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 Arag&#243;n. The work of RSA was supported by a grant from the Asociaci&#243;n Espa&#241;ola Contra el C&#225;ncer (AECC) PRDAR21487SOLE. The authors would like to acknowledge the use of Servicio General de Apoyo a la Investigaci&#243;n-SAI, Universidad de Zaragoza.

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 ...

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

The authors declare no conflicts of interest.

Since Otto Warburg reported that cancer cells shift their metabolism and potentiate &#34;aerobic glycolysis&#34;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 ...

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 &#34;Warburg effect&#34; and enables some cancer cells to supply their energetic demands for rapid growth and division, despite generating ATP less efficiently than the aerobic process3,4,5. 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.&#160;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 cells9,10,11,12,13,14,15,16.
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 methods18,19. This methodology allows the use of any nuclei donor regardless of the availability of their corresponding &#961;0 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).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3500XL Genetic Analyzer&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>4406016</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Corning</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpFlSTR Identifiler Plus PCR Amplification Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>4427368</td>
			<td></td>
		</tr>
		<tr>
			<td>Anode Buffer Container 3500 Series</td>
			<td>Applied Biosystems</td>
			<td>4393927</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>PanReac</td>
			<td>131015</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay</td>
			<td>Biorad</td>
			<td>5000002</td>
			<td></td>
		</tr>
		<tr>
			<td>Cathode Buffer Container 3500 Series</td>
			<td>Applied Biosystems</td>
			<td>4408256</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks</td>
			<td>TPP</td>
			<td>90076</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>PanReac</td>
			<td>131026</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Gibco</td>
			<td>10131027</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Teflon pestle</td>
			<td>Deltalab</td>
			<td>196102</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>ATCC</td>
			<td>CCL-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniProtean Tetra4 Gel System</td>
			<td>BioRad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Sigma-Aldrich</td>
			<td>Custom products</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide Solution 30%</td>
			<td>PanReac</td>
			<td>A3626</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>P7181</td>
			<td></td>
		</tr>
		<tr>
			<td>POP-7</td>
			<td>Applied Biosystems</td>
			<td>4393714</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAmp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51306</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine-6G</td>
			<td>Sigma-Aldrich</td>
			<td>R4127</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum Fetal Bovine</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>SspI</td>
			<td>New England Biolabs</td>
			<td>R3132</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>PAN biotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S3089</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>PanReac</td>
			<td>P14030b</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td></td>
		</tr>
	</tbody>
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transmitochondrial cybrid generation using cancer cell lines

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 (&#961;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.

Author Spotlight: Transmitochondrial Cybrid Generation Using Cancer Cell Lines  

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

This research aims to better understand the contribution of mitochondrion in cancer progression and metastasis. The transmitochondrial cybrid generation can help us to elucidate how and at what level mitochondrial genotypes are involved in the aggressiveness of a tumor cell. One of the technologies to obtain information on mitochondrial role in cancer is cybrid generation.We combine this tool with a variety of other methods that include donative electrophoresis, respiration analysis and other mitochondrial functional assays, flow cytometry, proteomics, et cetera. The plasticity of cancer cells together with the interaction networks between nuclear and mitochondrial genes, increases the complexity of our experiments and data interpretation. This is one of the reasons why is necessary to develop a new experimental approaches.One of the most exciting findings from our research has been the discovery of two mitochondrial DNA mutations associated with detachment of cells from the play dish resembling metastasis generation and making the cells completely dependent on aerobic glycolysis, and also making them more tumorigenic and metastatic in in vivo models. This mitochondria exchange protocol can be applied to suspension growing cancer cells. The chronology can overcome the limitations of traditional approaches where the nucleation process and the complete removal of mitochondrial DNA from mitochondrial recipient cell lines often become a real challenge.

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...

Watch this Scientific Journal Video about Transmitochondrial Cybrid Generation Using Cancer Cell Lines at JoVE.com

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.

