neutrophil extracellular traps在癌细胞粘附及EGFR驱动肺癌细胞Notch 1介导的上皮-间充质转化中的动态作用

中性粒细胞胞外陷阱（NETs）是由活化的中性粒细胞释放的复杂结构，可能调节转移级联中的不同步骤。本研究旨在探讨NETs如何调节癌细胞的粘附特性，以及细胞暴露于NETs是否能激活上皮-间充质转化（EMT）程序，从而增强肿瘤细胞的迁移和侵袭能力。不同的癌细胞系被用于固相粘附试验，使用覆盖有NET的培养板，加入或不加入抗α5β1或CCDC25受体的抗体。孵育1-4小时后，粘附细胞以总细胞数的百分比表达。为检测EMT的发生，细胞在NETs中处理长达48小时，然后通过免疫印迹分析E-cadherin、vimentin、Snail、Slug、Zeb 1和Twist 1的水平，以及Notch 1和切割型Notch 1的水平。未处理和NET处理的细胞被用Transwell和FBS作为趋化因子的24孔板迁移试验。结果显示，癌细胞对NET覆盖板的粘附率在30%至92.7%之间，且显著高于未涂覆板。加入抗α5β1或CCDC25抗体显著降低了细胞对NET的粘附。EGFR驱动的癌细胞系长时间暴露于NETs导致Notch 1的激活（通过上调和裂解）和EMT标志物的表达增强，从而确认了EMT程序的激活。伴随的上皮表型丧失导致驱动癌基因的表达显著下降。与未处理细胞相比，NET处理细胞的迁移能力显著增强。我们的研究揭示了NETs的动态作用，它们可能为许多癌细胞在远端部位的结合提供富含DNA和纤维连接蛋白的环境，长时间暴露于NETs通过激活Notch 1信号通路触发EMT，继而增强癌细胞的迁移和侵袭能力。此外，本研究还提供了免疫/炎症微环境如何直接调节癌细胞对致癌基因靶向药物敏感性的示例。

Neutrophil extracellular traps (NETs) are complex structures released by activated neutrophils that may modulate different steps of the metastatic cascade. The aim of our study was to investigate how NETs can modulate the adhesion properties of cancer cells and whether cell exposure to NETs can activate the epithelial-to-mesenchymal transition (EMT) program thus enhancing the migratory and invasive properties of tumor cells.Different cancer cell lines were subjected to a solid-phase adhesion assay using NET-coated plates with or without the addition of antibodies against α5β1 or CCDC25 receptor. After 1-4 h of incubation, adherent cells were expressed as the percentage of total cell number. To test EMT occurrence, cells were treated with NETs for up to 48 h and then the levels of E-cadherin, vimentin, Snail, Slug, Zeb 1 and Twist 1 along with levels of Notch 1 and cleaved Notch 1 were determined by western blotting. Untreated and NET-treated cells were subjected to migration assays using 24-multiwell plates with transwell and FBS as chemoattractant.Cancer cell adhesion to NET-coated plates varied between 30% and 92.7% and was significantly higher than that obtained in uncoated plates. The addition of antibodies against α5β1 or CCDC25 caused a strong reduction of cell adhesion to NETs. The prolonged exposure of EGFR-driven cancer cell lines to NETs caused the activation of the EMT program through the upregulation and cleavage of Notch 1 and was confirmed by the enhanced expression of EMT markers. The consequent loss of the epithelial phenotype induced a strong reduction of the expression of the oncogene driver. Cell migration was significantly enhanced in NET-treated cells as compared to untreated cells.Our findings reveal the dynamic role of NETs that may provide a DNA and fibronectin rich environment for binding of many cancer cells at distant sites where the prolonged exposure to NETs triggers the EMT through the activation of Notch 1 signaling pathway with the subsequent enhancement of migratory and invasive properties of cancer cells. Furthermore, our findings provide an example of how an immune/inflammatory microenvironment may directly modulate the sensitivity of cancer cells to oncogene targeted agents.