Near Infrared Spectroscopy (NIRS) as a Method for Measuring Oxidative Capacity of Skeletal Muscle Mitochondria in Breast Cancer and All Gynecological Cancer Patients
Conditions: Breast Cancer, Gynecologic Cancers, Mitochondrial Function
Sponsor: University of South Carolina

Near Infrared Spectroscopy (NIRS) as a Method for 
Measuring Oxidative Capacity of Skeletal Muscle 
Mitochondria in Breast Cancer and all Gynecological 
Cancer Patients Protocol 
 
6/24/22 
 

Participants.  Participants recruited for this study will be breast cancer or gynecological cancer 
patients (N=40) who agree to participate in an exercise study examining the effects of 
chemotherapy on muscle mitochondrial oxidative capacity, a measure of skeletal muscle health.  
A written informed consent will be obtained from the participants before data collection.  This 
study has been submitted for review by the Institutional Review Board at the Prisma Office of 
Research Compliance and Administration (ORCA).  We pledge to abide by all applicable 
institutional and governmental regulations regarding the ethical use of human volunteers for the 
duration of this research. 
This study focuses on breast cancer or gynecological cancer patients who will undergo a 
standard of care chemotherapy treatment as directed by their oncologist.  
Other eligibility requirements for this study are as follows:  
• 
Patients diagnosed with breast cancer or gynecological cancer without distance 
metastasis 
• 
Ages > 20 years old  
• 
Able to perform exercise on a stationary cycle ergometer at moderate intensities for a 
maximum of 15 minutes 
• 
Hemoglobin values greater than 10 at baseline 
• 
ALT and AST values less than 2.5X the upper limit of normal by institutional standards 
• 
Godin-Shepard Leisuretime Physical Activity Questionnaire (GLTEQ) score of 14 or 
greater will be included. A score of less than 14 is considered insufficiency 
active/sedentary. 
The exclusion criteria include patients with:  
• 
Clinically advanced cardiovascular disease 
• 
Clinically advanced pulmonary disease 
• 
Disease requiring continuous oxygen supplementation 
• 
Greater than 2 centimeters or more of subcutaneous adipose tissue on the anterior thigh 
• 
Inability to walk or stand 
• 
Movement disorders 
• 
Spinal cord injuries 
• 
Autoimmune disorders 
• 
Pregnant or breastfeeding 
• 
Mini-Mental State Examination (MMSE) 
Participants for this study will be recruited through informative pamphlets at the Center for 
Integrative Oncology Survivorship, Prisma Health System’s Cancer Institute at Greenville 
Memorial Hospital and affiliated physician offices.  
Experimental Design. We will use an observational, 2-way, treatment x time, within-subjects 
research design to compare skeletal muscle oxidative capacity of the vastus lateralis muscle, 
during stationary cycling, across breast cancer or gynecological cancer patients at baseline and 
across their chemotherapy regimen. Muscle mitochondrial oxidative capacity of the Vastus 

Lateralis muscle will be non-invasively evaluated using continuous wave, Near Infrared 
Resonance Spectroscopy (NIRS, PortaMon by Artinis Medical Systems BV in The Netherlands).  
Study protocol. The NIRS participant testing will be performed in the University of South 
Carolina School of Medicine Greenville’s Human Performance Laboratory located within Cancer 
Institute Faris Road facility. Testing will take approximately 30 minutes per session, with a 
maximum of 180 minutes of participation per patient. The independent variables in this study 
are drug regimen and number of chemotherapy treatments. The dependent variables in this 
study will be the change in oxygenation and deoxygenation status of the vastus lateralis as 
measured by the NIRS device, indicative of mitochondrial oxidative capacity, after a bout of 
cycling. Patients will be asked to avoid exercise and use of certain substances (tobacco, 
alcohol, caffeine, and contraindicated medications) 24 hours prior to testing. 
 
The NIRS device consists of three LED transmitters that transmit light waves into muscle tissue 
and one high sensitivity PIN diode receiver, equipped with ambient light protection, which 
assesses light received from the transmitters.  The three transmitters each emit light at two 
wavelengths. These wavelengths correspond to the absorption wavelengths of oxyhemoglobin 
(O2Hb) and deoxyhemoglobin in the skeletal muscle tissue (approximately 760 nm and 850 
nm). These wavelengths of light will be received from three separate transmitter distances 
(30mm, 35mm, and 40mm) at 10 Hz.  The ΔO2Hb and Δ tissue saturation index (TSI%) will be 
calculated as the change from the last 30-s average of the work interval to the last 30-s average 
of the recovery interval. Oxygen uptake (VO2) will be obtained by using open-circuit spirometry 
(Parvo Medics, Inc, Salt Lake City, Utah). Cycling power will be determined via lactate threshold 
curves generated for each participant.  
Participants will be asked to perform exercise in shorts or equivalent. Participants will first sit at 
rest in a chair, as the NIRS device is applied to the vastus lateralis. The device will have double 
sided adhesive tape to lightly secure its position to the skin. The device will be wrapped in a 
black cloth to fully secure its position, limit device mobility, and block ambient light in the 
laboratory. Participants will then stand and walk to the cycle with the NIRS secured to the 
participant’s leg. Participants will sit on the bike for a 2-minute rest while wearing a fitted mask 
that comfortably measures the participant’s rate and amount of oxygen uptake (VO2) and other 
ventilatory measurements at rest and during exercise.   
 
