Susceptibility of rhabdomyosarcoma cells to macrophage-mediated cytotoxicity

The prognosis of advanced stage rhabdomyosarcoma (RMS) is still sobering. In recent years, outcome has not been further improved by conventional therapy. Therefore, novel treatment options such as macrophage-directed immunotherapy have to be investigated. The aim of this study was to analyze the phagocytosis of RMS cells by macrophages and to modulate the susceptibility using monoclonal antibodies and cytotoxic drugs.   Expression of the macrophage activating ligand calreticulin and CD47, the counterpart of the inhibitory receptor SIRPα, was analyzed with Affymetrix mRNA expression arrays and immunohistochemistry on 11 primary RMS samples. Results were verified in two RMS cell lines using flow cytometry and immunocytochemistry. Macrophage cytotoxic activity was quantified by a MTT colorimetric assay in co-culture experiments of RMS cells with monocyte-derived, GM-CSF stimulated macrophages. Gene expression analysis and immunohistochemistry revealed a high expression of CD47 and calreticulin in alveolar and embryonal RMS tissue specimens. Extracellular expression of CD47 on RMS cell lines was confirmed by flow cytometry, whereas calreticulin was exclusively detected in the endoplasmatic reticulum. After co-culturing of RMS cells with macrophages, viability dropped to 50-60%. Macrophage-mediated cytotoxicity was not influenced by a blocking antibody against CD47. However, susceptibility was significantly enhanced after pre-treatment of RMS cells with the anthracycline drug doxorubicin. Furthermore, translocation of calreticulin onto the cell surface was detected by flow cytometry. The immunologic effect of doxorubicin may improve the efficacy of adoptive cellular immunotherapy and chemotherapy of childhood RMS.