Mesothelin Expression And Functionl And Molecular Targeted Magnetic Resonance Imaging In Malignant Pleural Mesothelioma

Objective(s):1.To identify the mesothelin(MSLN)expression levels of malignant pleural mesothelioma(MPM)cell lines and xenograft models,and explore the potential regulatory mechanism of MSLN on the migration and invasion of MPM.2.To construct and prepare MSLN antibody and Fe3O4@SiO2 coupled nanoprobe,and evaluate and analyze the feasibility of MRI in vivo and in vitro molecular imaging.3.To explore the potential application value of multimodal functional MRI and MSLN targeted MRI nanoprobes in vivo and in vitro molecular targeted imaging to detect MPM.Methods:1.Western Blotting detects MSLN protein expression level of cell lines and tumor tissues in the MeT-5A(normal immortalized mesothelial cells,normal control group),A549(human lung adenocarcinoma,negative control group),H2452(epithelial MPM),H226(epithelial MPM)and MSTO-211H cells(biphasic MPM),RT-PCR detection of cell mRNA expression level.Scratch test and Transwell migration test detect cell migration ability;Transwell invasion test detects cell invasion ability;tumor tissue Western Blotting,MSLN immunohistochemistry and serum SMRP test to verify the expression level of MSLN in vivo tissue level.After using lentivirus-mediated H226(epithelial MPM)to overexpress MSLN,Western Blotting and RT-PCR verified the changes in MSLN expression in cells.Compare the protein expression of N-Cadherin,MMP7 and MMP9,cell migration and invasion ability among wild-type H226(negative control group),NC(transferred to empty vector,blank control group)and MSLN overexpression group(experimental group).Observe and compare the tumor growth curve and survival period of different tumor-bearing mice.2.The anti-MSLN antibody and Fe3O4@SiO2-NH2 material were connected by chemical coupling method to synthesize MSLN targeting MRI nanoprobe.Use transmission electron microscope(TEM)to observe the shape and size of the nanoprobe;MRI analyzer to detect the lateral and longitudinal relaxation time of the nanoprobe;X-ray photoelectron spectroscopy(XPS)to determine element calibration;ICP detection to determine the iron ion concentration of the nanoprobe;Serum and normal saline respectively dilute the nanoprobe to evaluate its stability.Prussian blue staining was used to observe the binding of tumor cells and probes in vitro;CCK8 was used to detect the cytotoxicity of nanoprobes;HE staining was used to observe the pathological changes of the main organs of nude mice after probe injection.T2WI and T2mapping imaging were performed after the probe was bound to the cells in vitro,and the main organs of nude mice after the probe injection were analyzed by ICP iron ion concentration to analyze the binding ability of the probe and tumor cells in vivo and in vitro.3.Nude mouse xenograft models of human lung adenocarcinoma cells A549(negative control group),epithelial MPM H226(experimental group),and biphasic MPM MSTO-211H(experimental group)were established.A total of 81 nude mice,27 in each group.On the 14th,28th and 42nd day after tumor inoculation,anatomical imaging T1WI and T2WI,T1 mapping,T2 mapping,BOLD and IVIM-DWI functional imaging were performed with a 3.0 T MRI scanner.Measure and compare the T1 value,T2 value,R2,T2*,apparent diffusion coefficient(ADC),true diffusion coefficient(D),pseudo diffusion coefficient(D*)and perfusion fraction(f)at 3 time points(9 animals/time point)between H226 group and MSTO-211H group.Evaluate the differences in parameters between different time points,and analyze the correlation between MRI multi-parameters and histological features related to prognosis(including necrosis score,NF;microvessel density,MVD).After nanoprobes with different iron ion concentration gradients are combined with cells,MRI scan is performed to observe the change trend of T2 signal intensity and T2 value in vitro.And after A549,MSTO-211H and H226 tumor-bearing mice were injected with normal saline,non-targeted nanoprobe and MSLN targeted nanoprobe,respectively,T2WI and T2 mapping imaging of tumor tissues in vivo were performed to analyze the relative T2 signal intensity and T2 value changes of different tumor types,different nanoprobes and different periods.Results:1.The expression of MSLN protein and mRNA of H226 cells was higher than that of other tumor cells(p<0.05).MSTO-211H migration