Subjects for this study included BC survivors and HC’s. Twenty-four BC survivors (at least 30 days but no more than 1 year since final treatment received) and fifteen HC completed testing. Thirteen BC survivors and thirteen HC were age-matched and included in the study. The BC survivors were all between 45-75 years old and had stage I-III BC. The HC were at least 42 years old with no history of cancer. The HC were sedentary prior to enrolling in the study, as defined by not exercising more than 30 minutes or more at a moderate intensity twice a week. The study was approved by the University of North Carolina at Chapel Hill’s Institutional Review Board. Additionally, participants signed informed consent prior to participating in the study.
Study Procedure
There were three testing visits at the beginning of the study. Then 16-weeks of aerobic and resistance training three
times per week for 1 hour per visit at the Get Real and Heel training facility (located at UNC Chapel Hill) under the supervision of trained exercise
specialist followed by three testing visits at the
Figure 1. Overall experimental timeline: including 3 days of pre-training exercise testing, 16 weeks of combined aerobic and resistance training, and then 3 days of post-training exercise. testing.
end of the 16-week exercise intervention. During the first visit, subjects completed a Medical History form, a Physical Activity Readiness Questionnaire, and a 12-lead electrocardiogram (ECG; which was sent to a study team physician for approval) prior to beginning of physical activity evaluations.
General vital signs were recorded using appropriate, standardized equipment and techniques in the Exercise Oncology Research Laboratory (EORL) at the University of North Carolina at Chapel Hill. Height (cm) and weight (kg) were recorded using the Health-o-Meter 402KL (Rye, NY) stadiometer and physicians scale, respectively. Blood pressure (mmHG) was recorded manually using standard sphygmomanometers and stethoscopes at the beginning of every visit. Heart rate (bpm) was recorded continuously throughout testing using a Polar (Polar Electro Inc., Lake Success, NY) chest strap and watch telemetry system during every visit.
Subjects completed a familiarization session on an electronically braked cycle ergometer. Familiarization included being appropriately adjusted in the cycle ergometer and also fitted for a mask to be used for indirect calorimetry assessment of peak oxygen uptake (VO2peak) during the cardiopulmonary exercise test (CPET). After the subject was sized to the cycle ergometer, the distance from the knob under the bike seat to the middle of the pedal was measured in centimeters with a tape measure and recorded so the same set up was used during post intervention testing.
Cardiorespiratory function was assessed using a CPET on day two of pre-training/post- training testing. The protocol used for the assessment of VO2peak included a continuous incremental ramp protocol following standard ACSM exercise testing guidelines procedures (Swain, Brawner, & Medicine, 2014). Breath by breath metabolic analysis using Parvo Medics TureMax 2400 Metabolic System (Parvo Medics, Salt Lake City, UT) was used during the CPET for the assessment of VO2peak. The CPET began with the subject sitting quietly on the cycle ergometer for
three minutes while research team collected resting metabolic data. The first stage of the CPET began with a two-minute warm up (unloaded at 0 watts), followed by a three-minute loaded warm- up phase (20 watts). After the two warm-up stages the workload (wattage) continuously increased 15 watts per minute until the termination of the test. HR and RPE (6-20) was continually monitored and recorded throughout the testing. Termination of the test was determined by subject reaching volitional exhaustion and signaling to stop the test. Other parameters to terminate the test included: VO2 plateau (VO2 decrease with an increase in exercise intensity) or an abnormal subject response to the test.
The third day of pre-training/post-training exercise testing occurred 1-7 days after day two of testing. It included a 45-minute moderate intensity exercise bout on the cycle ergometer at 60% of the peak wattage from the subject’s exercise test on day two. During the third day, the subjects was overnight fasted and was instructed to follow pre-assessment guidelines. Upon arrival to the lab subjects laid quietly for 10 minutes resting, and then a peripheral venous catheter indwelling venous catheter (BD Bioscience, Franklin Lanes, NJ, USA) was inserted into the antecubital vein of the forearm for repeat blood sampling using sterile technique by trained personnel (non-effected side for BC survivors). Approximately 50 mL of blood was drawn into blood collection tubes (inverted several times) containing EDTA (BD Bioscience, Franklin Lanes, NJ, USA), Sodium Heparin (BD Bioscience, Franklin Lanes, NJ, USA), and Serum (BD Bioscience, Franklin Lanes, NJ, USA) throughout the duration of the test day.
