Figure 3. Susceptibility of rhabdomyosarcoma cell lines to cytotoxicity by macrophages. (A) Rh30 and RD cells were co-cultured with macrophages for 96 h at an E:T ratio of 2.5:1. Indicated wells were pre-incubated with 10 mg/ml blocking antibody to CD47 for 30 min. Pictures show tumor cell free areas in cultures with macrophages. (B) Cell lysis was quantified by measuring the cell free area in the tumor cell layer by an image analyzer system. (Fig. 2A). Therefore, we intended to induce the transport of calreticulin to the RMS cell surface by using doxorubicin. The IC50 for both RMS cell lines was determined to be 0.1 mg/ml (Fig. 5A). A lower concentration (0.01 mg/ml) was also tested in flow cytometry and co-culture experiments, which on the one hand had an effect on RMS cell viability (80% viability in an 96 h assay), but on the other hand was low enough, to avoid massive drug dependent cell death and to assess phagocytosis effects. Cell surface presentation of calreticulin, depending on the used doxorubicin concentration, was detected by flow cytometry (Fig. 5B). A higher expression was measured in RD cells com- pared with Rh30. Thereupon, after treatment with the higher Figure 4. Effect of CD47 antibody on the cytotoxicity of macrophages against rhabdomyosarcoma cells. Co-cultures of Rh30 and RD cells with macrophages (E:T 2:1) were performed after incubating the tumor cells for 30 min with an anti-CD47 antibody (10 mg/ml). Cell viability was then determined by MTT assay 24 h later. Data represent means ± SD of relative cell viability from triplicates. Experiments without antibody were set at 100% viability. concentration of doxorubicin (0.1 mg/ml) the co-culture experi- ments with macrophages revealed an efficient phagocytosis of RD cells up to 90% (Fig. 6B). When additionally treated with the anti-CD47 mAb (10 mg/ml), this effect could not be enhanced (viability RD 15%). In Rh30 cells, a lysis of 65% was achieved after treatment with the IC50 concentration of doxorubicin (0.1 mg/ml). After co-culture with macrophages, the Rh30 cell viability dropped without reference to the addition of the CD47 antibody to 35% and 40%, respectively (Fig. 6A). Viability of both RMS cell lines was half-reduced after treatment with the lower dose of doxorubicin (0.01 mg/ml) and co-culture with macrophages (viability of Rh30 55% and of RD 50%). These results were no further influenced by the addition of the anti- CD47 mAb (viability of Rh30 65% and of RD 55%). Four different treatment options served as controls: (A) Co-culturing with macrophages reduced the viability of Rh30 cells to 60% and RD cells to 55%. (B) After antibody treatment and co-culture with macrophages 65% of Rh30 and 60% of RD cells remained viable. (C) Exclusive treatment with the previously evaluated IC50 of doxorubicin (0.1 mg/ml) resulted in a drop of cell viability to 55% (Fig. 5A). (D) Treatment with the lower tested drug concentration (0.01 mg/ml) had no effect on the morphology and viability of Rh30 cells (95%). However, survival of RD cells dropped to 85% by doxorubicin alone. In summary, phagocytosis of RMS cells was significantly enhanced by retrieving calreticulin using the IC50 concentration of doxorubicin (two-way ANOVA, p , 0.001). The cause-effect relationship between doxorubicin treatment, CRT exposure and enhanced susceptibility to cytotoxic effects mediated by macrophages was demonstrated using a blocking peptide to calreticulin. This peptide abrogated phagocytosis of lymphoblast cell lines by macrophages.19 In co-cultures of RMS cells and macrophages, the enhanced phagocytosis achieved by 282 OncoImmunology Volume 1 Issue 3