Figure 7. RMS cells Rh30 (A) and RD (B) were incubated with human macrophages in culture media alone or with calreticulin-blocking peptide (4 mg/ml) for 24 h, after which cell viability was analyzed by MTT assay. Pre-treatment of RMS cells with 0.01 and 0.1 ng/ml doxorubicin resulted in increased phagocytosis compared with control cells. Blockade of calreticu- lin on RMS cells partially abrogated phagocytosis. Cellular stress, including apoptosis, induces the expression of many stress proteins, among them calreticulin.32 This leads to increased amounts of calreticulin on the cell surface.33 However, the exact transport mechanism of calreticulin remains unclear. Anthracyclines are able to induce the translocation of calreticulin from the endosplasmatic reticulum onto the cell surface. The presented results show a dose-dependent calreticulin presentation after doxorubicin treatment. For colon carcinoma cells, a con- centration of 25 mM doxorubicin, converted 14.5 mg/ml, was described in the same incubation period.15 For RMS cells, a measureable extracellular calreticulin expression and a significantly increased phagocytosis was demonstrated after incubation with 0.1 mg/ml doxorubicin. Furthermore, a different response of RME compared with RMA cells was observed. Under equal doxorubicin concentrations, the RME cells showed a higher extracellular calreticulin presentation and accordingly a more efficient phagocytosis compared with RMA. The role of calreticulin in the doxorubicin enhanced phagocytosis was demonstrated using a blocking peptide, which reversed the activity of macrophages, although CD47 was expressed at high density. A complete blockage can be expected in the absence of CD47 as demonstrated for mutant lymphomas.19 We could clearly demonstrate that macrophages are able to phagocyte allogeneic RMS cells under conditions of cell-to-cell contact. After GM-CSF activation of macrophages and co- culturing with RMS cells, viability dropped to 50–60% without any additional treatment. After activation of calreticulin, the efficient phagocytosis of RME cells was enhanced to 90%. This promising effect in vitro has to be verified in vivo. Doxorubicin may improve the efficacy of adoptive cellular immunotherapy and chemotherapy protocols. So far, doxorubicin is used in combination with ifosfamide and vincristine in the therapy protocol for the high risk soft tissue sarcoma group (CWS-2002 P). Material and Methods Gene expression analysis. The gene expression data used in this study were part of a microarray analysis described previously.34 All patients were treated within the trials of the Cooperative Soft Tissue Sarcoma Study (CWS) Group of the Society of Pediatric Oncology and Hematology (GPOH). Mean age of patients was 6.4 y (range 1 to 15 y, 95% CI of mean 4.2 to 8.8). Histological analysis revealed 5 RMA and 6 RME. All cases received central pathological review and patients were treated according to the uniform protocols. Only samples with tumor cell content of at least 80% were included for analysis. Metastases were present at the time of surgery in all patients suffering from RMA and in none in RME patients. Analysis of tissue samples was approved by the local ethical committee (CWS 2002-P: 418/ 2004V). As controls, data of skeletal muscle biopsies from 8 patients acquired on the same array platform were used.35 The expression of CD47 (probesets 211075_s_at and 213857_s_at on the Affymetrix HG-U133 Plus 2.0 microarray) and calreticulin (212953_x_at and 214315_x_at) was compared with the housekeeping gene GAPDH (212581_x_at and 213453_x_at) for each sample as fraction of the signal log ratio. Comparison of relative expression in RMA, RME, and control tissue was performed by Student’s t-test. Cell lines and culture conditions. The RMA cell line Rh30 (DSMZ, ACC-489) as well as the RME cell line RD (ATCC, CCL-136) were cultured in DMEM medium (Biochrom) supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, and 1% L-glutamine (Biochrom) in a humidified atmosphere containing 5% CO2 at 37°C. All cells were tested to be mycoplasma negative. Cell identity was proven by SLR analysis of the DNA profile using PowerPlex 16 (Promega). Flow cytometry analysis. Trypsinized cells were incubated for 30 min in FACS-buffer (PBS with 2% FBS, 2 mM EDTA, 0.005% NaN3; Sigma-Aldrich) containing anti-CD47 mAb (B6H12; BD Biosciences). Intracellular flow cytometry of calreticulin was performed with cells fixed in 3.7% formaldehyde solution (Merck), and permeabilized by 0.2% Tween 20 in PBS (Sigma-Aldrich). Primary antibody (anti-calreticulin, FMC 75; Enzo Life Sciences) was added for 30 min diluted in FACS-buffer. This was followed by a washing step with FACS-buffer and cells were labeled with FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch). Data was acquired with FACSCalibur (Becton Dickinson) and analyzed by FCS Express (De Novo Software). 284 OncoImmunology Volume 1 Issue 3