This study is fully supported by the Division of Intramural Research of the National Institute of Nursing Research of the NIH, Bethesda, Maryland.

The current study (NCT00852111) was approved by the Institutional Review Board (IRB) of the National Institutes of Health (NIH), Bethesda, Maryland. Participants enrolled in this study were euthymic men, 18 years of age or older, who were diagnosed with non-metastatic prostate cancer with or without prior prostatectomy and were scheduled to receive external beam radiation therapy (EBRT). Potential participants were excluded if they had a progressive disease that could cause significant fatigue, had psychiatric disease wi...

<ol>
	<li>Berger, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-Related+Fatigue,+Version+2.2015.">Cancer-Related Fatigue, Version 2.2015.</a> Journal of the National Comprehensive Cancer Network. 13 (8), 1012-1039 (2015).</li><li>Feng, L. R., Dickinson, K., Kline, N., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+phenotyping+approaches+lead+to+dissimilar+biologic+profiles+in+men+with+chronic+fatigue+following+radiation+therapy.">Different phenotyping approaches lead to dissimilar biologic profiles in men with chronic fatigue following radiation therapy.</a> Journal of Pain and Symptom Management. 52 (6), 832-840 (2016).</li><li>Feng, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Predictors+of+Fatigue+in+Men+With+Non-Metastatic+Prostate+Cancer+Receiving+External+Beam+Radiation+Therapy.">Clinical Predictors of Fatigue in Men With Non-Metastatic Prostate Cancer Receiving External Beam Radiation Therapy.</a> Clinical Journal of Oncology Nursing. 19 (6), 744-750 (2015).</li><li>Feng, L. R., Wolff, B. S., Lukkahatai, N., Espina, A., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploratory+Investigation+of+Early+Biomarkers+for+Chronic+Fatigue+in+Prostate+Cancer+Patients+Following+Radiation+Therapy.">Exploratory Investigation of Early Biomarkers for Chronic Fatigue in Prostate Cancer Patients Following Radiation Therapy.</a> Cancer Nursing. 40 (3), 184-193 (2017).</li><li>Myhill, S., Booth, N. E., McLaren-Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+fatigue+syndrome+and+mitochondrial+dysfunction.">Chronic fatigue syndrome and mitochondrial dysfunction.</a> International Journal of Clinical and Experimental Medicine. 2 (1), 1-16 (2009).</li><li>Pernas, L., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mito-Morphosis:+Mitochondrial+Fusion,+Fission,+and+Cristae+Remodeling+as+Key+Mediators+of+Cellular+Function.">Mito-Morphosis: Mitochondrial Fusion, Fission, and Cristae Remodeling as Key Mediators of Cellular Function.</a> Annual Review of Physiology. 78 (1), 505-531 (2016).</li><li>Vecchiet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+characterization+of+somatic+parietal+tissues+in+humans+with+chronic+fatigue+syndrome.">Sensory characterization of somatic parietal tissues in humans with chronic fatigue syndrome.</a> Neuroscience Letters. 208 (2), 117-120 (1996).</li><li>Jones, D. E. J., Hollingsworth, K. G., Taylor, R., Blamire, A. M., Newton, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+in+pH+handling+by+peripheral+muscle+and+potential+regulation+by+the+autonomic+nervous+system+in+chronic+fatigue+syndrome.">Abnormalities in pH handling by peripheral muscle and potential regulation by the autonomic nervous system in chronic fatigue syndrome.</a> Journal of Internal Medicine. 267 (4), 394-401 (2010).</li><li>Feng, L. R., Suy, S., Collins, S. P., Saligan, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+TRAIL+in+fatigue+induced+by+repeated+stress+from+radiotherapy.">The role of TRAIL in fatigue induced by repeated stress from radiotherapy.</a> Journal of Psychiatric Research. 91, 130-138 (2017).</li><li>Karamercan, M. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+mitochondrial+alterations+after+cardiac+surgery+involving+cardiopulmonary+bypass:+Clinical+correlations.">Leukocyte mitochondrial alterations after cardiac surgery involving cardiopulmonary bypass: Clinical correlations.</a> Shock. 21 (4), 315-319 (2004).</li><li>Straub, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+and+immune+system+prompt+energy+shortage+in+chronic+inflammation+and+ageing.">The brain and immune system prompt energy shortage in chronic inflammation and ageing.</a> Nature Reviews Rheumatology. 13, 743 (2017).</li><li>Kuhnke, A., Burmester, G., Krauss, S., Buttgereit, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetics+of+immune+cells+to+assess+rheumatic+disease+activity+and+efficacy+of+glucocorticoid+treatment.">Bioenergetics of immune cells to assess rheumatic disease activity and efficacy of glucocorticoid treatment.</a> Annals of the Rheumatic Diseases. 62 (2), 133-139 (2003).</li><li>Kramer, P. A., Ravi, S., Chacko, B., Johnson, M. S., Darley-Usmar, V. 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Fatigue in cancer patients is a debilitating condition that is not well defined or characterized1. Diagnosis of fatigue entirely relies on subjective reporting and there is no current diagnostic standard or treatment for this condition, largely due to a lack of understanding in its pathobiology2. Of the proposed mechanisms underlying fatigue in cancer patients, impairment in mitochondrial function is one of the most therapeutically targetable pathways. Therefore, we develop...

