Study On The Role And Mechanism Of Exosomes Derived From Prostate Cancer Cells In Mediating T Cell Exhaustion

Background:Prostate Cancer(PCa)is a common male malignant tumor.At present,the treatment effect of advanced metastatic castration-resistant prostate cancer(m CRPC)is not ideal,and the mechanism of PCa progression and metastasis needs to be further clarified.T cells are the main effector cells that mediate adaptive immune responses in the body and play an important role in the occurrence and prevention of various immune diseases.Under normal circumstances,T cells will begin to be activated and proliferate in large numbers after being stimulated by external antigents.Subsequently,T cells acquire effect functions to produce cytokines and granzymes that mediate cytotoxicity,thus killing cancer cells.However,when T cells continue to be stimulated by tumor antigents in the tumor microenvironment,the surface inhibitory receptors will continue to be highly expressed,resulting in the decline of T cell function,entering a“fatigue”state,and eventually becoming exhuasted T cells,which is the best time for tumor cells to escape from immunity.In recent years,it has been found that tumor cells can participate in this immune escape mechanism by secreting exosomes.The exosomes from tumor cells can promote the occurrence and development of tumors by promoting the formation of tumor microenvironment,mediating the information exchange between tumor cells and immune cells,inhibiting the activity of immune cells,and increasing the ability of tumor cells to invade and migrate.However,it is unclear whether prostate cancer cells can also affect the immune function of T cells by secreting exosomes,thus promoting the progression and metastasis of prostate cancer.Therefore,this experiment aims to explore the effect and mechanism of exosomes from prostate cancer cells on T cell exhaustion,and provide a new data for the study of the immune escape of prostate cancer,which is of great significance for the immunotherapy and drug research of PCa.Objective:This paper aims to study whether the exosomes of prostate cancer cells can induce T cell exhaustion,and then preliminarily explore the molecular mechanism underlying the exosomes of prostate cancer cells mediating T cell exhaustion.Methods:1.Cultured prostate cancer cell lines PC-3 and RM-1,collected their respective conditional mediums,and isolated PC-3 exosomes(PC-3-exos)and RM-1 exosomes(RM-1-exos)by Millipore ultrafiltration.Using a transmission electron microscope to observe the shape of the exosome,nanoparticle tracking analysis technology to detect the particle size of the exosome,the surface marker proteins of the exosomes were identified by western blot and flow cytometry;2.Established a T cell induction system in vitro:extracted human peripheral blood single nuclear cells(PBMC),CD8~+T cells were sorted by magnetic beads,and the purity of sorted cells was identified by flow cytometry.CD8~+T cells were cultured with GT-T551 H3 complete medium containing 5μg/m L CD3,2μg/m L CD28 and 20ng/m L IL-2.The experiment was divided into three groups:CD8~+T cells normally without any treatment as the control group;CD8~+T cells were induced by PC-3-exos(100μg)as PC-3-exos group;CD8~+T cell were induced by exosomes from PC-3 cells terated with GW4869 as GW4869 group;PC-3-exos were labelled with PKH67 and added to CD8~+T cells for co-culture,immunofluorescence was used to detecte the absorption of PC-3-exos by CD8~+T cells at 6,12,24,and 48h respectively;3.After 3 days of co-culture of PC-3-exos and CD8~+T cells,CFSE marker was used to detect the proliferation of each group of CD8~+T cells;Annexin V-FITC/PI double-labeled apoptosis kit was used to detect the number of apoptosis of each group of CD8~+T cells;CCK-8 was used to detect the killing effect of each group of CD8~+T cells on PC-3 cells;Flow cytometry was used to detect the expression of CD8~+T cells surface exhaustion indicators PD-1 and TIM-3 in each group;q PCR was used to detect the expression of PD-1,TIM-3,LAG-3 and CTLA-4 in each group;ELISA kit was used to detect the expression of IL-2,IL-4,IL-6,IL-10,TNF-α,IFN-γ,TGF-βin each group of CD8~+T cells;4.Established a mouse model of T cell exhaustion:BALB/C mice were divided into 3 groups,including RM-1 cells,RM-1 cells+RM-1-exos,RM-1 cells+10μmol/L GW4869 group.These cells from above groups were injected under the right armpital skin respectively,every two days and lasted for 28 days.After that,the mice were sacrificed aseptically,and the tumor tissues were took out,isolated into a single-cell suspension.The magnetic beads were used to sort the tumor infiltrated CD8~+T cells,and the expression of the tumor infiltrated CD8~+T cells surface exhaustion indicators PD-1 and TIM-3 in each group of mice were detected by flow cytometry;5.QPCR was used to detect the expression of miR-100-5p in PC-3-exos and RM-1-exos;PC-3-exos were used to induce CD8~+T cells,and q PCR was used to detect the expression of miR-100-5p and Fox O1 in CD8~+T cells before and after induction;6.The micro RNA target gene database was used to predict the binding site of miR-100-5p and m TOR;After the transfection of miR-100-5p inhibitor and mimic,q PCR was used to detect the expression of the target gene m TOR;After the transfection of miR-100-5p inhibitor,q PCR was used to detect the expression of Fox O1 and PD-1 in each group of CD8~+T cells,Flow cytometry was used to detect the expression changes of surface exhaustion indicators PD-1 and TIM-3 of each group of CD8~+T cells,Annexin V-FITC/PI double-labeled apoptosis kit was used to detect the apoptosis of CD8~+T cells in each group.Results:1.Under the transmission electron microscope,it was observed that the exosomes showed a concave spherical shape.The nanoparticle tracking analysis technology revealed that the diameter of the exosome was about 100nm.Two marker proteins CD63 and TSG101 in exosomes were identified by western blot,and the expression of CD9 marker protein in exosomes was identified by flow cytometry;2.With the increase of time,the absorption of PC-3-exos by CD8~+T cells was also increasing,and the absorption rate reached 85%at 48 hours;3.After CD8~+T cells were induced with PC-3-exos,the cell proliferation ability was weakened,the number of apoptosis was increased,the ability of killing tumor cells PC-3 was weakened,the expression of cell surface exhaustion indicators PD-1and TIM-3 was significantly increased,the levels of IL-2,IL-4,IL-6,TNF-αand IFN-γwere decreased,while secretory inhibitory cytokines IL-10 and TGF-βwere increased;4.In the mouse model,The RM-1-exos group promoted the tumor growth compared with the control,and the expression of the tumor infiltrated CD8~+T cells surface exhaustion indicators PD-1 and TIM-3 were significantly increased;5.The expression of miR-100-5p was high in PC-3-exos and RM-1 exos;After CD8~+T cells were induced with PC-3-exos,the expression of miR-100-5p was increased in cells,and the expression of Fox O1 was also increased in cells;6.The database predicted that miR-100-5p has a binding site with the target gene m TOR 3′UTR;Overexpressed miR-100-5p reduced the expression of m TOR,while the inhibition of miR-100-5p promoted the expression of m TOR,and led to the decrease of the expression of Fox O1 and PD-1,and the number of apoptosis of CD8~+T cells.Conclusion:The exosomes of prostate cancer cells can be absorbed by CD8~+T cells and further induces the exhaustion of CD8~+T cells,possibly through inhibiting the expression of its target gene m TOR by transporting miR-100-5p to CD8~+T cells,and then up-regulating the expression of Fox O1 to promote the expression of inhibitory receeptor PD-1,and eventually inducing CD8~+T cells exhaustion.