Evaluating the Role of Mitochondrial Function in Cancer-related Fatigue

Summary

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.

Abstract

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.

Introduction

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 setting^3,4.

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 muscles^8,9, 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 conditions^11,12. Mitochondria in circulating immune cells are particularly sensitive to changes in immune functions and immune signaling molecules such as cytokines^13,14,15. 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 setting^5,14,18. 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 inhibitors^19,20,21,22. 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.

Protocol

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 within the past five years, had uncorrected hypothyroidism or anemia, or had a second malignancy. Individuals who used sedatives, steroids, or non-steroidal anti-inflammatory agents were also excluded. Healthy control blood samples were obtained at the NIH Department of Transfusion medicine from healthy donors under an IRB-approved protocol (NCT00001846). All participants are recruited at the Magnuson Clinical Research Center at the NIH. Signed written informed consents were obtained prior to study participation.

1. Mitochondrial Function Measurement Preparation (Day 1 of the Experiment)

1. Hydrate Sensor Cartridge (see Table of Materials; approximate duration: 5 min).
   1. Remove Sensor Cartridge from package. Add 200 μL Calibrant solution into each well of the utility plate, then fill each moat with 400 μL Calibrant solution.
   2. Return Sensor plate to the utility plate, which now has Calibrant solution in it. Hydrate the Cartridge in a non-CO₂ 37 °C incubator overnight.
      NOTE: Sensor Cartridge needs to be hydrated for a minimum of 4 h and a maximum of 72 h.

2. Clinical Sample Preparation (Day 2 of the Experiment)

1. Measure fatigue using the 13-item Functional Assessment of Chronic Illness Therapy - Fatigue (FACIT-F)^2,23,24 at baseline (prior to EBRT initiation), midpoint and completion of EBRT, and 1-year post-EBRT.
   1. Use a 0 - 4 scale for each item response, where a 0 represents "not at all" and a 4 indicates that the respondent relates to the corresponding statement "very much." Total scores should range from 16 - 53, with lower scores reflecting high fatigue intensity.
   2. Define fatigue as a FACIT-F score lower than 43, with a FACIT-F score of ≥43 indicating absence of or not clinically-meaningful fatigue².
      NOTE: A FACIT-F score of 43 best divides fatigue scores of cancer patients and the general population².
   3. Include the following 13 items in the FACIT Fatigue Scale: 1) I feel fatigued; 2) I feel weak all over; 3) I feel listless ("washed out"); 4) I feel tired; 5) I have trouble starting things because I'm tired; 6) I have trouble finishing things because I am tired; 7) I have energy; 8) I am able to do my usual activities; 9) I need to sleep during the day; 10) I am too tired to eat; 11) I need help doing my usual activities; 12) I am frustrated by being too tired to do the things I want to do; and 13) I have to limit my social activity because I am tired.
2. Isolate PBMC from freshly collected blood samples (approximate duration: 1 h).
   1. Collect 8 mL of blood into a Mononuclear Cells Preparation Tube (see Table of Materials).
      NOTE: Blood samples should be processed within 2 h of collection. The quality of PBMCs may be compromised if blood samples are processed more than 2 h after sample collection.
   2. Centrifuge at 1,750 x g for 30 min at room temperature (18 - 25 °C). Transfer the cloudy layer to a 15 mL conical tube. Add up to 15 mL of PBS and invert 5 times.
   3. Centrifuge at 300 x g for 15 minutes at 4 °C. Carefully remove and discard the supernatant without disturbing pellet. Re-suspend the pellet by adding up to 10 mL PBS, and invert 5 times.
   4. Centrifuge at 300 x g for 10 min at 4 °C. Discard liquid supernatant and re-suspend cells in 1 mL PBS. Transfer the PBMCs to a 1.5 mL microfuge tube.
   5. Centrifuge at 610 x g for 10 minutes. Carefully remove supernatant and resuspend pellet in complete RPMI-1640 (RPMI-1640 supplemented with 10% FBS, 10 mM Penicillin/Streptomycin).
3. Coat cell plates for non-adherent cells (approximate duration: 30 min).
   1. Prepare cell and tissue adhesive solution (see Table of Materials) by diluting the stock solution in 0.1 M sodium bicarbonate pH 8.0. The working solution should be at 3.5 μg/cm² of surface area.
      NOTE: This solution contains polyphenolic proteins extracted from the marine mussel, Mytilus edulis. These proteins are key components of the glue secreted by the mussel to anchor itself to surfaces. We find that Cell-Tak works the best with PBMCs.
   2. Add 100 μL of the diluted adhesive solution to each well and incubate for at least 20 min at room temperature. Wash three times with DI water and air dry.

