Pylaeva Ekaterina, Spyra Ilona, Bordbari Sharareh, Lang Stephan, Jablonska Jadwiga
Department of Otorhinolaryngology, University Hospital, University of Duisburg-Essen.
Department of Otorhinolaryngology, University Hospital, University of Duisburg-Essen;
J Vis Exp. 2019 Jul 20(149). doi: 10.3791/59807.
The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc.
中性粒细胞在肿瘤发生调节中的作用日益受到关注。这些细胞具有异质性，根据肿瘤微环境的不同，可具有促肿瘤或抗肿瘤能力。在肿瘤环境中调节中性粒细胞功能的重要细胞因子之一是I型干扰素。在干扰素存在的情况下，中性粒细胞获得抗肿瘤特性，包括细胞毒性或刺激免疫系统。相反，缺乏干扰素信号会导致显著的促肿瘤活性，其特征是强烈刺激肿瘤血管生成。最近，我们证明中性粒细胞的促血管生成特性取决于这些细胞中烟酰胺磷酸核糖转移酶（NAMPT）信号通路的激活。抑制肿瘤相关中性粒细胞中的这一信号通路会导致其产生强大的抗血管生成表型。在此，我们展示了我们新建立的模型，该模型可用于体内评估经操控的肿瘤相关中性粒细胞（TANs）的致瘤潜力。简而言之，可以从携带肿瘤的干扰素缺陷小鼠中分离出促血管生成的肿瘤相关中性粒细胞，并通过阻断NAMPT信号将其重极化至抗血管生成表型。随后可使用主动脉环试验评估这些细胞的血管生成活性。可将抗血管生成的TANs转移到携带肿瘤的野生型受体小鼠中，并监测肿瘤生长14天。在第14天处死小鼠，取出肿瘤并进行切割，评估其血管化情况。总体而言，我们的方案提供了一种新工具，可在体内评估原代细胞（如肿瘤相关中性粒细胞）的血管生成能力，而无需使用人工中性粒细胞系模型。
