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 two differences in the sequence of their mt-Nd2 gene which can be used to confirm the purity of mtDNA once the cybridization process has been finished¹³. In this case, the purity of the nuclear background was confirmed by antibiotic sensitivity, since, contrary to L929dt cells, L929 were resistant to geneticin.

1. Mitochondrial depletion by rhodamine 6G treatment (recipient cells)

NOTE: The first step for successful cybrid generation is to completely and irreversibly abolish mitochondrial functions in the recipient cells. For this purpose, it is necessary to previously determine, for each cell line, the appropriate concentration and treatment duration with rhodamine 6G. This adequate concentration should be just below drug-induced cell death (the highest that does not kill the cells during the treatment). Perform the following once the optimal conditions are defined.

1. Seed 10⁶ cells in a 6-well plate using complete culture medium (see Table 1 for details) and treat them daily with the optimal concentration of rhodamine 6G for 3-10 days, depending on the cell line. Traditional concentrations range from 2 to 5 µg/mL for 3-10 days^20,21. In this study, for L929-derived cells, 2.5 µg/mL rhodamine 6G for 7 days was the selected treatment^13,22.
2. To keep the cells alive, supplement the cell culture medium with 50 µg/mL of uridine and 100 µg/mL of pyruvate and renew it every 24 h.
3. After treatment and prior to fusion, change the medium of rhodamine 6G-treated cells to complete cell culture medium without rhodamine 6G, and leave them in this medium for 3-4 h in an incubator at 37 °C with 5% CO₂.
   NOTE: The rhodamine 6G toxic effect is irreversible, and cells with non-functional mitochondria should not recover²⁰. Thus, after treatment, cells that do not receive functional mitochondria should die even in uridine-supplemented culture medium. Therefore, no nuclear selection should be necessary. However, a control fusion of rhodamine 6G-treated cells without mitochondria is recommended.

2. Expansion and mitochondrial isolation (donor cells)

1. Expand the mitochondria donor cells during the time lapse of rhodamine 6G treatment to obtain around 25 x 10⁶ cells.
2. On day 7, harvest exponentially growing cells in a 50 mL tube and collect them by centrifugation at 520 x g for 5 min at room temperature (RT). Wash the cells 3x with cold phosphate-buffered saline (PBS) and sediment them by centrifugation at 520 x g for 5 min at RT. From now on, perform all the steps of mitochondrial extraction at 4 °C, using cold reagents and keeping the tubes with cells or mitochondria on ice.
3. After the third centrifugation, discard the supernatant by aspiration using a glass pipette coupled to a vacuum pump and resuspend the packed cells in a volume of hypotonic buffer equal to 7x the cell pellet volume. Then, transfer the cell suspension into a homogenizer tube and let the cells swell by incubating them on ice for 2 min.
4. Break the cell membranes by performing 8 to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rpm.
   NOTE: The step of cell membrane disruption can vary between cell types; thus, it has to be optimized for each cell type.
5. Add the same volume of hypertonic buffer to the cell suspension (7x the cell pellet volume) to generate an isotonic environment.
6. Transfer the homogenate into a 15 mL tube and centrifuge it in a fixed rotor at 1,000 x g for 5 min at 4 °C. Then, collect only 3/4 of the supernatant leaving a large margin from the pellet, to avoid contamination with nuclei or intact cells, and transfer it to another tube. Repeat the same process twice. Note that the supernatant must be kept and the pellet discarded.
7. Save the mitochondrial fraction (supernatant). Transfer it into 1.5 mL tubes and centrifuge at maximum speed (18,000 x g) for 2 min at 4 °C.
8. Discard the supernatant and wash the mitochondria-enriched pellet with buffer A, combining the content of the two tubes into one and centrifuging at the same conditions as described in step 2.7. Repeat the same process until all the material is in only one tube.
9. Make an additional wash with 300 µL of buffer A and quantify the mitochondrial protein concentration using the Bradford assay²³. For each cybridization assay, the optimal amount of mitochondria for the transfer procedure has to be determined (in our case, a concentration between 10-40 µg of mitochondrial protein per 10⁶ cells).
10. Simultaneously, prepare the rhodamine 6G-pretreated cells for the fusion by collecting them in a 15 mL tube and centrifuging at 520 x g for 5 min at RT. Note that the pellet acquires a neon pink color due to rhodamine 6G treatment.
11. To ensure that both mitochondrial function abolishment in receptor cells as well as organelle purification from donors have been properly performed, seed a small number of rhodamine 6G-treated cells and isolated mitochondria using the complete culture medium in a 6-well plate and culture them for a month to check that no surviving cells remain in any of the wells (Figure 2).
12. In parallel, evaluate the absence of nuclei contaminants in the mitochondrial fraction by immunodetection of nuclear proteins (i.e., lamin beta, histone H3, etc.) or by quantitative polymerase chain reaction (qPCR) amplification of a nuclear gene (i.e., SDH, 18S rRNA, etc.).
    NOTE: To avoid contaminations, it is recommended to perform all the steps in aseptic conditions working under a laminar flow hood.

