Figure 2. CD47 and calreticulin expression in rhabdomyosar- coma cell lines. (A) High expression of CD47 (black lines) but no extracellular expression of calreticulin was detected on both Rh30 and RD cells by flow cytometry. The control staining without primary antibody is plotted on each panel as gray histogram. Dead cells were gated out by their position in forward and site scatter. (B) Expression of calreticulin shown by intracellular flow cytometry analysis and immunocytochemistry in the endoplasmatic reticulum of RMS cells. Secondary FITC- labeled antibody and Hoechst 33342 for nuclear staining were used. Controls were incubated without primary antibody. with the array analysis obtained previously (Fig. 2B). The low signal in flow cytometry might be a result of an ineffective permeabilization of the endoplasmatic reti- culum. The “don’t eat me” signal CD47 was strongly expressed in RMS tissue samples of both histological subtypes and on the cell surface of RMS cells. Therefore, we next modulated the macrophage cytotoxic activity in vitro using inhibitory antibodies. Cytotoxic activity of macrophages modulated by anti- CD47. The ability of human macrophages to phagocyte allogeneic RMS cells in co-cultures at an E:T ratio of 2.5:1 was monitored for up to 96 h. After 24 h, a few poly- nucleated cells were observed in the confluent grown tumor cell culture (pictures not shown). Distinct holes in the cell layer caused by phagocytosis were also observed and photos were taken after 96 h (Fig. 3A). No cell free areas were detected in the control samples. The most and largest holes in the tumor cell monolayer were present in the cultures treated with the anti-CD47 mAb (Fig. 3B). When treated without antibody, Rh30 cells were more efficiently phagocyted than RD cells with a cell free area of 4600 mm2 compared with 2500 mm2. Blocking CD47 with an anti-CD47 mAb increased the phagocytosis of both RD cells and Rh30 cell cultures (17900 mm2 and 12700 mm2, respectively). In relation to the whole area of 151810 mm2, a 5% increase in phagocytosis of Rh30 was detected and the increment for RD was 10%. By measuring cytotoxicity in co-cultures, we observed a specific phagocytosis of both RMS cell lines again (Fig. 4). After co-culturing with macrophages at an E:T ratio of 2:1 for 24 h, 70% of Rh30 and 45% of RD cells remained viable. When additionally treated with the anti-CD47 mAb, cell viability did not change significantly (Rh30: 80%; RD: 60%). As a control, exclusive antibody treat- ment had no effect on Rh30 cell viability. In contrast, cell viability of RD cells dropped to 85%. Taken together, the phagocytosis of RMS cells could not be increased by blocking of the inhibitory ligand CD47 using mAbs in this short period of time. Therefore, we next investigated other mechanisms to shift the balance between activating and inhibiting signals in favor of phagocytosis. Retrieving calreticulin to enhance phagocytosis. Macrophages can be activated by the interaction of calreticulin and LRP. Preliminary data has shown that RMS cells do not express calreticulin on their surface www.landesbioscience.com OncoImmunology 281