This protocol describes a simple and rapid method for measuring tumor mitochondrial function. The protocol is broadly applicable to clinical translational oncology, and will help enhance diagnostic and mechanistic interpretation of the role of mitochondria in the onset and progression of cancer. The main advantage of this technique is the application of tissue homogenization to simplify preparation and maximize the yield of mitochondria while mitigating the limitations of diffusion.
This technique offers potential applications in clinical and diagnostic settings with a quantitative assessment of tumor mitochondrial function using freshly biopsied or excised tumor tissue. Begin by placing a glass homogenizer containing one milliliter of mitochondrial respiration medium, or MiR05, in a tightly-fitting glass pestle on wet ice. Place the tissue in one milliliter of biopsy preservation solution, or BIOPS, in an ice-cold Petri dish.
Cut the tumor into small pieces of approximately 5 to 10 milligrams each, and place the remaining tumor pieces back into 10 milliliters of BIOPS kept on ice. After blotting the tissue sections carefully on a filter paper, place them on a small plastic tared weigh boat and record the initial wet weight. Place the unused pieces back into the 10-milliliter conical tube of BIOPS for continued preservation.
Submerge the tissue sections into an ice cold homogenizer containing MiR05 and record the remaining weight on the weigh boat. Using a glass pestle with a clearance range of 0.09 to 0.16 millimeters, gently disrupt the tumor tissue by completing five to seven down and up strokes. For each stroke, rotate the pestle in a clockwise/counter-clockwise motion three times while pushing the pestle down, and three additional times while pulling the pestle back up.
Allow the tissue to settle to the bottom of the homogenizer in between strokes, but avoid bringing the pestle fully above the fluid volume to prevent foaming. Pour the homogenate into a 15-milliliter conical tube and place it on ice. Pipette one to three milliliters of fresh MiR05 over the pestle and into the homogenizer to wash any remaining tissue homogenate.
Pour the MiR05 wash into the conical tube containing the homogenate. Repeat this step two to three times to ensure complete transfer of the homogenate. To accurately calculate the tissue homogenate concentration, carefully inspect the homogenizer and the homogenate for remaining non-homogenized material, which includes connective tissue.
To remove the non-homogenized material from the homogenizer that cannot be reached with tweezers, after adding MiR05 to the homogenizer, aspirate the volume, including the tissue, with a pipette and move the contents to a Petri dish. Remove the non-homogenized material settled in large pieces at the bottom of the conical tube from the homogenate by aspirating them with a pipette, and place them on the cap of the conical tube. Remove the tissue from the Petri dish or conical tube cap with tweezers, and blot it on filter paper.
Add the remaining homogenate from the conical tube cap back into the conical tube. Cap the tube, then invert to mix. Reinspect the homogenizers and homogenate for any additional non-homogenized material, and repeat the steps to remove non-homogenized material if needed.
Weigh and record the mass of the non-homogenized material recovered from the homogenizer. Inspect the homogenate preparation for any grossly-intact tissue transferred, and removed the non-homogenized pieces and weigh as necessary. Subtract the tissue weight recovered from the weigh boat, homogenizer, and homogenate from the initial wet weight to calculate the final sample weight.
Using the final sample weight, add additional MiR05 to bring homogenate to the desired concentration. Once the homogenate is weighed and prepared, proceed to assay as soon as possible. Keep the sample stored on wet ice until it is transferred to the instrument.
Once the instrument is calibrated and the sample is prepared, remove the stoppers with a twisting motion and aspirate the MiR05 from the chambers, avoiding the membrane exposed inside the chamber. Mix the homogenate well, and add 2.25 milliliters of the homogenate to the chamber. Suppose one homogenate is being added to multiple chambers, pipette one milliliter of the homogenate at a time into each chamber while mixing the homogenate to ensure equal tissue distribution.
Click on F4 to name and timestamp the event, and then click on Okay. Fill a 50-milliliter syringe with oxygen from an oxygen tank with a regulator and gas tubing using a blunt 18-gauge needle. Inject the oxygen directly into the chambers to hyperoxygenate them to approximately 500 micromolar oxygen.
