RESEARCH PAPER OncoImmunology 1:3, 279–286; May/June 2012; G 2012 Landes Bioscience Susceptibility of rhabdomyosarcoma cells to macrophage-mediated cytotoxicity Delia Herrmann,† Guido Seitz,† Jörg Fuchs and Sorin Armeanu-Ebinger* Department of Pediatric Surgery and Urology; University Children’s Hospital Tübingen; Tübingen, Germany †These authors contributed equally to this work. Keywords: rhabdomyosarcoma, macrophages, phagocytosis, immunotherapy, CD47, calreticulin Abbreviations: RMS, rhabdomyosarcoma; RME, embryonal RMS; RMA, alveolar RMS; LRP, lipoprotein-related protein; SIRPa, signal regulatory protein alpha; mAb, monoclonal antibody; CWS, Cooperative Soft Tissue Sarcoma Study; GPOH, Group of the Society of Pediatric Oncology and Hematology; PBMCs, peripheral blood mononuclear cells; ALL, acute myeloid leukaemia 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 immuno- therapy 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 SIRPa, 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. Introduction Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. It represents 3–4% of all pediatric cancers1 and 7–8% of all solid malignant tumors in children.2 There exist two main histopathological subtypes of this malignancy, embryonal RMS (RME) and alveolar RMS (RMA) with completely different tumor biology.3 RMS tumors are currently treated by multimodal therapies, including surgery, radiotherapy and systemic chemo- therapy.4 Major treatment problems are metastatic invasion, local tumor recurrence, and multidrug resistance.5 Therefore, it is not only necessary to develop novel strategies to destroy cancer cells efficiently, but also to attempt a stimulation of the immune system in order to control residual tumor cells. Macrophages play an important role in the defense against tumors.6,7 They have the capacity to recognize and destroy tumor cells through several different mechanisms, including secretion of tumor necrosis factor-a,8 nitric oxide,9 interleukin-1β,10 and reac- tive oxygen intermediates.11 Furthermore, macrophages are involved in the antibody-dependent cellular cytotoxicity in thera- peutic approaches with recombinant antibodies.12 Macrophage cytotoxicity relies on the balance between activating stimuli and suppressive signals. One well known signal for engulfment by phagocyting cells is a change in the composition of phospholipids on the target cells.13 Furthermore, the interaction of calreticulin with the low-density lipoprotein-related protein (LRP) plays also an important role in apoptotic cell removal, resulting in an activation of the macrophages.14 Calreticulin is an intracellular calcium-binding protein and anthracyclines are able to elicit it’s translocation onto the cell surface.15 On the other hand, the interaction between CD47 and the signal regulatory protein a (SIRPa) is a key function to protect viable cells from phagocytosis.16 CD47, a widely distributed inhibitory receptor on macrophages that can *Correspondence to: Sorin Armeanu-Ebinger; Email: sorin.armeanu-ebinger@med.uni-tuebingen.de Submitted: 07/25/11; Revised: 10/27/11; Accepted: 10/31/11 http://dx.doi.org/10.4161/onci.18612 www.landesbioscience.com OncoImmunology 279