with 2.5106 Daudi-luc cells (10 mice per group). myeloma and potentially other hematological tumors. Co-cultures of mouse m and Daudi cells in the presence of 6.7?nM DARA or F(ab)2 fragments thereof, E:T ratio of 1 1:1 (A, B) or 3:1 (C). (A) Double positive (DP) m were characterized as F4/80+calcein+CD19C and the percentage DP macrophages was calculated as described in Materials and Methods. (B) The percentage eliminated target cells was calculated from the number of remaining F4/80- cells as described in Materials & Methods. Each bar shows mean SEM, results from a representative experiment are shown (= 3). (C) Time-lapse imaging microscopy, bright field images of a mouse Amentoflavone m (arrow) that sequentially engulfed 5 individual Daudi cells (numbers) over a period of 800?s. The images are representative for observations in multiple impartial phagocytosis experiments (= 3) (****< 0.0001 Bonferroni's multiple comparison test). Threshold CD38 expression level for phagocytosis induction To explore the effect of CD38 expression levels on phagocytosis induction by DARA, we set up a flow cytometric assay with mouse m and leukemic target cells with variable levels of CD38 expression (Table 1). In a pilot experiment, we found that the MM cell lines UM9 and L363, with relatively low CD38 Amentoflavone expression (50,000100,000 and 100,000150,000 molecules/cell, respectively) were not susceptible to DARA-dependent phagocytosis. However, uptake into m and substantial elimination of target cells was consistently observed for CD38-transduced UM9-CD38 and L363-CD38 variants with high levels of CD38 expression (350,000600,000 and 450,000800,000 molecules/cell, respectively). These results suggest that DARA-dependent phagocytosis is related to CD38 expression levels. However, it is difficult to define a threshold level of CD38 expression that allows efficient DARA-dependent phagocytosis, as phagocytosis was also consistently observed in Wien-133 cells that express relatively low CD38 levels (Table 1). In addition, large differences, especially in the percentage of eliminated target cells, were observed between cell lines with comparable CD38 expression levels (e.g., Daudi and Raji, Table 1). Thus, additional factors are likely to determine the efficacy of DARA-dependent phagocytosis. Table 1. DARA-dependent m-mediated phagocytosis of human multiple myeloma and lymphoma cell lines Phagocytosis of Daudi cells by mouse m in the presence of 6.7?nM mAb, E:T ratio of 1 1:1. (A) Double-positive (DP) m were characterized as F4/80+calcein+CD19C and the percentage DP macrophages was calculated as described in Materials and Methods. (B) Percentage eliminated target cells was calculated using the number of remaining F4/80- cells as described in Materials & Methods. Each bar shows mean SEM, results from a representative experiment (= 3) (**< 0.01, ****< 0.0001 Bonferroni's multiple comparison test). In a subcutaneous Daudi-luc tumor xenograft model, DARA-K322A provided significantly stronger inhibition of tumor growth than DARA-IgG2-K322A (Fig. 3A), indicating an important contribution of phagocytosis to the in vivo Rabbit Polyclonal to CEP57 efficacy of DARA. Furthermore, in the intravenous leukemic Daudi-luc xenograft model, in which mice were treated at the time of tumor challenge, DARA-K322A also exhibited a Amentoflavone significantly stronger tumor growth inhibition compared to DARA-IgG2-K322A (Fig. 3B). Upon therapeutic treatment in this leukemic Daudi-luc xenograft model, DARA-K322A also showed better potency than DARA-IgG2-K322A (treatment with 0.5?mg/kg at day 14), as shown in Fig. S2. These data demonstrate that phagocytosis contributes to the in vivo mechanism of action of DARA. Open in a separate window Physique 3. (A) Kaplan-Meier plot showing time to tumor progression (cutoff set at a tumor volume > 800?mm3) for mice that had been inoculated s.c. with 20 106 Daudi-luc cells (8 mice per group). Subsequently, mice were treated i.p. with 250?g mAb per mouse (12.5?mg/kg) at day 0. Tumor progression was significantly reduced in DARA-K322A-treated mice compared to DARA-IgG2-K322A treatment (< 0.004 Mantle-Cox log-rank test at time to progression). (B) Kaplan-Meier plot showing time to tumor progression (cutoff set at bioluminescence > 50 000 cpm) for mice that had been inoculated i.v. with 2.5106 Daudi-luc cells (10 mice per group). Subsequently, mice were treated i.p. with 10?g mAb per mouse (0.5?mg/kg) at day 0. Tumor progression was significantly reduced in DARA-K322A-treated vs. DARA-IgG2-K322A-treated mice (< 0.001 Mantle-Cox log-rank test at time to progression). Patient MM tumor cells are efficiently phagocytosed by.