Department of Advanced Biomedical Sciences, University "Federico II", Naples, Italy.
Institute of Biostructures and Bioimaging, National Research Council, Naples, Italy.
Front Immunol. 2024 Oct 4;15:1470620. doi: 10.3389/fimmu.2024.1470620. eCollection 2024.
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.
中性粒细胞胞外诱捕网（NETs）是由激活的中性粒细胞释放的复杂结构，可能调节转移级联的不同步骤。我们研究的目的是探讨 NETs 如何调节癌细胞的黏附特性，以及细胞暴露于 NETs 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 NET 包被的平板进行不同的癌细胞系固相黏附测定，平板可添加或不添加针对α5β1 或 CCDC25 受体的抗体。孵育 1-4 小时后，以总细胞数的百分比表示黏附细胞。为了检测 EMT 的发生，用 NETs 处理细胞长达 48 小时，然后通过 Western blot 检测 E-钙黏蛋白、波形蛋白、Snail、Slug、Zeb1 和 Twist1 以及 Notch1 和裂解 Notch1 的水平。未经处理和 NET 处理的细胞在含有 FBS 作为趋化剂的 24 孔多微孔板中进行迁移测定。
癌细胞在 NET 包被平板上的黏附率在 30%至 92.7%之间，明显高于未包被平板上的黏附率。添加针对α5β1 或 CCDC25 的抗体可强烈降低细胞对 NET 的黏附。EGFR 驱动的癌细胞系长时间暴露于 NET 会通过 Notch1 的上调和裂解激活 EMT 程序，并通过 EMT 标志物的增强表达得到证实。上皮表型的丧失继而导致致癌基因驱动因子的表达显著降低。与未经处理的细胞相比，NET 处理的细胞的迁移明显增强。
我们的研究结果揭示了 NETs 的动态作用，它可以为远处许多癌细胞的结合提供富含 DNA 和纤维连接蛋白的环境，而 NETs 的长时间暴露通过 Notch1 信号通路的激活触发 EMT，从而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示例，说明免疫/炎症微环境如何直接调节癌细胞对致癌基因靶向药物的敏感性。
