Figure 5. Viability and calreticulin expression of rhabdomyosarcoma cells pre-treated with doxorubicin. (A) Rh30 and RD cells were incubated for 96 h with increasing doses of doxorubicin. Data are means ± SD of triplicate viability measurements using MTT assay. Experiments without doxorubicin were set at 100% viability. (B) Extracellular calreticulin expression on Rh30 and RD cells was measured by flow cytometry after incubation for 24 h with 0.01 mg/ml (dark gray line) or 0.1 mg/ml doxorubicin (dashed line). No calreticulin on the cell surface was detected without doxorubicin (black line). The control staining without primary antibody is plotted on each panel as gray histogram. doxorubicin could be partially blocked by the blocking peptide (Fig. 7). Therefore, the effect of doxorubicin on the phagocytosis of RMS seems to be mediated to some extent by calreticulin exposure. Discussion Effective antibody targeting of CD47 has been demonstrated for several human cancers including acute myeloid leukemia (ALL),20 bladder cancer,21 and non-Hodgkin lymphomas.22 The thera- peutic effect of blocking CD47 with antibodies is mediated primarily through macrophage phagocytosis23 and was observed for ALL in vitro and in tumor engraftment in vivo.24 Targeting of tumor cells with antibodies against CD47 has previously Figure 6. Phagocytosis of rhabdomyosarcoma cells after pre-treatment with doxorubicin. As indicated, Rh30 (A) and RD cells (B) were incubated with 0.01 mg/ml or 0.1 mg/ml doxorubicin (Doxo) for 24 h and CD47 antibody for 30 min. Macrophages were added at an E:T ratio of 2:1 and cell viability was assessed using MTT assay 24 h later. Data are means ± SD of relative viability from three experiments. Experiments without antibody and doxorubicin were set at 100% viability. been reported to directly induce apoptosis.25-28 Now the idea is supported, that targeting CD47 may lead to a disruption of the CD47-SIRPa interaction, thereby preventing a phagocytic inhibitory signal.20 CD47 was found to be highly expressed not only on tumor cells but also on normal tissues.29 However, potential toxic effects with a CD47 antibody therapy could be excluded.24 Selective targeting of tumor cells is probably due to a high cell surface expression of calreticulin. Indeed, calreticulin is expressed in all cells, but only a low presentation was profiled on the cell surface of healthy tissues. By contrast, for several human cancers a high extracellular expression was demonstrated.18 This data led us to speculate, that targeting CD47 will be a promising tool against a wide range of human tumors, including RMS. Our data revealed a high expression of CD47 on RMS tissues and cells, which may play a role in the immune evasion of RMS. Furthermore, a significant association between the higher expression of CD47 and the more aggressive RMA subtype was found in gene expression analysis. Interaction of RMS cells with GM-CSF activated macrophages required more than four days of incubation to reveal a notable phagocytosis. Enhanced cytotoxi- city was only obtainable by increasing the proportion of macrophages (data not shown). Usually, such assays with human acute myeloid leukemia stem cells revealed a fast engulfment of tumor cells within two hours of co-culture with stimulated macrophages.20 We therefore conclude that RMS cells are less susceptible to phagocytosis by macrophages. To enhance phagocytosis, we tried to disrupt the CD47-SIRPa interaction with a mAb demonstrably able to specifically block CD47.30 In our short-term assay of 24 h, no enhancement of phagocytosis was observed. This might be due to low activating signals for the phagocytes. Besides the exposure of GM-CSF and phosphatidylserine, which may interact with several phagocyte receptors, calreticulin functions on tumor cells as a ligand for the activating phagocyte receptor LRP/CD91.13,16,31 Furthermore, annexin-1 is under active investigation.26 www.landesbioscience.com OncoImmunology 283