and invasion ability(migration rate,number of migrating cells,OD value of migrating cells,number of invasive cells,OD value of invasive cells)is stronger than other tumor cells(p<0.05).The expression of MSLN in tumor tissues and serum was detected by Western Blotting,immunohistochemistry and Elisa.As the tumor grew,the expression of MSLN in MPM tumors increased,and the H226 group was higher than the MSTO-211H group(all p<0.05).In addition,the tumor growth rate of MSTO-211H group was faster than that of H226 group,and the survival time of tumor-bearing mice in MSTO-211H group was shorter than that of H226 group(p<0.05).After lentivirus transfection to H226,the MSLN protein and mRNA of H226 were higher than those of wild type and NC group(all p<0.05),and the protein expression of N-Cadherin,MMP7 and MMP9 increased,and migration rate,the number of migrating cells,the number of invasion cells and the OD value of crystal violet staining increased,and the survival time was shortened(all all p<0.05).2.MSLN targeting MRI nanoprobe was successfully synthesized.A large ratio of transverse relaxation rate/longitudinal relaxation rate indicates that the probe is a good MRI negative contrast agent;XPS detection shows that the anti-MSLN antibody is successfully coupled to Fe3O4@SiO2 nanoprobe.In addition,the nanoprobe has good dispersion and stable properties.Prussian blue staining showed that MSLN nanoprobe could label H226 cells.CCK8 detection showed that the nanoprobe did not significantly reduce the proliferation of A549 cells(lung adenocarcinoma cells)and H226 cells,and had no obvious toxicity to the cells.After the nanoprobe was injected,the histological performance of the brain,liver,lung,spleen,heart and kidney of nude mice was no different from that of the normal saline group,indicating that the nanoprobe had no obvious toxic and side effects on the tissue.MRI imaging after nanoprobe in vitro cell binding showed that the T2 signal and T2 value of H226 group were lower than those of A549 group,and the difference was statistically significant(p<0.05).ICP detected the distribution level of iron ions in the organs.The concentration of iron ions in the tumor tissues of the H226 group was higher than that of the A549 group,and the difference was statistically significant(p<0.05).3.In MPM functional MRI,compared to the H226 group,T2 and T2*were higher and ADC,D,D*,and f were lower in the MSTO-211H group(p<0.05).Serial measurements in the H226 group showed that both ADC and D decreased significantly at 28 and 42 days compared to 14 days(p<0.05),while f significantly increased at 28 and 42 days(p<0.05).A significant increase in the D*was found at 28 days and 42 days vs.14 days in the two groups.Moderate correlations were found between ADC and tumor volume,NF(r=-0.53,p=0.000 and r=-0.508,p=0.000),and there are positive correlation between f and tumor volume,NF and MVD in the H226 group.Only f showed significant negative correlation with tumor volume,NF and MVD,with moderate relationship(r=-0.560,p=0.009;r=-507,p=0.001;r=-0.554,p=0.000;respectively)in the MSTO-211H group.After H226 cells were combined with MSLN targeting nanoprobes with different iron ion concentration gradients,the T2 signal and T2 value continued to decrease,and when the iron ion concentration was 60 ?g/ml,the T2 signal and T2 value of the H226 group were lower than MSTO-211H group,H2452,MeT-5A and A549 group(all p<0.05).In 3 different tumor growth periods,1.5-3.5 hours after injection of non-targeted and MSLN targeted nanoprobes in H226 group,the relative T2 signal intensity and T2 value of tumor decreased,and the degree of decrease was greater than that of MSTO-211H group(all p<0.05).Conclusion(s):1.MPM cells express MSLN,and there are differences in expression in different tumor histological subtypes and growth stages;and MSLN promotes tumor migration and invasion ability by regulating N-Cadherin,MMP7 and MMP9,and shortens the survival period.2.The synthesized MSLN targeting nanoprobe has low toxicity,good stability,can specifically bind to MPM cells,and has a good effect on specifically reducing T2 signal intensity and T2 value.3.Functional MRI parameters and MSLN targeting nanoprobes are helpful for MPM diagnosis and histological subtype analysis,can indicate tumor progression,and provide information and basis for indirect evaluation of MPM migration,invasion and survival.