Fatigue is a prevalent and distressing condition that has a negative impact on the quality of life of cancer patients1. To this date, cancer fatigue remains poorly defined and relies only on subjective reporting by patients2. Therefore, there is an urgent need to identify an easily adaptable diagnostic laboratory test to objectively characterize fatigue in the clinical setting3,4.
Multiple underlying mechanisms, including mitochondria dysfunction, have been proposed to cause fatigue5. Mitochondria are the powerhouse organelles, providing 95% of cellular energy needs via oxidative phosphorylation, and play an important role in calcium signaling, apoptosis, immune signaling, and regulation of other intracellular signaling events6. Accordingly, impaired mitochondrial bioenergetics and defects in energy production may contribute to fatigue. Supporting this hypothesis, previous studies have observed mutations in mitochondrial DNA in patients with chronic fatigue syndrome7. While it remains unclear whether the pathophysiological origin of fatigue lies within the central nervous system or peripheral tissues, such as skeletal muscles8,9, there is currently no direct method to accurately assess mitochondrial dysfunction related to fatigue in live, respiring cells.
Using peripheral blood mononuclear cells (PBMCs) to study mitochondrial function offers several advantages. First, PBMCs are readily available in the clinical setting using standard phlebotomy and can be isolated quickly using basic laboratory techniques. Second, blood collection is less invasive than collecting other tissues such as a muscle biopsy. Thus, blood samples can be collected from the same patient repeatedly over time, which facilitates longitudinal assessment of treatment effects. Interestingly, mitochondrial function in PBMCs appeared to be well correlated with kidney mitochondrial status in an animal model10. Furthermore, immune cell mitochondria have been used as a proxy for detecting systemic changes under different disease conditions11,12. Mitochondria in circulating immune cells are particularly sensitive to changes in immune functions and immune signaling molecules such as cytokines13,14,15. For example, it has been observed that PBMCs from patients with acute rheumatic inflammatory diseases exhibit high baseline oxygen consumption14. In contrast, oxygen consumption was reduced in PBMCs isolated from patients with systemic inflammatory conditions including sepsis16. Under inflammatory conditions, free radicals produced by dysfunctional mitochondria may further contribute to elevated oxidative stress and prolonged inflammation17. The central role of mitochondria in energy production as well as in oxidative stress suggests the potential utility of using mitochondrial function as a proxy for studying fatigue in cancer patients 13.
Previous studies examining mitochondrial function utilized biochemical techniques, mitochondrial membrane potential measurement, or isolation of specific cell populations that may not be readily adaptable in the clinical setting5,14,18. In recent years, the development of extracellular flux assays has allowed researchers to easily and accurately examine changes in oxygen consumption rate (OCR) in response to automated injections of respiratory inhibitors19,20,21,22. However, most of these studies are designed for specific cell types and the large high-throughput format may not be applicable in a clinical setting. In this manuscript, we describe an optimized protocol for examining mitochondrial function for clinical use.

