trigger a signal transduction cascade resulting in inhibition of phagocytosis, serves as the ligand for SIRPa.17 Overexpression of inhibitory molecules like CD47 is a common mechanism of tumor cells to escape phagocytosis. A high expression of calreticulin or blocking of CD47 by mono- clonal antibodies may shift the balance between activating and inhibiting signals in favor of phagocytosis.18 The aim of this study was to describe the distribution of CD47 and calreticulin in human RMS and to analyze the cytotoxic activity of GM-CSF activated macrophages in combination with a CD47-blocking monoclonal antibody (mAb). Furthermore, we evaluated the effect of doxorubicin on the interaction between RMS cells and macrophages. both in Rh30 and RD cells. In these cell lines calreticulin seems to be compartmentalized to the endoplasmatic reticulum, as revealed by immunocytochemistry. In contrast to flow cytometry analysis, the expression of calreticulin was high, which is in turn consistent Results Expression of CD47 and calreticulin on RMS tissues and cells. In a first attempt to evaluate the role of CD47 and calreticulin in RMS, expression of these genes was examined in RMS tissue samples by micro- array analysis. CD47 inhibits phagocytosis and its gene was expressed in the 11 RMS tissue samples analyzed (Fig. 1A). With a fold change of 1.2, a significant higher expression was observed in RMA compared with RME tissues (p = 0.02; Student’s t-test) and skeletal muscle biopsy samples (p = 0.002; Student’s t-test). The macro- phage activating gene calreticulin showed a very high expression in the array analysis of both RMA and RME, independent of the histological subtype (median, 0.7). Compared with muscle control tissues, a significant higher expression was found in RMS (p , 0.0001; Student’s t-test). Accordingly, we detected high expression levels of the proteins CD47 and calreticulin on RMS tissue slices by immunofluores- cence analysis (Fig. 1B). In immunofluor- escence staining, fewer differences between RMA and RME samples were observed with regard to the expression of CD47. When examining CD47 on RMS cell lines by flow cytometry, the protein was observed on the cell surface of Rh30 and RD. Both showed a high expression of CD47 as revealed by staining with an anti-human CD47 mAb (Fig. 2A). Calreticulin could not be detected on the cell membrane. However, intracellular flow cytometry ana- lysis revealed a low expression of calreticulin Figure 1. Expression of CD47 and calreticulin in rhabdomyosarcoma and muscle tissue samples. (A) Gene expression is shown for each tissue sample enrolled in the microarray expression analysis relative to GAPDH. (***Student’s t-test p , 0.0001). (B) Immunofluorescence analysis of representative RMS tissue sections using CD47 and calreticulin mAbs followed by the red fluorescent secondary antibody and Hoechst 33342 for nuclear blue staining. As control served RMS tissues incubated without primary mAb. 280 OncoImmunology Volume 1 Issue 3