This research aims to better understand the contribution of mitochondria 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 mitochondria genes, increases the complexity of our experiments and data interpretation. This is one of the reasons why it's 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 to maregenic and metastatic in in vivo models. This mitochondrial 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.
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 520 G for five minutes.
Note that the pellet acquires a neon pink color due to Rhodamine 6G treatment. 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 520 G for five minutes. Wash the cells with cold phosphate buffered saline and sediment them by centrifugation at room temperature and 520 G 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 re-suspend 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 eight to 10 strokes in the homogenizer coupled to a motor-driven pestle rotating at 600 rotations per minute. Into the cell homogenate, add the hypertonic 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 in a fixed rotor at 1, 000 G and four degrees Celsius for five minutes. Then collect only three-fourths 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, 000 G 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.
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 520 G 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 cybrid 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.