Visit #1. Researchers will determine the participant’s lactate threshold curve and ventilatory 
threshold during a graded exercise test (Table 1). The lactate threshold is a measure of the rate 
at which capillary blood lactate measurements rise as an effect of increased exercise intensity. 
The blood lactate curve will be used to determine an individualized power wattage for 
subsequent exercise testing of “on-off kinetics” (see below description). Capillary blood lactate 
levels will be obtained by finger stick for point of care testing. Ventilatory threshold will be 
measured using indirect calorimetry via a metabolic cart that captures expired gasses (O2 and 
CO2) through a comfortable silicone mask worn by the participant.  
 
A baseline blood lactate level will be obtained at rest, then the participant will be instructed to 
cycle for three minutes beginning at 10W. After the 3-minute stage is complete, the power 

output will increase by 15 watts. The participant will cycle for 3 minutes at each stage until their 
lactate threshold reaches 4.0 mmol/L. Once the blood lactate reaches 4.0 mmol/L, one final, 
three-minute stage will be performed. A final lactate reading will be obtained at the completion 
of this exercise stage. The power output that is equivalent to 1.1x the wattage at which their 
lactate threshold exceeded 4.0 mmol/L will be used as the exercise intensity (“on-kinetics”) for 
subsequent visits accompanying chemotherapy treatments. 
 
Subsequent Visits. Participants will return for each subsequent visit within a week before their 
next chemotherapy infusion as long as they are able to safely continue the exercise protocol 
(Table 2). The NIRS device will be secured to the participant’s leg similar to the first visit and the 
participants will put on the mask to measure ventilation. Participants will begin pedaling for a 
warmup at 0 watts for 2 minutes. After the two-minute warm up period is completed, the power 
output will increase to the previously calculated wattage of 1.1x LT. The participant will cycle at 
a self-selected cadence at this power wattage for a duration of 2 minutes (“on-kinetics”). At the 
end of each on-kinetics stage, participants will be asked to rate their level of effort during 
exercise, Rate of Perceived Exertion (RPE). The participant will then stop pedaling and will 
remain in recovery, sitting on the bike for 2 minutes (“off-kinetics” stage). With 5 seconds 
remaining in the “off-kinetics” stage, the participant will start pedaling at the same self-selected 
cadence for their next “on-kinetics” stage, followed by another 2-min “off-kinetics” period. This 
cycle of exercise followed by rest, known as on-off kinetics, will repeat for a total maximum of 3 
times, or until the participant reaches volitional fatigue. 
 
Collection of surveys and biometrics at each visit. We will be administering MoCA, BFI, Physical 
Activity Intake, PROMIS Global Health, and GLTPAQ surveys prior to participant engagement in 
cycling exercises.  We will also be administering BFI, PROMIS Global Health, and Physical 
Activity Follow-up surveys after each individual study session to assess changes in cancer 
related fatigue associated with chemotherapy treatment that may develop throughout the study 
time period.  
 
Patients will undergo routine, standard of care blood draws for a CBC and metabolic testing per 
their managing physician with an additional tube, during the same venipuncture, for a study 
covered aliquot for biomarkers related to mitochondrial health for correlative research. 
 
For biomarker analysis, exosomes from patient serum (approximately 250 uL samples) will be 
isolated using an ExoQuick ULTRA EV Isolation System (System Biosciences). Exosomes will 
then be detected with Western blotting with exosome specific antibodies. Exosome samples will 
be sent for RNASeq analysis of RNA within the isolated exosomes at System Biosciences. 
Bioinformatics analysis of the RNAs identified from the RNASeq will then be used to determine 
if there are specific RNAs present in patient blood associated with changes in mitochondrial 
function. In addition, selected mRNAs associated with mitochondrial function will be assessed 
(expression levels determined) with RT-qPCR using aliquots of the isolated serum exosomes. 
  
 

Statistical Analysis. ANOVA one way with tukey post-hoc analysis to detect differences 
between drug regimen groups at each time point (baseline, all subsequent chemotherapy 
treatments) for time constant response and change in mitochondrial function for mitochondrial 
oxidative capacity. One-way ANOVAs will be used to additionally detect differences between 
different timepoints from baseline to final chemotherapy treatment for each individual 
chemotherapy regimen.  
 
Table 1. 
  
 
 
 
 
 
 
 
 
 
Table 2.