The overall goal of this procedure is to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This method can help answer key questions in the supportive cancer care field such as whether mitochondrial dysfunction is associated with fatigue in patients with cancer after receiving radiation therapy. The main advantage of this technique is that it is less invasive and can be easily used in the clinic to identify patients at risk for toxicities from cancer therapy.
This technique not only advances our understanding of the role of mitochondrial dysfunction in cancer related fatigue bit it can also improve assessment and can optimize its management. Though this method can provide insight into the underlying mechanisms of cancer related fatigue it can also be applied to other fatiguing disease conditions such as metabolic and uri inflammatory conditions. Generally it can be challenging for individuals new to this method to inject respiratory inhibitors into each port of the sensor cartridge properly without prior visual demonstration.
Demonstrating this procedure will be Mr.Quang Nguyen a postbac fellow from our lab. To hydrate the sensor cartridge first remove it from the package. Then add 200 microliters of calibrant solution to each well of the utility plate.
Also fill each moat with 400 microliters of calibrant solution. Next return the sensor plate to the utility plate and start hydrating the cartridge in a non carbon dioxide incubator at 37 degrees Celsius overnight. Measure the level of fatigue based on the 13-item functional assessment of chronic illness therapy.
Fatigue at baseline, midpoint, at completion of EBRT and one year post EBRT. First prepare the assay medium for the mitochondrial function measurement. After preparing the medium warm it up to 37 degrees Celsius and adjust the pH to 7.4.
It is important to ensure that the assay media is prepared on the day of the experiment and is warmed up to 37 degrees before adjusting the PH to 7.4. Next seed the PBMCs in each well to achieve 80 to 90 percent confluency. Then spin the plate at 200 times g for 2 minutes for the cells to adhere to the bottom of the wells.
After the spin remove the media from each well and rinse the wells once with assay media. Add 180 microliters of assay medium in each well including the background wells. Then incubate the plate at 37 degrees Celsius in a non carbon dioxide incubator for 45 to 60 minutes.
To reconstitute the drugs add 252 microliters of assay medium to the drug to generate a 50 micromolar stock solution of oligomycin. Add 288 microliters of assay medium to the drug to generate a 50 micromolar stock solution of FCCP. Finally add 216 microliters of assay medium to generate a 25 micromolar stock solution of antimycin a/rotenone.
After reconstituting all three drugs vortex the drug suspension for one minute and then dilute the working solutions. Then pipette 20 microliters of 10 micromolar oligomycin in port A to a final concentration of one micromolar in each well. Next pipette 22 microliters of 10 micromolar FCCP in port B to a final concentration of one micromolar in each well.
Finally pipette 25 microliters of 5 micromolar antimycin a/rotenone in port C to a final concentration of 0.5 micromolar in each well. Then select the Mito Stress Test on the extracellular flux instrument. Next insert the sensor cartridge following the instrument prompt.
The instrumental will automatically perform sensor calibration. Once the calibration is done insert the cell plate for the instrument to finish the rest of the assay. To prepare the cell proliferation assay solution add 5.76 microliters of nucleic acid stain and 28.8 microliters of background suppressor to 1.405 milliliters of phosphate buffered saline.
After the Mito Stress Test is over add 180 microliters of 2x working solution directly in the medium in each well. Then incubate the cells with the cell proliferation assay solution at 37 degrees Celsius for 45 to 60 minutes. Quantify the number of live cells on a plate reader at 508 nanometers excitation and 527 nanometers emission followed by normalizing the OCR data.
On performing FCCP titration one micromolar is the optimal drug concentration that is used to assess the PBMC mitochondrial function. Investigation of mitochondrial respiration and freshly isolated PBMCs shows that OCR starts to decrease at three hours and continues until eight hours. However the maximal respiration elicited by FCCP injection also does not exceed the basal OCR level.
Here normalization of the mitochondrial function where the cells are isolated from two healthy individuals are shown. In fact the OCR normalized to live cells represented by the green fluorescence DNA binding dye is similar in both the healthy individuals. In fact no significant difference in the basal OCR is observed between a healthy and a fatigued subject.
However the fatigued subject shows decreased maximal oxygen consumption and spare respiratory capacity in comparison to the control. No differences are found in non mitochondrial and ATP related oxygen consumption or coupling efficiency between the healthy and the fatigued subjects. Once mastered this technique can be completed in four hours if it is performed properly.
While attempting this procedure it's important to remember to first determine the optimal cell density and FCCP concentrations. Following this procedure other methods like isolating different cell populations can be performed in order to guide future mechanistic investigations focused on specific cell types. After its development this technique paved the way for researchers in the field of supportive care to explore the role of mitochondrial dysfunction in fatigued cancer patients.
After watching this video you should have a good understanding of how to measure mitochondrial function and clinical samples that can also be used to proactively identify patients at risk for developing cancer treatment related toxicities including fatigue. Don't forget that working with respiratory inhibitors such as oligomycin and rotenone can be extremely hazardous. Precautions such as wearing personal protection equipment should always be taken while performing this procedure.
