中性粒细胞胞外陷阱（NET）是由活化的中性粒细胞释放的复杂结构，可以调节转移级联的不同步骤。我们研究的目的是研究 NET 如何调节癌细胞的粘附特性，以及细胞暴露于 NET 是否可以激活上皮间质转化 (EMT) 程序，从而增强肿瘤细胞的迁移和侵袭特性。使用添加或不添加针对 α5β1 或 CCDC25 受体的抗体的 NET 包被板对细胞系进行固相粘附测定。孵育1-4小时后，贴壁细胞表示为总细胞数的百分比。为了测试 EMT 的发生，用 NET 处理细胞长达 48 小时，然后通过蛋白质印迹法测定 E-钙粘蛋白、波形蛋白、Snail、Slug、Zeb 1 和 Twist 1 的水平以及 Notch 1 和 cleaved Notch 1 的水平。使用 Transwell 和 FBS 作为化学引诱剂的 24 多孔板对未处理和 NET 处理的细胞进行迁移测定。癌细胞对 NET 包被板的粘附在 30% 到 92.7% 之间变化，并且显着高于未包被板中获得的粘附力。添加针对 α5β1 或 CCDC25 的抗体会导致细胞对 NET 的粘附强烈减少。 EGFR驱动的癌细胞系长时间暴露于NETs，通过Notch 1的上调和裂解引起EMT程序的激活，并通过EMT标志物表达的增强得到证实。随后上皮表型的丧失导致癌基因驱动基因表达的强烈减少。与未处理的细胞相比，经 NET 处理的细胞的细胞迁移显着增强。我们的研究结果揭示了 NET 的动态作用，它可能为许多远处部位的癌细胞结合提供富含 DNA 和纤连蛋白的环境，在这些部位，长期暴露于 NET 会引发癌症EMT 通过激活 Notch 1 信号通路来增强癌细胞的迁移和侵袭特性。此外，我们的研究结果提供了一个例子，说明免疫/炎症微环境如何直接调节癌细胞对癌基因靶向药物的敏感性。版权所有 © 2024 Dimitrov、Maddalena、Terlizzi、Altobelli、Pellegrino、Mehmood、De Rosa、Iommelli 和 Del Vecchio。

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.Copyright © 2024 Dimitrov, Maddalena, Terlizzi, Altobelli, Pellegrino, Mehmood, De Rosa, Iommelli and Del Vecchio.