Fig. 2

DSP107 enhances macrophage-mediated phagocytosis of B cell lymphoma cell lines as a single agent and in combination with RTX. A) Representative pictures of M1 macrophage-mediated phagocytosis of SUDHL10 cells untreated, treated with DSP107, RTX or RTX and DSP107. White arrows point macrophages with engulfed cancer cells, cancer cells (green) were labelled with CFSE and macrophages (red) were labelled with CD11b-AF594, scale bar 50 μm. B) Representative pictures of phagocytosis of SUDHL10 cells by M2c macrophages as in A. C) Phagocytosis of seven different B cell lines by allogeneic human M1 macrophages alone (red diamonds) or upon treatment with 10 nM DSP107 (blue diamonds). Each line represents the average phagocytic uptake calculated from 3 pictures of a single experiment, parametric paired t-test was used with respect to control sample versus each dose of DSP107, D) M1 macrophage-mediated phagocytosis of B cell lymphoma cells treated with 10 nM DSP107 and 1 μg/ml RTX (red diamonds) compared to RTX (blue diamonds) as in C, E) M2c-macrophage phagocytosis of SUDHL2 and SUDHL10 cells. Phagocytic uptake in the untreated sample (red diamonds) or upon treatment with 10 nM DSP107 (blue diamonds), parametric paired t-test was used with respect to control sample versus DSP107 treatment, F) Phagocytosis by M2c macrophages with 1 μg/ml RTX alone (red diamonds) and in combination treatment of 10 nM DSP107 and RTX (blue diamonds), as in E, G) Phagocytic index calculated for phagocytosis by M2 macrophages, parametric paired t-test was used with respect to medium control versus treatment with 10 nM DSP107, RTX or combination treatment, H) Comparison of DSP107, CD47 mAb and SIRPα:Fc, Phagocytosis assay by LPS/IFNγ stimulated macrophages on SUDHL10 (left) or SUDHL2 cells (right). DSP107, CD47 mAb or SIRPα:Fc for monotherapy (red bars) or combination treatment with RTX (blue bars) are used at an equal concentration of 10 nM. Phagocytosis results were normalized to the highest phagocytic uptake percentage of the same donor for a proper comparison of phagocytosis between the drugs. I) Comparison of DSP107, CD47 mAb and SIRPα:Fc for induction of phagocytosis by M2c macrophages. Results were normalized as in H. J) SUDHL10 cell removal by M1 macrophages after 72 h of co-culture, parametric paired t-test was used with respect to medium control versus treatment with DSP107, RTX or combination treatment. K) Representative pictures of phagocytosis with DSP107, RTX and combination of DSP107 and RTX of primary patient-derived MCL cells with autologous macrophages, cancer cells (green) were labelled with CFSE and macrophages (red) were labelled with CD11b-AF594, scale bar 50 μm. L) Analysis of primary malignant MCL cells by autologous macrophages (from one donor). Three pictures per condition were counted and phagocytosis was calculated as the number of cancer cells per 100 macrophages. Across the figure, t-test values: * = p < 0.05, ** = p < 0.01, *** = p < 0.001, all error bars in the figure represent SD