Fontanil Tania, Mohamedi Yamina, Cal Santiago, Obaya Álvaro J
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Asturias, Spain.
Facultad de Medicina, Instituto Universitario de Oncología (IUOPA), Universidad de Oviedo, Oviedo, Asturias, Spain.
Methods Mol Biol. 2018;1731:133-143. doi: 10.1007/978-1-4939-7595-2_13.
Proteases play crucial roles in all steps of tumor progression including cancer cell migration. In fact, uncontrolled proteolytic activity could lead to the degradation of different components of the extracellular matrix which facilitates dissemination of tumor cells. However, numerous studies have revealed that proteases may also exert tumor-protective actions which could impede progression of malignant cells. Consequently, it is crucial to distinguish those situations in which proteases promote tumor growth from those in which exhibit tumor-suppressive effects. In this regard, analysis of the influence of a particular protease on the capacity of a cell line to migrate can be employed as an approach to better understand its involvement in tumorigenesis. Different experimental designs have been developed to investigate cell migration. Herein, we describe a barrier assay to monitor cell migration, which overcomes some disadvantages of traditional methods such as the Boyden chamber or the wound healing assays. The version of the barrier assay explained in this chapter allows to examine cell migration through the analysis of the closure of a premade 500 μm wound. This method also facilitates comparison between two different situations in a given cell line (i.e., gene up- or downregulation) in the same assay and under the same conditions. Additionally, migration can be monitored and measured using a time lapse microscope which facilitates further analysis through different softwares.
蛋白酶在肿瘤进展的所有步骤中都起着关键作用，包括癌细胞迁移。事实上，不受控制的蛋白水解活性可能导致细胞外基质的不同成分降解，从而促进肿瘤细胞的扩散。然而，大量研究表明，蛋白酶也可能发挥肿瘤保护作用，这可能会阻碍恶性细胞的进展。因此，区分蛋白酶促进肿瘤生长的情况与表现出肿瘤抑制作用的情况至关重要。在这方面，分析特定蛋白酶对细胞系迁移能力的影响可作为一种方法，以更好地了解其在肿瘤发生中的作用。已经开发了不同的实验设计来研究细胞迁移。在此，我们描述一种用于监测细胞迁移的屏障试验，该试验克服了传统方法（如博伊登室或伤口愈合试验）的一些缺点。本章所解释的屏障试验版本允许通过分析预先制作的500μm伤口的闭合情况来检测细胞迁移。该方法还便于在同一试验和相同条件下比较给定细胞系中的两种不同情况（即基因上调或下调）。此外，可以使用延时显微镜监测和测量迁移情况，这便于通过不同软件进行进一步分析。
