Abstract Background Macrophages play important roles in phagocytosing tumor cells. However, tumors escape macrophage phagocytosis in part through the expression of anti-phagocytic signals, most commonly CD47. In Ewing sarcoma (ES), we found that tumor cells utilize dual mechanisms to evade macrophage clearance by simultaneously over-expressing CD47 and down-regulating cell surface calreticulin (csCRT), the pro-phagocytic signal. Here, we investigate the combination of a CD47 blockade (magrolimab, MAG) to inhibit the anti-phagocytic signal and a chemotherapy regimen (doxorubicin, DOX) to enhance the pro-phagocytic signal to induce macrophage phagocytosis of ES cells in vitro and inhibit tumor growth and metastasis in vivo. Methods Macrophages were derived from human peripheral blood monocytes by granulocyte–macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Flow cytometry- and microscopy-based in-vitro phagocytosis assays were performed to evaluate macrophage phagocytosis of ES cells. Annexin-V assay was performed to evaluate apoptosis. CD47 was knocked out by CRISPR/Cas9 approach. ES cell-based and patient-derived-xenograft (PDX)-based mouse models were utilized to assess the effects of MAG and/or DOX on ES tumor development and animal survival. RNA-Seq combined with CIBERSORTx analysis was utilized to identify changes in tumor cell transcriptome and tumor infiltrating immune cell profiling in MAG and/or DOX treated xenograft tumors. Results We found that MAG significantly increased macrophage phagocytosis of ES cells in vitro ( p &lt; 0.01) and had significant effect on reducing tumor burden ( p &lt; 0.01) and increasing survival in NSG mouse model ( p &lt; 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner ( p &lt; 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro ( p &lt; 0.01) and significantly decreased tumor burden ( p &lt; 0.01) and lung metastasis ( p &lt; 0.0001) and extended animal survival in vivo in two different mouse models of ES ( p &lt; 0.0001). Furthermore, we identified CD38, CD209, CD163 and CD206 as potential markers for ES-phagocytic macrophages. Moreover, we found increased M2 macrophage infiltration and decreased expression of Cd209 in the tumor microenvironment of MAG and DOX combinatorial therapy treated tumors. Conclusions By turning “two keys” simultaneously to reactivate macrophage phagocytic activity, our data demonstrated an effective and highly translatable alternative therapeutic approach utilizing innate (tumor associated macrophages) immunotherapy against high-risk metastatic ES.

Combinatorial macrophage induced innate immunotherapy against Ewing sarcoma: Turning “Two Keys” simultaneously