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This research was funded by grant number PID2019-105128RB-I00 to RSA, JMB, and AA, and PGC2018-095795-B-I00 to PFS and RML, both funded by MCIN/AEI/10.13039/501100011033 and grant numbers B31_20R (RSA, JMA, and AA) and E35_17R (PFS and RML) and funded by Gobierno de Aragon. The work of RSA was supported by a grant from the Asociacion Espanola Contra el Cancer (AECC) PRDAR21487SOLE. The authors would like to acknowledge the use of Servicio General de Apoyo a la Investigacion-SAI, Universidad de Zaragoza.

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Transmitochondrial Cybrid Generation Using Cancer Cell Lines

Publicated to:Jove-Journal Of Visualized Experiments. 2023 (193): e65186- - 2023-03-01 2023(193), DOI: 10.3791/65186

Authors: Soler-Agesta, Ruth; Marco-Brualla, Joaquin; Fernandez-Silva, Patricio; Mozas, Pilar; Anel, Alberto; Moreno Loshuertos, Raquel

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Abstract

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 (.0, 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.

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