and find the best efficacy target ratio;3.Under the best effect target ratio,use CBA kit combined with flow cytometry to detect Th cytokines IL-2,IFN-?,TNF,IL-4,IL-5,IL-10 in the effect target experiment;4.Use the ELISA method to detect granzyme B in the efficacy target experiment to determine the basic law of CAR-T cytotoxicity.Results: Part 1:1.Flow cytometry showed that among 5 ovarian cancer cell lines of different pathological types,4 cells had high expression of uPAR protein;ES-2 cells were uPAR strongly positive cell lines with an expression rate of 95.2%;A2780 was uPAR negative cell line.2.Construction of stably transfected uPAR-overexpressing A2780 cells and its control cells was completed and overexpression of uPAR up-regulates the expression of uPA and PAI-1;construction of uPAR gene knockout ES-2 cells and its control cells was completed.The uPAR protein and mRNA levels of the stably transfected cell lines were significantly different from those of the blank group and the control group.3.Scratch experiments and Transwell chamber experiments show that overexpression of uPAR promotes the migration of ovarian cancer cells;on the contrary,knockdown of uPAR inhibits the migration of ovarian cancer cells.4.uPAR enhances the metastatic capacity of ovarian cancer cells,up-regulates mesenchymal cell phenotype proteins N-cadherin and vimentin,down-regulates epithelial cell phenotype protein E-cadherin,and promotes epithelial-mesenchymal transition process.Part 2:1.In the mouse abdominal cavity tumor model,after the knockdown of uPAR,the ability of the ovarian cancer cell line ES-2 to form abdominal cavity tumors and produce ascites was reduced;2.After the knockdown of uPAR ES-2 cells formed a mouse ovarian cancer model,their uPAR negative expression characteristic was successfully retained.HE staining showed that the tumors formed by uPAR-negative cells had densely arranged cells,deep stained nuclei,and the cell morphology was oval.The tumors formed by uPAR-positive cells had disordered cell arrangement and the cell morphology was spindle-shaped.Part 3:1.The extracellular recognition region of uPAR-CAR-T cells uses ATF as the natural binding fragment of uPAR and named as ATF-CAR-T cells.CD28-CD137-CD3? was used as the intracellular region;2.The method of exogenous addition of CD3,CD28 antibodies,and IL-2 factors was used to expand and activate human peripheral blood-derived lymphocytes in vitro.The proportion of CD3 positive was 99.0%,and the proportion of CD8 positive increased significantly.3.The transduction efficiency of CAR lentivirus for T lymphocytes is 61.0%.Part 4:1.1.CAR-T cells have a significant cytotoxicity effect on uPAR-positive ovarian cancer cells,but not on uPAR-negative ovarian cancer cells,indicating that CAR-T cells play a role of target antigen specificity;using different ratios of target ratio 1: 1,5: 1,10: 1,20: 1,it was found that the cytotoxicity effect of CAR-T is effector cell dose-dependent.When the effector target ratio is 10: 1,the lysis rate of ES-2 by ATF-CAR-T cells is 65.2%±2.7%.When the effector target ratio is 20: 1,the lysis rate of ES-2 by ATF-CAR-T cells can reach 70.6% ± 1.7%.Considering the balance between cost and effect,10: 1 is the most suitable effector target ratio.2.When CAR-T specifically kills uPAR-positive cells,the secretion of IL-2,IFN-?,and TNF cytokines and the release of granzyme B are significantly increased.Conclusions:1.Successfully constructed uPAR stable overexpression and knockdown ovarian cancer cell lines;determined that overexpression of uPAR promoted the upregulation of uPA;overexpression of uPAR gene enhanced the metastatic capacity of ovarian cancer cells;conversely,knockdown of uPAR gene caused ovarian cancer reduced transfer capacity;2.uPAR promotes the metastasis of ovarian cancer cells by affecting the epithelial-mesenchymal transition.uPAR overexpression promotes the generation of mesenchymal cell phenotype and inhibits epithelial cell phenotype.Conversely,knockdown of uPAR has the opposite effect3.In a mouse abdominal ovarian cancer model,knockdown of uPAR of ovarian cancer leads to a reduction of abdominal lesions and a decrease in ascites formation;4.Effector target killing experiments show that CAR-T cells have a cytotoxity effect on ovarian cancer cells,and this effect has uPAR target antigen specificity and CAR-T cell dose dependency,laying a foundation for the development of clinical extrails;5.When CAR-T cells exert a killing effect,the release of IFN-?,TNF,IL-2 and granzyme B is significantly increased.The feasibility of anti-uPAR-CAR-T cells in the treatment of uPAR-positive ovarian cancer was confirmed. |
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