<table border="0" cellpadding="0" cellspacing="0" style="width:876px;" width="875">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:331px;">CPT Mononuclear Cells Preparation Tube&#160;</td>
			<td style="width:123px;">BD Biosciences</td>
			<td style="width:165px;">362761</td>
			<td style="width:257px;">For isolating PBMCs following phlebotomy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RPMI-1640&#160;</td>
			<td>Corning</td>
			<td>10-040</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>For making growth media for PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell-Tak</td>
			<td>Corning</td>
			<td>354240</td>
			<td>Cell and Tissue adhesive solution; allows suspension cells to adhere to the surface</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XF Calibrant Solution</td>
			<td>Agilent</td>
			<td>103059-000</td>
			<td>For hydrating cartridges</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XFp Fluxpak (miniplates and sensor cartridges)</td>
			<td>Agilent</td>
			<td>103022-100</td>
			<td>Contains XFp cell culture miniplates and sensor cartridges</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">XF base media</td>
			<td>Agilent</td>
			<td>103335-100</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">45% cell culture D-(+)-Glucose solution</td>
			<td>Corning</td>
			<td>25-037-CI</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium pyruvate solution</td>
			<td>Corning&#160;</td>
			<td>25-000-CI</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">L-glutamine solution</td>
			<td>ThermoFisher</td>
			<td>25030081</td>
			<td>For making XF assay media</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XFp Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103010-100</td>
			<td>Contains oligomycin, FCCP, antimycin A/rotenone</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CyQUANT Direct Cell Proliferation Assay</td>
			<td>ThermoFisher</td>
			<td>C35011</td>
			<td>For quantification of live cells and data normalization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Seahorse XFp Analyzer</td>
			<td>Agilent</td>
			<td>S7802AEA</td>
			<td>For measuring mitochondrial function in live cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cytation 5 Cell Imaging Multi-Mode Reader (or any instrument that can quantify fluorescent cells in a plate)</td>
			<td>BioTek</td>
			<td>BTCYT5PV</td>
			<td>For quantification of live cells and data normalization</td>
		</tr>
	</tbody>
</table>

evaluating the role of mitochondrial function in cancer-related fatigue

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to objectively measure the severity of this condition. Here we describe an optimized method for assessing mitochondrial function of PBMCs collected from fatigued cancer patients. Using a compact extracellular flux system and sequential injection of respiratory inhibitors, we examined PBMC mitochondrial functional status by measuring basal mitochondrial respiration, spare respiratory capacity, and energy phenotype, which describes the preferred energy pathway to respond to stress. Fresh PBMCs are readily available in the clinical setting using standard phlebotomy. The entire assay described in this protocol can be completed in less than 4 hours without the involvement of complex biochemical techniques. Additionally, we describe a normalization method that is necessary for obtaining reproducible data. The simple procedure and normalization methods presented allow for repeated sample collection from the same patient and generation of reproducible data that can be compared between time points to evaluate potential treatment effects.

The Mito Stress Test relies on measuring oxygen consumption rate (OCR) after sequential injection of various respiratory inhibitors to map a complete mitochondrial profile. OCR measurements after each drug injection can be used to calculate the following parameters related to mitochondrial health: Basal OCR is first measured before any drug injection to assess oxygen consumption needed to meet resting level ATP demand. Basal respiration is calculated by subtracting non-mi...

Watch this Scientific Journal Video about Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue at JoVE.com

Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Our goal was to develop a practical protocol to evaluate mitochondrial dysfunction associated with fatigue in cancer patients. This innovative protocol is optimized for clinical use involving only standard phlebotomy and basic laboratory procedures.

Fatigue is a common and debilitating condition that affects most cancer patients. To date, fatigue remains poorly characterized with no diagnostic test to ...

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.

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JoVE Journal

Cancer Research

9.0K visualizações.  National Institutes of Health. 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.