Transmitochondrial Cybrid Generation Using Cancer Cell Lines

Summary

This protocol describes a technique for cybrid generation from suspension-growing cancer cells as a tool to study the role of mitochondria in the tumorigenic process.

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 (ρ⁰, 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.

Introduction

Reprogramming energy metabolism is a hallmark of cancer¹ that was observed for the first time by Otto Warburg in the 1930s². Under aerobic conditions, normal cells convert glucose into pyruvate, that then generates acetyl-coA, fuelling the mitochondrial machinery and promoting cellular respiration. Nevertheless, Warburg demonstrated that, even under normoxic conditions, most cancer cells convert pyruvate obtained from the glycolysis process into lactate, shifting their way to obtain energy. This metabolic adjustment is known as the "Warburg effect" and enables some cancer cells to supply their energetic demands for rapid growth and division, despite generating ATP less efficiently than the aerobic process^3,4,5. In recent decades, numerous works have supported the implication of metabolism reprogramming in cancer progression. Hence, tumor energetics is considered an interesting target against cancer¹. As a central hub in energetic metabolism and in the supply of essential precursors, mitochondria play a key role in these cell adaptations that, to date, we only partially understand.

In line with the above, mitochondrial DNA (mtDNA) mutations have been proposed as one of the possible causes of this metabolic reprogramming, which could lead to an impaired electron transport chain (ETC) performance⁶ and would explain why some cancer cells enhance their glycolytic metabolism to survive. Indeed, it has been reported that mtDNA accumulates mutations within cancer cells, being present in at least 50% of tumors⁷. For example, a recent study carried out by Yuan et al. reported the presence of hypermutated and truncated mtDNA molecules in kidney, colorectal, and thyroid cancers⁸. Moreover, many works have demonstrated that certain mtDNA mutations are associated with a more aggressive tumor phenotype and with an increase in the metastatic potential of cancer cells^{9,10,11,12,13,14,15,16}.

Despite the apparent relevance of the mitochondrial genome in cancer progression, the study of these mutations and their contribution to the disease have been challenging due to limitations in the experimental models and technologies currently available¹⁷. Thus, new techniques to understand the real impact of mitochondria DNA in cancer disease development and progression are needed. In this work, we introduce a protocol for transmitochondrial cybrid generation from suspension-growing cancer cells, based on the repopulation of rhodamine 6G-pretreated cells with isolated mitochondria, that overcomes the main challenges of traditional cybridization methods^18,19. This methodology allows the use of any nuclei donor regardless of the availability of their corresponding ρ⁰ cell line and the transfer of mitochondria from cells that, following the traditional techniques, would be difficult to enucleate (i.e., non-adherent cell lines).

Protocol

NOTE: All culture media and buffer compositions are specified in Table 1. Prior to cybrid generation, both mitochondrial and nuclear DNA profiles from the donor and recipient cells must be typed to confirm the presence of genetic differences in both genomes between cell lines. In this study, a commercially available L929 cell line and its derived cell line, L929dt, which was spontaneously generated in our laboratory (see13 for more information) were used. These cell lines present …

Representative Results

After following the above-presented protocol, a homoplasmic cybrid cell line with a conserved nuclear background but with a new mitochondria genotype should be obtained, as represented in the schematics in Figure 1 and Figure 2. The purity of the mitochondrial and nuclear DNA present in the cybrids can be confirmed by RFLP, as shown in Figure 3, and by nuclear DNA genotyping analysis, as shown in Figure 4</stron…

Discussion

Since Otto Warburg reported that cancer cells shift their metabolism and potentiate "aerobic glycolysis"^3,4 while reducing mitochondrial respiration, the interest in the role of mitochondria in cancer transformation and progression has grown exponentially. In recent years, mutations in the mtDNA and mitochondrial dysfunction have been postulated as hallmarks of many cancer types²⁵. To date, numerous studies have analyzed the mtDNA …

Materials

3500XL Genetic Analyzer	ThermoFisher Scientific	4406016
6-well plate	Corning	08-772-1B
Ammonium persulfate	Sigma-Aldrich	A3678
AmpFlSTR Identifiler Plus PCR Amplification Kit	ThermoFisher Scientific	4427368
Anode Buffer Container 3500 Series	Applied Biosystems	4393927
Boric acid	PanReac	131015
Bradford assay	Biorad	5000002
Cathode Buffer Container 3500 Series	Applied Biosystems	4408256
Cell culture flasks	TPP	90076
DMEM high glucose	Gibco	11965092
EDTA	PanReac	131026
Ethidium Bromide	Sigma-Aldrich	E8751
Geneticin	Gibco	10131027
Homogenizer Teflon pestle	Deltalab	196102
L929 cell line	ATCC	CCL-1
MiniProtean Tetra4 Gel System	BioRad	1658004
MOPS	Sigma-Aldrich	M1254
PCR primers	Sigma-Aldrich	Custom products
Polyacrylamide Solution 30%	PanReac	A3626
Polyethylene glycol	Sigma-Aldrich	P7181
POP-7	Applied Biosystems	4393714
Pyruvate	Sigma-Aldrich	P5280
QIAmp DNA Mini Kit	Qiagen	51306
Rhodamine-6G	Sigma-Aldrich	R4127
Serum Fetal Bovine	Sigma-Aldrich	F7524
SspI	New England Biolabs	R3132
Streptomycin/penicillin	PAN biotech	P06-07100
Sucrose	Sigma-Aldrich	S3089
TEMED	Sigma-Aldrich	T9281
Tris	PanReac	P14030b
Uridine	Sigma-Aldrich	U3750