Study On Serum Metabolomics Biomarkers And Exosomes In DLBCL

ObjectiveDiffuse large B-cell lymphoma(DLBCL)represents the most prevalent subtype of nonHodgkin lymphoma(NHL).DLBCL is distinguished by remarkable heterogeneity in clinical manifestations,biological features,responses to treatment and outcomes.During the rituximab era,the limitations of international prognosis index(IPI)in DLBCL prognosis are increasingly prominent.It is crucial to look for new lymphoma biomarkers and explore the mechanism of malignant biological behavior in DLBCL.The research in the paper is divided into three parts: Part 1.To find DLBCL serum metabolomics biomarkers and study the value of the biomarker in DLBCL diagnosis,prognosis prediction and efficacy judgment;Part 2.The influence of lymphoma cellderived exosomes on DLBCL biological malignant behavior was researched;Part 3.The proteomics analysis was performed on the serum exosomes from DLBCL patients.MethodsPart 1.First,serum samples of 91 DLBCL patients and 91 healthy volunteers in test set was analyzed by UPLC-QTOF-MS to find metabolomics biomarkers in DLBCL.The serum samples of 148 DLBCL patients at different treatment phases were then collected.LC-MS/MS was used to detect the serum biomarkers level of DLBCL patients,retrospective analysis was performed on the clinical data of DLBCL patients,and the importance of the biomarker on the DLBCL diagnosis,curative effect judgment and prognosis prediction was analyzed by statistical method.Part 2.Exosomes were isolated from the supernatants of cell culture of diffuse large Bcell lymphoma cell line Su-DHL-4 and Burkitt lymphoma cell line Raji by Total Exosome Isolation(from cell culture media).The morphology and size of exosomes were observed by transmission electron microscopy,the expressions of exosome markers,ALIX and Tsg101 were detected by Western blot,and the size distributions of exosomes were measured by Zetaview nanoparticle tracing analyzer.The proliferation curve was drawn by trypan blue exclusion method to detect cell proliferations of SuDHL-4 and Raji after co-cultured with lymphoma-derived exosomes.The half maximal inhibitory concentration(IC50)of doxorubicin(DOX)on lymphoma cells was detected by CCK-8 method.The capabilities of lymphoma cells to migrate and invade were detected by Transwell chamber assay.Part 3.Exosomes were isolated from serum of DLBCL patients and healthy volunteers by Total Exosome Isolation(from serum).Proteomics detection was performed on exosomes derived from serum samples of healthy volunteers and DLBCL patients with different outcomes and from risk group.Principal components analysis,KEGG Pathway analysis,GO analysis were conducted to find the differential proteins.ResultsPart 1.By metabolomics technology analysis,DLBCL expressed a special metabolic fingerprint,and a novel biomarker,Phenylalanyl-tryptophan(Phe Trp)was found closely related to DLBCL.The serum Phe Trp concentration before treatment of DLBCL patients was obviously lower than the healthy volunteers(P<0.001)and Phe Trp had a potential diagnosis valve on DLBCL with a diagnosed AUC of 0.945,sensitivity of 90.54% and specificity of 85.92%;the serum Phe Trp level of untreated DLBCL was in negative correlation with IPI score and staging(P <0.001);low Phe Trp at diagnosis was related to the poor prognosis of DLBCL,and was an independent prognosis factor of PFS;IPI combined with Phe Trp obtained better prognosis prediction value for DLBCL;Phe Trp level at diagnosis could predict the curative effect on DLBCL patients(AUC=0.650,P<0.001);Phe Trp and 18F-FDGPET/CT had similar effect on the prediction of prognosis and curative effect for DLBCL patients.Part 2.Transmission electron microscopy showed that the isolated exosomes were bilayer membrane vesicles with typical structure.Western blot showed that the isolated exosomes expressed exosome marker proteins,ALIX and Tsg101.Zetaview particle size analysis showed that the size of the extracted exosomes ranged from 30 to 150 nm.Lymphoma cell lines Su-DHL-4 and Raji cells were co-cultured with different concentrations of exosomes Su-EXO and Raji-EXO.The proliferations of lymphoma cell lines were significantly enhanced by co-culture with lymphoma-derived exosomes in a dose-dependent manner(48 h,40 μg/ml,P < 0.01).The susceptibility of Su-DHL-4 cells to DOX were significantly reduced after co-cultured with Su/DOX-EXO(P < 0.05),which were exosomes derived from the DOX-resistant cell line Su-DHL-4/DOX.Transwell chamber assay showed that lymphoma cell-derived exosomes,Su-EXO and Raji-EXO,could significantly enhance the migration and invasiveness of lymphoma cell lines Su-DHL-4 and Raji(migration,P < 0.05 for Su-DHL-4,P < 0.001 for Raji;invasion,P < 0.05 for Su-DHL-4,P < 0.01 for Raji).Part 3.Proteomics analysis showed that,by comparing high-risk DLBCL with poor outcomes(Group A)with low-risk DLBCL with good outcomes(Group B),there were 7 specifical proteins in Group A and one in Group B;by comparing Group A with healthy contrast group(Group C),Group A had 9 specificity protein and Group B had 2;by comparing Group B with Group C,Group B had one specificity protein.Through comparison between Group A and Group B,Group A had 44 types of protein upregulation and 31 kinds of protein down-regulation;through comparison between Group A and Group C,Group A had 51 proteins up-regulation and 19 proteins downregulation;through comparison between Group B and Group C,Group B had 24 proteins up-regulation and 8 proteins down-regulation.KEGG pathway analysis,GO analysis showed that obvious difference occurs in PI3K/AKT signal pathway,ECM receptor interaction signal pathway and PPARs signal pathway,and a series of differential proteins were founf,include VWF,TNC,COL6A3,VTN,FN1,etc.Conclusion1.The novel biomarker based on metabolomics,Phe Trp played an important role in DLBCL diagnosis,prognosis prediction and curative effect judgment.2.Lymphoma cell-derived exosomes can promote the proliferation of lymphoma cells,reduce the sensitivity of lymphoma cells to chemotherapeutic drug doxorubicin,and enhance the capabilities of lymphoma calls on migration as well as invasion.It suggested that exosomes interfere with the malignant biological behaviors of lymphoma cells,and might be a potential target for the treatment of refractory/relapsed aggressive lymphoma.3.DLBCL patients from different risk group had significant unique characteristics in proteomics,suggested that protein in serum exosomes were related to the heterogeneity of efficacy and prognosis of DLBCL patients.