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...

transmitochondrial-cybrid-generation-using-cancer-cell-lines

Research

JoVE Journal

Cancer Research

Mitochondrial Depletion by Rhodamine 6G for Transmitochondrial Cybrid Generation

To perform mitochondrial depletion of the mitochondrial recipient cell line, seed 1 million cells in a six-well plate using complete culture medium containing 10%FBS and Penicillin-Streptomycin. Treat the cells daily with the optimal concentration of Rhodamine 6G for three to seven days, depending on the type of cell line. To keep the cells alive, supplement the cell culture medium with 50 micrograms per milliliter of uridine and 100 micrograms per milliliter of pyruvate.After treatment and prior to fusion with a mitochondria isolated from the donor cell line, remove the medium of Rhodamine 6G treated cells and add complete cell culture medium without Rhodamine 6G. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for three to four hours. Finally, to prepare the Rhodamine 6G pretreated cells for the fusion, collect them in a 15-milliliter tube, and centrifuge the tube at room temperature and 520G for five minutes.Note that the pellet acquires a neon pink color due to Rhodamine 6G treatment.

Mitochondrial Isolation for Transmitochondrial Cybrid Generation

Once around 25 million mitochondria donor cells are obtained on day seven, harvest the exponentially grown cells in a 50-milliliter tube and collect them by centrifuging at room temperature and 520g for five minutes. Wash the cells with cold phosphate-buffered saline and sediment them by centrifugation at room temperature in 520g for five minutes. Now onwards, perform the whole mitochondrial extraction at four degrees Celsius using cold reagents and keep the cells or mitochondria on ice.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 seven times the cell pellet volume. Then transfer the cell suspension into a homogenizer tube and let the cells swell by incubating them on ice for two minutes. Break the cell membranes by performing 8 to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rotations per minute.Into the cell homogenate, add the hypotonic buffer equal to seven times the volume of the initial pellet to generate an isotonic environment. Transfer the homogenate into a 15-milliliter tube and centrifuge it in a fixed rotor at 1, 000g and four degrees Celsius for five minutes. 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.After repeating the process twice, transfer the supernatant containing the mitochondrial fraction into 1.5-milliliter tubes. Centrifuge the tubes at the maximum speed of 18, 000g for two minutes at four degrees Celsius. Discard the supernatant and wash the mitochondria-enriched pellet with buffer A.Combine the content of the two tubes into one and centrifuge as demonstrated before.Repeat the process until all the material is in only one tube. Wash the pellet obtained from the last centrifugation using 300 microliters of buffer A.Quantify the mitochondrial protein concentration using the Bradford assay. Before fusion with the mitochondrial recipient cell line, evaluate the absence of nuclei contaminants in the mitochondrial fraction by immunodetection of nuclear proteins.Alternatively perform quantitative polymerase chain reaction or QPCR amplification of a nuclear gene.

Fusion and Transmitochondrial Cybrid Generation

For transmitochondrial cybrid generation, use the mitochondrial recipient cells that have been depleted to mitochondria by Rhodamine 6G treatment, and perform fusion with mitochondria isolated from the mitochondria donor cells. Ensure proper mitochondrial function abolishment in recipient cells and organelle purification from donor cells by seeding a small number of Rhodamine 6G-treated cells and isolated mitochondria in complete culture medium in a six-well plate. Check for no surviving cells remaining in the wells after one month of culture.To proceed with the fusion, carefully add the Rhodamine 6G-treated cells to the isolated mitochondria pellet. Then centrifuge at 520g for five minutes to allow the cells to mix with the mitochondria. Add 100 microliters of 50%polyethylene glycol and gently resuspend the pellet for 30 seconds Then allow the suspension to rest untouched for another 30 seconds.Finally, transfer the mix into a six-well plate with fresh complete cell culture medium, and place it in the incubator at 37 degrees Celsius with 5%carbon dioxide. Usually, after about one week, transmitochondrial cybrids should start growing, giving rise to clones that can be individually selected or mixed in a pool prior to their analysis. The restriction fragments obtained from the restriction fragment length polymorphism, or RFLP, analysis of wild-type mitochondria differed from the mutant one.The RFLP analysis of the new transmitochondrial cell lines indicated that restriction fragments were identical to those obtained in their respective mitochondrial donors and different from those generated with the recipient cell lines. Further, the difference in a typical DNA nuclear genotyping profile of the two cell lines used in the hybridization process could also be used to confirm nuclear DNA purity.

1.5K Views.  University of Zaragoza.  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. 

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Summary