| Objective:Malignant pleural effusion(MPE)is a frequent complication of various cancers such as lung cancer,breast cancer,and gastric carcinoma,and often leads to pleural compression,dyspnea,decreased quality of life,and short survival.Although the treatment of MPE including pleurodesis,indwelling pleural catheters,and chemotherapeutics drugs infusion,which are suboptimal in terms of their efficacy and safety.Thus,approaches that can efficiently target MPE with minimal side effects are highly desirable.Recently,immunotherapies have shown the unprecedented success in clinical cancer treatment.Chimeric antigen receptor T cell immunotherapy and immune checkpoint inhibitor were approved to the clinical application,suggested that the coming of immunotherapy.However,the innate immunity in tumor therapy still lag out.Neutrophils are the most abundant immune cells in our body,and crucial in fighting against bacteria infections,however,whether neutrophils play a role in the anti-tumor processes remains unclear.Our previous study have shown that drug loaded microparticles have a good therapeutic effect on tumors therapeutic and effectively target in tumor repopulating cells.In clinical trial,robust neutrophils were collected in pleural cavity after drug loaded micropaticles pleural perfusion.The purpose of this study was to investigate the role of neutrophils in the treatment of malignant pleural effusion with drug-loaded microparticles.Methods:(1)Clinical trial:Patients diagnosed with non-small cell lung cancer with primary malignant pleural effusion.All patients were received pemetrexed(PEM,500mg/m~2)and cisplatin(DDP,75 mg/m~2)i.v.administration on day 1,and treated with 50ml MTX-MPs(5×10~7 MPs containing 25?g MTX)or 50ml saline on day 5,7,9,11,13 and 15 by intrathoracic injection.(2)Cells were isolated from MPE on day 5and 7,the change of cells were analyzed by flow cytometry.(3)In BALB/c mice ascite model and C57BL/6 mice MPE model,MTX-MPs were i.p.injected and the long time survival were analyzed.(4)1×10~5 Lewis were intrapleural injected to C57BL/6 mice or1×10~5H22 i.p injected to BALB/c mice,PKH26 labeled MPs were intropleural or introperitoneal injected to the mice,cells in pleural cavity or peritoneal cavity were collected,the uptake of MPs by tumor cells,macrophage,and neutrophils were analyzed by flow cytometry.(5)Macrophages,tumor cells and neutrophils were isolated from MPE,and cocultured with MTX-MPs for 2 hours,the supernatants were used for neutrophil recruitment in transwell assay.(6)MPE were collected at different time points and the change of neutrophils were analyzed by flow cytometry.(7)Macrophages were collected from MPE and cocultured with MTX-MPs for 2 hours,CXCL1 and CXCL2 were measured by realtime PCR and ELISA.(8)Cells were isolated from MPE on day 5 and day 7,FSC,SSC and phenotype of neutrophils were analyzed by flow cytometry,the expression of NOX2,arginase-1,MPO and i NOS were analyzed by realtime PCR.(9)Tumor cells from MPE or A549 cells were cocultured with neutrophils from MPE on day 5 and 7 in MTX-MPs group for 16 hours,the apoptosis of tumor cells and A549 cells were analyzed by flow cytometry,and neutrophil incubated with A549 cells in the presence or absence of NAC or DPI for 16hours,the apoptosis of tumor cells was measured by flow cytometry.(10)1×10~5 H22were i.p injected to BALB/c mice for 3 days,followed by MTX-MPs i.p.injection for18 hours,peritoneal neutrophils were collected and lysosomal biogenesis were analyzed.(11)Neutrophils were isolated from control and MTX-MP group,and neutrophils extracellular traps(NETs)releasing was analyzed by Scanning Electron Microscope(SEM),and then cocultured with CFSE labeled A549 cells at the presence or absence of NETs inhibitor Cl-amidine for 16 hours,the apoptosis of A549 cells were analyzed.(12)Neutrophils were isolated from MPE on day 5 and 7 in control and MTX-MP group,and the effect of neutrophils on endothelial cells permeability was analyzed.