ADAM17-mediated EGFR ligand shedding directs macrophage-promoted cancer cell invasion. ADAM17-mediated EGFR ligand shedding directs macrophage-promoted cancer cell invasion

Macrophages in the tumor microenvironment have a substantial impact on tumor progression. Depending on the signaling environment in the tumor, macrophages can either support or constrain tumor progression. It is therefore of therapeutic interest to identify the tumor-derived factors that control macrophage education. With this aim, we correlated the expression of A Disintegrin and Metalloproteinase (ADAM) proteases, which are key mediators of cell-cell signaling, to the expression of protumorigenic macrophage markers in human cancer cohorts. We identified ADAM17, a sheddase upregulated in many cancer types, as a protein of interest. Depletion of ADAM17 in cancer cell lines reduced the expression of several protumorigenic markers in neighboring macrophages in vitro as well as in mouse models. Moreover, ADAM17–/– educated macrophages demonstrated a reduced ability to induce cancer cell invasion. Using mass spectrometry–based proteomics and ELISA, we identified HB-EGF and AREG, shed by ADAM17 in the cancer cells, as the implicated molecular mediators of macrophage education. Additionally, RNA-Seq and ELISA experiments revealed that ADAM17-dependent HB-EGF ligand release induced the expression and secretion of CXCL chemokines in macrophages, which in turn stimulated cancer cell invasion. In conclusion, we provide evidence that ADAM17 mediates a paracrine EGFR-ligand-chemokine feedback loop, whereby cancer cells hijack macrophages to promote tumor progression. Overall design: Comparative gene expression profiling analysis of RNA-seq data for bone marrow derived macrophages co-cultured with 4T1 wt or 4T1 ADAM17 knock-out cells for 48h; Seq was performed by BGI

肿瘤微环境中的巨噬细胞对肿瘤进展具有重要影响。根据肿瘤内的信号传导微环境，巨噬细胞既可以促进肿瘤进展，也可以抑制肿瘤进展。因此，筛选可调控巨噬细胞教化过程的肿瘤源性因子具有重要的治疗价值。基于此研究目标，我们在人类癌症队列中，将作为细胞间信号传导关键介质的解整合素金属蛋白酶（A Disintegrin and Metalloproteinase, ADAM）家族蛋白酶的表达水平，与促瘤性巨噬细胞标志物的表达水平进行了关联分析。我们筛选出在多种癌症类型中表达上调的脱落酶ADAM17作为目标研究蛋白。在癌细胞系中敲除ADAM17后，无论是体外实验还是小鼠模型中，邻近巨噬细胞的多种促瘤性标志物表达水平均出现下调。此外，经ADAM17敲除细胞教育后的巨噬细胞，其诱导癌细胞侵袭的能力显著降低。通过基于质谱的蛋白质组学分析与酶联免疫吸附实验（ELISA），我们证实癌细胞中ADAM17所脱落的HB-EGF与AREG是调控巨噬细胞教化的关键分子介质。此外，RNA测序（RNA-Seq）与ELISA实验结果显示，依赖ADAM17的HB-EGF配体释放可诱导巨噬细胞表达并分泌CXCL趋化因子，进而促进癌细胞侵袭。综上，我们的研究证实ADAM17介导了一条旁分泌型EGFR配体-趋化因子反馈通路，癌细胞通过该通路劫持巨噬细胞以促进肿瘤进展。实验整体设计：将骨髓来源巨噬细胞与4T1野生型（wt）或4T1 ADAM17敲除细胞共培养48小时后，对其RNA-seq数据进行比较基因表达谱分析；测序工作由BGI完成。