Loosely insert the stoppers and wait to close until the oxygen reaches approximately 480-micromolar. With a twisting motion, slowly close the chamber and allow the respiration to equilibrate for approximately 15 to 20 minutes. Fill the stopper's central capillary with MiR05 if necessary.
Use dedicated microsyringes to inject substrates, uncouplers, and inhibitors into fully closed chambers to name and timestamp the events for each chamber in real time. Select F6 to adjust the oxygen concentration and oxygen slope negative scales as needed. After each injection, wash the syringe three times in water for water-soluble compounds, and 70%ethanol for ethanol-dissolved compounds.
Add five microliters of 0.8-molar malate and proceed immediately to the next injection. Wash the injection syringe three times in water. Immediately add five microliters of 1-molar pyruvate, and wait for the respiration to stabilize.
After washing the syringe, add 10 microliters of 0.5-molar ADP and wait for the ADP response to stabilize. Wash the syringe and add five microliters of 2-molar glutamate and wait for the respiration to stabilize. Wash the syringe, then add five microliters of 4-millimolar cytochrome-C, and wait for the respiration to stabilize.
After washing the syringe, add 20 microliters of 1-molar succinate and wait for the respiration to stabilize. Titrate 0.5-to 1-microliter increments of 1-millimolar FCCP and wait for the respiration to stabilize after each injection. Continue until there is no additional increase in respiration.
Wash the injection syringe three times in 70%ethanol. Add two microliters of 150-micromolar rotenone and wait for the respiration to stabilize. Add another one microliter of rotenone to ensure there is no further inhibition.
If there is a decrease in respiration, continue with additional injections until there is no decrease in respiration, then wash the injection syringe three times in 70%ethanol. Add two microliters of 125-micromolar antimycin A and wait for the respiration to stabilize. Add another one microliter of antimycin A to ensure there is no further inhibition.
If there is a decrease in respiration, continue with additional injections until there is no decrease in respiration. Wash the syringe three times with 70%ethanol. Check the oxygen concentration of the chamber.
If the concentration is below 125-micromolar, reoxygenate to room air, or mildly hyperoxygenate to ensure that oxygen does not limit the respiratory flux. Add five microliters of 0.8-molar ascorbate. Wash the syringe three times in 70%ethanol.
Immediately add 10 microliters of 0.2-molar TMPD, and wait for an increase in respiration. After washing the syringe with ethanol, add 25 microliters of 4-molar sodium azide immediately when the respiratory flux of ascorbate TMPD plateaus. End the by clicking on File, Save and disconnect.
It was observed that 40 milligrams per milliliter of tissue homogenate resulted in rapid oxygen depletion. The oxygen consumption slowed substantially with 30 and 20 milligrams per milliliter of tissue homogenate, but still decreased rapidly in a short time in the absence of substrates or ADP. The 10 milligrams per milliliter concentration of tissue homogenate resulted in the optimum oxygen consumption rate.
A SUIT protocol was used to evaluate NADH-and succinate-linked oxidative phosphorylation and electron transfer, as well as Complex IV activity. Cytochrome C release was assessed in the presence of pyruvate and malate, or succinate and rotenone, both of which revealed that the homogenate preparation did not damage the mitochondria. It was also found that NADH-linked oxidative phosphorylation was negligible in EO771 tumors.
Titration of FCCP to drive maximal electron flow revealed that in EO771 tumors, phosphorylation, rather than oxidation, was limiting to respiration. The tumor homogenate respiratory profiles were similar to those of non-implanted, digitonin-permeabilized EO771 cells, except for diminished maximal electron transfer supported by N-and S-linked substrates in the tumor. The respiratory kinetics of succinate were further evaluated by stepwise titrations of subsaturating ADP until the maximal rate was achieved.
The half-maximal concentration of ADP in the presence of succinate and rotenone was 37.5-micromolar, whereas the maximal rate was approximately 10.5 picomoles per second, per milligram. Instruments set up in routine care, as well as tissue handling, are of critical importance for the success of these experiments. Following these procedures, other mass-or marker-specific approaches to rate normalization can be applied based upon the question of interest.
Common measures include total protein quantification and enzymatic activity of citrate synthase, but vary based upon the model system, statistical design, and research question.