3. Fusion and cybrid generation

1. To proceed with the fusion, carefully add 10⁶ of the rhodamine 6G-treated cells to the isolated mitochondria pellet (10-40 µg of mitochondrial protein) and centrifuge at 520 x g for 5 min to allow the cells to mix with the mitochondria.
2. Add 100 µL of polyethylene glycol (PEG, 50%) and gently resuspend the pellet for 30 s. Then, allow to rest untouched for another 30 s.
3. Finally, transfer the mix into a 6-well plate with fresh complete cell culture medium and place in the incubator at 37 °C with 5% CO₂. After a few days (usually 1 week), transmitochondrial cybrids should start growing (Figure 2), giving rise to clones that can be individually selected or mixed in a pool prior to their analysis.

4. Verification of both mitochondrial and nuclear background

NOTE: Once the new cell line has been established and cells begin to grow exponentially, the purity of their mitochondrial and nuclear DNAs must be verified. Thus, the original cell lines should harbor different mutations or polymorphisms within their genomes to make them recognizable.

1. Total DNA isolation
   1. Isolate the genomic DNA of all cell lines used for cybrid generation by employing a commercial genomic DNA extraction kit (see Table of Materials) or by performing a standard protocol using phenol-chloroform-isoamyl alcohol extraction and alcohol precipitation²⁴.
2. mtDNA evaluation (Figure 3)
   NOTE: Several techniques, such as sequencing, restriction fragment length polymorphism (RFLP) analysis, or allele-specific qPCR, can be performed to analyze the purity of mtDNA. To confirm the presence of mtDNA sequence variations by RFLP, follow the next protocol steps.
   1. Amplify an mtDNA fragment containing the nucleotide change by PCR.
      1. Here, L929dt cells present an mtDNA mutation at position 4206, within an mt-Nd2 gene (m.4206C>T) that is absent in L929 cells. To confirm the presence of this substitution in the transmitochondrial cybrids, amplify a 397 bp fragment by PCR using a standard protocol and the following primers: 1) 5'-AAGCTATCGGG
         CCCATACCCCG-3' (positions 3862-3884) and 2) 5'-TAATCAGAAGTGGAATGGGGCG -3' (positions 4236-4258).
   2. Analyze the presence of the desired nucleotide substitution by RFLP, using a specific endonuclease that recognizes the sequence change and generates a different cut pattern for both cell lines.
      NOTE: When the L929dt mtDNA variant is present (4206T), the amplicon obtained in step 4.2.1 contains two restriction sites for SspI (see Table of Materials) that produces three DNA fragments of 306 bp, 52 bp, and 39 bp. The restriction site that generates the 52 and 39 bands is disrupted when the wild-type (WT) version C4206 is present, and a new band of 91 bp appears. Therefore, an internal control for full digestion for SspI is included in the analysis. The digestion reaction is performed at 37 °C, following the manufacturer's instructions.
   3. Separate the restriction fragments by electrophoresis and compare the band pattern.
      1. Once the digestion is performed, analyze the obtained restriction fragments by electrophoresis in 10% polyacrylamide-Tris-borate-EDTA (TBE) gels (see Table 1 for 1x TBE composition). Run the electrophoresis at 80 V for 1 h at RT. Visualize the DNA fragments after gel staining in a solution of ethidium bromide in 1x TBE for 15 min at RT (see Table 1 for gel staining solution composition).
3. Nuclear DNA genotyping
   NOTE: Nuclear DNA genotyping must be performed using the previous DNA extraction sample employed for RFLP analysis.
   1. Amplify 16 loci (D21S11, CSF1PO, vWA, D8S1179, TH01, D18S51, D5S818, D16S539, D3S1358, D2S1338, TPOX, FGA, D7S820, D13S317, D19S433, and AMEL) by using a pool of commercial-specific oligonucleotides (see Table of Materials).
   2. Perform electrophoresis by using a genetic analyzer in order to separate the fragments previously obtained.
      NOTE: Once amplified, the different loci are analyzed by electrophoresis using a capilar system (see Table of Materials). The fragments are separated in a 50 cm long capilar filled with a commercial polymer. Cathode and anode buffers are also commercially available, as shown in the Table of Materials. Electrophoresis is performed at 19.5 kV for 20 min at 60 °C.
   3. Use bioinformatical tools to determine the alleles corresponding to each amplified locus (see Table of Materials).
   4. Compare the data obtained in step 4.3.3 with the nuclear DNA profile database (Table 1), to check if the nuclear DNA profile matches with the mitochondria receptor cell line profile (Figure 4).

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

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

Name	Company	Catalog Number	Comments
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