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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 < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 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.
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 < 0.01) and had significant effect on reducing tumor burden (p < 0.01) and increasing survival in NSG mouse model (p < 0.001). The csCRT level on ES cells was significantly enhanced by DOX in a dose- and time-dependent manner (p < 0.01). Importantly, DOX combined with MAG significantly enhanced macrophage phagocytosis of ES cells in vitro (p < 0.01) and significantly decreased tumor burden (p < 0.01) and lung metastasis (p < 0.0001) and extended animal survival in vivo in two different mouse models of ES (p < 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.
摘要：
背景：巨噬细胞在吞噬肿瘤细胞中发挥重要作用。然而,肿瘤通过抗吞噬信号的表达部分逃避巨噬细胞吞噬，最常见的是CD47。在尤因肉瘤（ES）中，我们发现肿瘤细胞利用双重机制通过同时过表达CD47和下调细胞表面钙网蛋白（csCRT）来逃避巨噬细胞清除，促吞噬信号。这里,我们研究了CD47阻断的组合(magrolimab，MAG）抑制抗吞噬信号和化疗方案（阿霉素，DOX）增强促吞噬信号，在体外诱导ES细胞的巨噬细胞吞噬，在体内抑制肿瘤生长和转移。
方法：巨噬细胞通过粒细胞-巨噬细胞集落刺激因子(GM-CSF)和巨噬细胞集落刺激因子(M-CSF)来源于人外周血单核细胞。进行基于流式细胞术和显微镜的体外吞噬作用测定以评估ES细胞的巨噬细胞吞噬作用。进行膜联蛋白-V测定以评估细胞凋亡。通过CRISPR/Cas9方法敲除CD47。使用基于ES细胞的和基于患者来源的异种移植物(PDX)的小鼠模型来评估MAG和/或DOX对ES肿瘤发展和动物存活的影响。RNA-Seq结合CIBERSORTx分析用于鉴定MAG和/或DOX处理的异种移植肿瘤中肿瘤细胞转录组和肿瘤浸润免疫细胞谱的变化。
结果：我们发现MAG在体外显著增加ES细胞的巨噬细胞吞噬(p<0.01)，在NSG小鼠模型中具有显著的降低肿瘤负荷(p<0.01)和提高存活率(p<0.001)的作用。DOX对ES细胞的csCRT水平呈剂量和时间依赖性（p<0.01）。重要的是,在两种不同的ES小鼠模型(p<0.0001)中,DOX联合MAG在体外显著增强ES细胞的巨噬细胞吞噬(p<0.01)并显著降低肿瘤负荷(p<0.01)和肺转移(p<0.0001)和延长动物体内存活。此外，我们将CD38,CD209,CD163和CD206鉴定为ES-吞噬巨噬细胞的潜在标志物.此外，我们发现MAG和DOX联合治疗肿瘤的肿瘤微环境中M2巨噬细胞浸润增加和Cd209表达降低。
结论：通过同时转动“两个键”来重新激活巨噬细胞吞噬活性，我们的数据表明,利用先天(肿瘤相关巨噬细胞)免疫疗法治疗高危转移性ES是一种有效且高度可翻译的替代治疗方法.
方法：巨噬细胞通过粒细胞-巨噬细胞集落刺激因子(GM-CSF)和巨噬细胞集落刺激因子(M-CSF)来源于人外周血单核细胞。进行基于流式细胞术和显微镜的体外吞噬作用测定以评估ES细胞的巨噬细胞吞噬作用。进行膜联蛋白-V测定以评估细胞凋亡。通过CRISPR/Cas9方法敲除CD47。使用基于ES细胞的和基于患者来源的异种移植物(PDX)的小鼠模型来评估MAG和/或DOX对ES肿瘤发展和动物存活的影响。RNA-Seq结合CIBERSORTx分析用于鉴定MAG和/或DOX处理的异种移植肿瘤中肿瘤细胞转录组和肿瘤浸润免疫细胞谱的变化。
结果：我们发现MAG在体外显著增加ES细胞的巨噬细胞吞噬(p<0.01)，在NSG小鼠模型中具有显著的降低肿瘤负荷(p<0.01)和提高存活率(p<0.001)的作用。DOX对ES细胞的csCRT水平呈剂量和时间依赖性（p<0.01）。重要的是,在两种不同的ES小鼠模型(p<0.0001)中,DOX联合MAG在体外显著增强ES细胞的巨噬细胞吞噬(p<0.01)并显著降低肿瘤负荷(p<0.01)和肺转移(p<0.0001)和延长动物体内存活。此外，我们将CD38,CD209,CD163和CD206鉴定为ES-吞噬巨噬细胞的潜在标志物.此外，我们发现MAG和DOX联合治疗肿瘤的肿瘤微环境中M2巨噬细胞浸润增加和Cd209表达降低。
结论：通过同时转动“两个键”来重新激活巨噬细胞吞噬活性，我们的数据表明,利用先天(肿瘤相关巨噬细胞)免疫疗法治疗高危转移性ES是一种有效且高度可翻译的替代治疗方法.
