Role Of Stat3Activation In Cell-cell Interaction Between B-cell Lymphoma And Macrophage:the In Vitro Study

BackgroundMacrophages that have infiltrated into tumor tissues are referred to as tumor-associated macrophages (TAMs) and are now of much interest because of their functions in the tumor microenvironment. Recent studies revealed the possible involvement of TAMs, especially anti-inflammatory M2TAMs, in several malignant tumors, including hematological malignancies. In Hodgkin lymphoma, diffuse large B-cell lymphoma, and angioimmunoblastic T-cell lymphoma, the number or ratio of CD163-positive M2TAMs is positively associated with worse clinical prognosis.Stat3is one of the important signal molecules related to angiogenesis, immunosuppression, cell survival, and cell proliferation, higher Stat3activation in tumor cells is significantly associated with lower survival rates of patients with several malignant tumor types. Our previous studies revealed that Stat3activation in primary central nervous system lymphoma, renal cell carcinoma, ovarian cancer, and glioma cells was strongly induced by co-culture with M2macrophages, however, detailed mechanisms regarding cell-cell interactions contributing to this Stat3activation remain unclear.BrdU(Bromodeoxyuridine) is a thymidine analog that incorporates DNA of dividing cells during the S-phase of the cell cycle. There is no endogenous BrdU exist in tissue cells, so BrdU can be used for monitoring cell proliferation indirectly. Objective:This study is designed to investigate the significance of Stat3activation for cell-cell interaction between B-cell lymphoma and macrophage using in vitro co-culture experiments.Methods:1. Co-culture experiments in vitro and impact on lymphoma cell proliferation Peripheral blood mononuclear cells were isolated from2healthy volunteer donors, and CD14-positive monocytes were isolated using CD14-microbeads. Monocytes cultured with GM-CSF for5days to induce differentiation to immature macrophages, then immature macrophages were stimulated with IFN-y for24h to induce mature M1macrophages. Monocytes cultured with GM-CSF and M-CSF for5days to induce differentiation to immature M2macrophages, then immature M2macrophages were stimulated with IL-10for24h to induce mature M2macrophages. Lymphoma cell lines and macrophages were co-cultured in DMEM supplemented with2%FBS in24-well plates. In in-direct co-culture experiments, transwell chamber dishes were used to separate the macrophages and lymphoma cells. After co-culture for3days, the BrdU incorporation assay and double-immunostaining was performed to evaluate tumor cell proliferation. Briefly, After culture with BrdU for3hours, cells were attached to slide glasses by Cytospin and fixed by acetone. CD204was stained as a pan-macrophage marker and visualized using a DAB substrate system. After washed in glycine buffer (pH2.2), cells were stained by anti-BrdU antibody and visualized using the HistoGreen solution.2. The effect of Stat3activation on lymphoma cell proliferation Lymphoma cells were transfected with siRNA against human Stat3using Lipofectamine RNAi MAX. Western blot was performed to evaluate the levels of Stat3in SLVL cells. After co-culture for3days, the BrdU incorporation assay and double-immunostaining was performed to evaluate tumor cell proliferation.3. The effect of C5a on the interaction between SLVL cells and macrophages Monoculture and co-culture conditioned medium was prepared and soluble factors from each were detected by cytokine array. Expression of C5a receptors was examined by RT-PCR. Western blot was performed to evaluate the levels of pStat3expression in SLVL cells. SLVL cells were cultured with human C5a for24h, then BrdU incorporation assay and immunostaining was performed to investigate its effects on cell proliferation. SLVL cells were cultured with M2macrophages and C5a receptor antagonist for24h, the BrdU incorporation assay and immunostaining was performed to evaluate tumor cell proliferation.Results:1. Significant proliferation of lymphoma cells was induced by direct co-culture with M2macrophagesFollowing co-culture of SLVL cells with immature, Ml, or M2macrophages for3days, the BrdU incorporation assay and double-immunostaining was performed to evaluate lymphoma cell proliferation. BrdU incorporation into SLVL cells was significantly induced by direct co-culture with M2macrophages. The size of SLVL cell nuclei was also enlarged by co-culture with M2macrophages, the longest length of nuclei in SLVL cells with or without co-culture was18.8±2.7μm and15.7μm respectively (P=0.003, n=20). When lymphoma cell proliferation in co-culture with the three macrophage types was compared, it was found to be elevated by all three, and to a notably greater extent by M2macrophages. Similar results were obtained using other B-cell lymphoma cell lines, such as Daudi cells and Raji cells. In addition, higher BrdU incorporation in SLVL cells was induced by direct co-culture with M2macrophages, as compared to indirect co-culture using the transwell culture system.2. Lymphoma cell proliferation induced by co-culture with macrophages was dependent on Stat3activationTo test the significance of Stat3activation in cell-cell interactions between lymphoma cells and macrophages, Stat3protein in SLVL cells was down-regulated by siRNA before macrophage co-culture experiments. Stat3down-regulation in co-cultured SLVL cells significantly suppressed BrdU incorporation.3. C5a was involved in cell-cell interactions between M2macrophages and lymphoma cellsUse of a cytokine array kit identified elevated C5a in M2macrophage supernatants and as this is considered to activate Stat3. SLVL cells expressed C5a receptors and significant Stat3activation was induced by co-culture with C5a recombinant protein. Cell proliferation and BrdU incorporation were also significantly induced by co-culture with C5a in SLVL cells. Lymphoma cell activation induced by C5a was suppressed by Stat3down-regulation. C5a receptor antagonist slightly lowered the BrdU incorporation in SLVL cells co-culture with M2macrophages, however, the data was statistically not significant.Conclusions:1. Stat3activation in lymphoma cells due to cell-cell interaction with M2macrophages is important for the development of the tumor microenvironment, Stat3inhibitors or compounds which deactivate M2macrophage could prove to be effective adjunctive therapeutic agents for malignant lymphoma patients.2. Stat3activation by macrophage-derived C5a might be one of mechanisms of cell-cell interaction, Use of C5a blockade as a novel therapeutic approach to patients with malignant lymphoma might warrant more investigation.