HBMSCs Promote The Growth Of Diffuse Large B-cell Lymphoma By Secreting IL-6 And Elevating IL-17A Levels In The Tumor Microenvironment

Diffuse large B-cell lymphoma(DLBCL),the most common subtype of non-Hodgkin’s lymphoma,is an aggressive lymphoma.Although the first-line chemotherapy regimen R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine and prednisone)has improved the prognosis of DLBCL patients in recent years,a considerable number of DLBCL patients have progressed or relapsed due to chemotherapy resistance,which is a major problem and challenge for clinical treatment.At present,the mechanism of drug-resistance and relapse in DLBCL patients is still unclear,which deserves further study.Many studies have found that rituximab resistance is related to mesenchymal stem cells.This study will clarify whether chemotherapy resistance and relapse in DLBCL patients are related to mesenchymal stem cells,and clarify the specific mechanism,so as to provide a new targeted therapy strategy for further solving the drug resistance and relapse in DLBCL patients.In this study,CCK-8 assay was used to detect the proliferation of DLBCL cell lines(SU-DHL-2 and SU-DHL-4 cell),and colony formation assay was used to detect the clone ability of DLBCL cell lines,and xenograft mouse model were used to investigate the effects of human bone marrow-derived mesenchymal stem cells(h BMSCs)on DLBCL growth.Immunohistochemistry,q RT-PCR,and ELISA were used to study the expressions of IL-6 and IL-17 A in DLBCL cell lines cocultures or tumor tissues.Flow cytometry was used to analyze Th17 cells and Treg cells expressions.Western blot analysis,microarray analysis,and bioinformatics analysis were used to analyze the pathways of IL-6 or IL-17 A mediated DLBCL growth.All statistical analyses were performed using SPSS 17.0.The results show that:(1)、h BMSCs promote the growth of DLBCL cells in vitro,and peripheral blood mononuclear cells(PBMCs)enhance these effects;h BMSCs significantly promote the proliferation and clonality of SU-DHL-2 and SU-DHL-4 cells;PBMCs significantly enhance the effects of h BMSCs on promoting SU-DHL-2 and SU-DHL-4 cells proliferation and clonality.(2)、h BMSCs secrete IL-6 into the co-culture supernatants,and the IL-6 level is higher in DLBCL tumor tissue than in benign tissue.(3)、h BMSCs promote DLBCL growth by secreting IL-6 in vivo.The tumor volumes are significantly larger in the MSC group and IL-6 group than in the Control group;IL-6 m RNA and protein levels are markedly higher in the MSC group and IL-6 group than in the Control group.(4)、 h BMSCs induce PBMCs differentiation into Th17 and Treg cells,thereby increasing IL-17 A and TGF-β levels in the co-culture supernatants.HBMSCs significantly increase the frequencies of Th17 and Treg cells in PBMCs,upregulating the relative expressions of RORγt,Foxp3,IL-17 A and TGF-β m RNA in PBMCs,increasing the levels of IL-17 A and TGF-β proteins in the supernatants.(5)、h BMSCs or IL-6 promote the growth of DLBCL cells by protecting them from spontaneous or drug-induced apoptosis,and IL-17 A reinforces these effects.HBMSCs and exogenous IL-6 and IL-17 A promote SU-DHL-4 cell proliferation,and a IL-6 or a IL-17 A abolishes these effects;IL-17 A increases the promoting effects of IL-6,whereas a IL-6 combined with a IL-17 A abolish these effects.(6)、 IL-6 or h BMSCs promote DLBCL cell growth by upregulating p-JAK and p-STAT3 via the JAK2/STAT3 signaling pathway.IL-6 significantly promote p-JAK2 and p-STAT3 in both SU-DHL-2 and SU-DHL-4 cells,whereas a IL-6 abrogates these effects;Similarly,h BMSCs upregulate p-JAK2 and p-STAT3 in SU-DHL-4 cells,whereas a IL-6 abolish these effects(7)、 IL-17 A promotes DLBCL cell growth by upregulating cyclin D2 via the PI3K/Akt signaling pathway.In conclusions,h BMSCs have a “dual effect” on promoting DLBCL growth by directly secreting IL-6 and indirectly upregulating IL-17 A levels.