3. Mitochondrial Function Measurement

1. Preparation of Assay Media (approximate duration: 10 min).
   NOTE: Assay media must be freshly prepared on the day of the experiment.
   1. Add L-glutamine, pyruvate, and glucose to base media (the same constituents as Dulbecco's Modified Eagle's Medium (DMEM), but without any sodium bicarbonate, glucose, glutamine, or sodium pyruvate) to make assay media. Warm media up to 37 °C, then adjust pH to 7.4.
      NOTE: Concentrations of L-glutamine, pyruvate, and glucose are usually the same as concentrations in normal growth media, but can be adjusted based on the assay.
2. Prepare PBMCs for the Mito Stress test (approximate duration: 2 h).
   1. Plate enough PBMCs from Step 2 into each well to reach 80 - 90% confluency. In our experience, 1.5 x 10⁵ cells/well yielded the most consistent results.
      NOTE: Wells A and H are background wells and should only contain assay media with no cells. In wells B to G, we recommend plating at least 3 - 6 wells per patient sample in order to account for outliers.
   2. Spin plates down at 200 x g for 2 min to allow cells to adhere to the bottom of the wells. Wash the cells once with Assay media. This step removes serum and sodium bicarbonate from the growth media.
      NOTE: In our experience, the washing step typically removes 20 - 30% of cells.
   3. Add 180 μL Assay media into each well, including background wells A and H. Incubate in a non-CO₂ 37 °C incubator for 45 - 60 min.
3. Running the Mito Stress Test
   1. Reconstitute drugs in the Mito Stress Kit with assay media as follows:
      1. Oligomycin: Add 252 μL assay media into the vial to generate a stock solution at 50 μM.
      2. FCCP: Add 288 μL of assay media into the vial to generate a stock solution at 50 μM.
      3. Antimycin A/Rotenone: Add 216 μL of assay media into the vial to generate a stock solution at 25 μM.
   2. Vortex reconstituted drugs for approximately 1 minute. Dilute into working solutions.
      NOTE: Concentrations of the working solutions should be titrated and pre-determined prior to the experiment depending on the cell type. For PBMCs, we find that 1 μM of Oligomycin, 1 μM FCCP, and 0.5 μM antimycin A/Rotenone work the best.
   3. Pipette the drugs into each port in the sensor cartridge sequentially:
      1. Port A: Pipette 20 μL of 10 μM Oligomycin, for a final concentration in each well of 1 μM.
      2. Port B: Pipette 22 μL of 10 μM FCCP, for a final concentration in each well of 1 μM.
      3. Port C: Pipette 25 μL of 5 μM antimycin A/rotenone for a final concentration in each well of 0.5 μM.
4. Select "Mito Stress Test" on an extracellular flux instrument. Follow instrument prompt and insert the sensor cartridge. The instrument will automatically perform sensor calibration. Insert cell plate after sensor calibration, and the instrument will finish the rest of the assay. Oxygen dynamics is measured at 530 nm (excitation)/650nm (emission), and proton concentration is measured at 470 nm (excitation)/ 530 nm (emission).

4. Normalization of Mitochondrial Function Data

1. Prepare Cell Proliferation Assay solution (approximate duration: 5 min).
   1. Add 48 μL nucleic acid stain (500x) and 240 μL background suppressor to 11.7 mL PBS (2x). The nucleic acid stain is a cell-permeant DNA-binding dye, and the background suppressor is a masking dye which blocks dead cells or cells with compromised cell membrane integrity from being stained. The combination of the DNA-binding dye and the background suppressor ensures that only live cells are stained.
   2. Add equal volume of 2x working solution (180 μL) directly into the medium in each well after the Mito Stress Test has completed.
2. Incubate the cells in the presence of cell proliferation assay solution in a 37 °C incubator for 45 - 60 min. Quantify the number of live cells (fluorescent) on a plate reader at 508 nm (excitation)/527 nm (emission) and normalize OCR data (approximate duration: 10 min).
   NOTE: in addition to the number live cells, users may also choose to normalize their data with the total number of cells, amount of nucleic acid, protein concentrations, etc.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CPT Mononuclear Cells Preparation Tube	BD Biosciences	362761	For isolating PBMCs following phlebotomy
RPMI-1640	Corning	10-040	For making growth media for PBMCs
Fetal bovine serum (FBS)	Corning	35-010-CV	For making growth media for PBMCs
Penicillin/Streptomycin	ThermoFisher	15140122	For making growth media for PBMCs
Cell-Tak	Corning	354240	Cell and Tissue adhesive solution; allows suspension cells to adhere to the surface
Seahorse XF Calibrant Solution	Agilent	103059-000	For hydrating cartridges
XFp Fluxpak (miniplates and sensor cartridges)	Agilent	103022-100	Contains XFp cell culture miniplates and sensor cartridges
XF base media	Agilent	103335-100	For making XF assay media
45% cell culture D-(+)-Glucose solution	Corning	25-037-CI	For making XF assay media
Sodium pyruvate solution	Corning	25-000-CI	For making XF assay media
L-glutamine solution	ThermoFisher	25030081	For making XF assay media
Seahorse XFp Mito Stress Test Kit	Agilent	103010-100	Contains oligomycin, FCCP, antimycin A/rotenone
CyQUANT Direct Cell Proliferation Assay	ThermoFisher	C35011	For quantification of live cells and data normalization
Seahorse XFp Analyzer	Agilent	S7802AEA	For measuring mitochondrial function in live cells
Cytation 5 Cell Imaging Multi-Mode Reader (or any instrument that can quantify fluorescent cells in a plate)	BioTek	BTCYT5PV	For quantification of live cells and data normalization