(13)1×10~5H22 were i.p injected to BALB/c mice for 3 days,followed by MTX-MPs i.p injection daily for 3 days,and then 1%Evans blue dye(5 mg/kg)was i.v.injected 1 hour before mice scarified.The extravascular Evans blue dye in pleural cavity was quantified spectrophotometrically.(14)1×10~5 Lewis cells were inoculated in the pleural cavity for10 days,followed by intrapleural injection of MTX-MPs.18 hours later,Sytox green was i.v.injected and the mice were scarified 2 hours later,the pleural vessels were analyzed by immunofluorescent staining.(15)In mice MPE model,MTX-MPs were intrapleural injected everyday with or without NETs inhibitor for 5 days,long time survival was analyzed.Results:(1)The count of CD45~-tumor cells were decreased,and CD45~+immune cells were increased after MTX-MPs treatment.(2)Robust neutrophils were collected in the effusions,while MDSCs,NK cells and macrophages were not altered.(3)The long time survival was increased after MTX-MPs treatment while it was decreased after neutrophils depletion.(4)Only macrophages take up MPs in Lewis induced MPE model and H22 induced hepatocarcinoma ascite model in 2 hour after MTX-MPs injection.(5)The supernatant of MTX-MPs treated macrophages demonstrated an attraction effect on neutrophils.(6)The number and proportion of neutrophils in the MPE started to increase 2 hours after MTX-MPs injection.(7)m RNA and protein level of CXCL1 and CXCL2 were upregulated in macrophage after 2 hours incubation with MTX-MPs.(8)The forward scatter(FSC)of neutrophils was reduced after MTX-MP treatment,and CD11b,CD54 and CD66b were upregulated,CD15 was downregulated.NOX2,MPO and i NOS were upregulated while arginase-1 was downregulated.(9)Neutrophils from MTX-MP-treated MPE on day 7 were able to kill tumor cells and A549 cells in vitro,and the use of ROS scavenger N-acetylcysteine(NAC)or NOX2 inhibitor diphenylene iodonium(DPI)could lower the ROS levels and also inhibit the killing of tumor cells by neutrophils.(10)We found that lysosomal biogenesis was enhanced in MTX-MP-treated neutrophils.(11)Considerable neutrophils extracellular traps(NETs)were found after MTX-MPs treatment,and blockade of ROS generation resulted in the inhibition of the NETs formation in MTX-MP-treated neutrophils.(12)We found that saline-treated neutrophils could not prevent FITC-Dextran from entering the bottom chamber by crossing the endothelial layer,however,MTX-MP treated neutrophils effectively prevented this process.(13)In the mice MPE model,we also found that Evans blue could be detected in the MPE of the saline-treated mice,but very little detected in the MTX-MP-treated mice.(14)The frozen section analysis showed that neutrophils anchored to blood vessels and abundant NETs were located between the endothelial gaps.(15)The therapeutic effect of MTX-MPs on pleural effusions was also inhibited and the mice survival was reduced when NETs inhibitor was used.Conclusions:In the treatment of MPE by MTX-MPs,robust neutrophils were collected in pelural cavity,and the increased neutrophils were positively correlated with MPE decreasing.It was further confirmed in animal models that a large number of neutrophils were recruited to the peritoneal cavity,and neutrophils recruitment is required for MTX-MPs treatment efficiency.In term of mechanism,the injection of MTX-MPs induces macrophages in the MPE to release CXCL1 and CXCL2 allowing for neutrophil recruitment.And the injection of MTX-MPs not only recruits neutrophils to the MPE of patients but also confers them an antitumor property.These neutrophils use ROS and NETs to kill tumor cells in the MPE of patients.Meanwhile,NETs released by neutrophils could seal off the damaged endothelium and inhibit vascular leakage.These findings reveal the potential for such cell-derived materials used to package drugs as an immunotherapeutic agent against MPE. |
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