Soler-Agesta Ruth, Marco-Brualla Joaquín, Fernández-Silva Patricio, Mozas Pilar, Anel Alberto, Moreno Loshuertos Raquel
Department of Biochemistry, University of Zaragoza.
Department of Biochemistry, University of Zaragoza; Institute for Biocomputation and Physics of Complex Systems, University of Zaragoza.
J Vis Exp. 2023 Mar 17(193). doi: 10.3791/65186.
In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more efforts are still needed to fully understand the link involving alterations in mitochondria and tumorigenesis, as well as to identify tumor-associated mitochondrial phenotypes. For instance, to evaluate the contribution of mitochondria in tumorigenesis and metastasis processes, it is essential to understand the influence of mitochondria from tumor cells in different nuclear environments. For this purpose, one possible approach consists of transferring mitochondria into a different nuclear background to obtain the so-called cybrid cells. In the traditional cybridization techniques, a cell line lacking mtDNA (ρ, nuclear donor cell) is repopulated with mitochondria derived from either enucleated cells or platelets. However, the enucleation process requires good cell adhesion to the culture plate, a feature that is partially or completely lost in many cases in invasive cells. In addition, another difficulty found in the traditional methods is achieving complete removal of the endogenous mtDNA from the mitochondrial-recipient cell line to obtain pure nuclear and mitochondrial DNA backgrounds, avoiding the presence of two different mtDNA species in the generated cybrid. In this work, we present a mitochondrial exchange protocol applied to suspension-growing cancer cells based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria. This methodology allows us to overcome the limitations of the traditional approaches, and thus can be used as a tool to expand the comprehension of the mitochondrial role in cancer progression and metastasis.
近年来，致力于确定线粒体与癌症之间联系的研究数量显著增加。然而，要全面理解线粒体改变与肿瘤发生之间的联系，以及识别与肿瘤相关的线粒体表型，仍需要付出更多努力。例如，为了评估线粒体在肿瘤发生和转移过程中的作用，了解不同核环境下肿瘤细胞中线粒体的影响至关重要。为此，一种可能的方法是将线粒体转移到不同的核背景中，以获得所谓的胞质杂种细胞。在传统的胞质杂交技术中，用来自去核细胞或血小板的线粒体重新填充缺乏线粒体DNA的细胞系（ρ，核供体细胞）。然而，去核过程需要细胞良好地粘附在培养板上，而在许多侵袭性细胞的情况下，这一特性会部分或完全丧失。此外，传统方法中发现的另一个困难是要从线粒体受体细胞系中完全去除内源性线粒体DNA，以获得纯净的核和线粒体DNA背景，避免在产生的胞质杂种中存在两种不同的线粒体DNA。在这项工作中，我们提出了一种适用于悬浮生长癌细胞的线粒体交换方案，该方案基于用分离的线粒体重新填充罗丹明6G预处理的细胞。这种方法使我们能够克服传统方法的局限性，因此可以用作一种工具来扩展对线粒体在癌症进展和转移中作用的理解。