Immediately following each blood collection, complete blood counts (CBC) were performed in duplicate with a maximal white blood cell difference of 0.1 cells/µL for each time point using a Sysmex Hematology Analyzer (XP-300, Kobe, Japan), and was placed on ice for the duration of the visit. Blood was drawn at the baseline, 0-hour (0h) (immediate end of 45-minute
exercise session), and at 1-hour (1h) after exercise (after 1-hour of seated rest). From the blood samples, 1) plasma and serum were isolated and stored at -80 until the end of the study for inflammatory biomarkers, and 2) peripheral blood mononuclear cells (PBMC) were isolated using density gradient centrifugation via SepMateTM-50 (Stemcell, Vancouver, BC Canada) as specified by the manufacturer and then cryopreserved with fetal bovine serum (FBS) and Dimethyl
sulfoxide (DMSO).
To stimulate the immune system, a standardized intermittent exercise protocol was used. The exercise test began with a 2-minute warm-up (1-minute unloaded (0 watts) and 1-min 30% of peak wattage obtained during CPET). Participants then completed 10 rounds of 3 minutes of cycling (at 60% of peak wattage obtained during the CPET) followed by 1.5 minutes of rest. In total it amounted to 30 minutes of cycling at 60% of peak wattage obtained during the CPET. Subjects were instructed that they could stand on the bike during the rest stages. Access to water, a fan, and music of choice was allowed. This exercise session has been previously shown to be well-tolerated by both breast (Evans et al., 2015) and prostate (Hanson et al., 2018) cancer survivors and induces an immune response. At the end of the 45-minute exercise session the 0- hour blood draw occurred with the participant seated on the cycle ergometer. Once blood pressure and heart rate return to baseline the participant was transferred to a chair and was given ad libitum access to water but no additional food or drink. The indwelling venous catheter was flushed with saline after the 0-hour draw and half-way through the rest time. After resting for 1-hour the final blood draw occurred.
Body Composition
Body Composition was measured using Dual X-ray Absorptiometry (DXA) (Discovery W, Hologic, Inc., Bedford, MA). Total body weight-mass (BW) and compositional aspects of lean
body mass (LBM), fat tissue mass (FM), and percentage body fat (% BF) were examined. The BW was measured using a high-grade analytical balance-scale (accuracy ± 10 grams).
Calculation of Plasma Volume
Hematocrit and hemoglobin values were used to calculate exercise induced plasma volume shifts by the equation developed by Dill and Costill (1974). Values were obtained from the complete blood count data pre-training and post-training (baseline to 0h shift and baseline to 1h shift). Plasma volume shifts were reported to estimate exercise induced fluid shifts (leukocyte concentration effect).
Blood Analysis
Frozen PBMCs were thawed in a 37°C warm water bath. Then 1mL of room temperature RPMI Complete Media (10% FBS, 1% Penicillin-Streptomycin in RPMI) was added in dropwise manner to the PBMCs. Cells were counted using a TC20 automated cell counted (verified via hemocytometer). Cell viability was verified using Trypan Blue (Bio Rad, CA, USA). Then PBMCs were allowed to rest for 2 hours at 37C and 5% CO2 (confirmed by pilot work). Cell viability was again assessed and then cells were stimulated with 2 ng/mL Phorbol 12-myristate 13-acetate
(PMA) and 1 ng/mL Ionomycin for 4h at 37C and 5% CO2. Cells were phenotyped using immunofluorescence labeling of cell surfaces with mouse anti-human monoclonal antibodies. The following antibodies were used: CD3 (APC-Cy7), CD4 (Amycan), CD8 (AF700), TCR Vα7.2 (PE), CD161 (QDot605). All antibodies were ordered from Biolegend. PBMCs were also intracellularly stained using mouse anti-human monoclonal antibodies in fixation and permeabilization buffer (BD Biosciences, NC USA) to quantify cytokine production [IFN-l
suspended in 300 µL of cell staining buffer for flow cytometry analysis. Cells were analyzed using flow cytometry (LSRII with Diva Software) (BD Biosciences, CA USA) and FlowJo CE software.
Figure 2. Representative Image of MAIT Cell Gaiting Strategy. A. Singlet cells. B. Lymphocytes. C. CD3+ T Cells. D. CD4/CD8 T Cells. E. MAIT Cells.
Experimental Standard
Samples were analyzed in two batches and a standard control was used to ensure stimulation for TNF-α and IFN-γ (Figure 3).
Figure 3: Standard Control for Cytokine Response. A. represents stimulated conditions for IFN- γ. B. represents the unstimulated comparison for IFN-γ. C. represents stimulated conditions for
TNF-α. D. represents the unstimulated comparison for TNF-α.
Get Real and Heel Exercise Training
The exercise training intervention occurred three times per week for approximately one hour for sixteen weeks. The program combined progressive aerobic and resistance exercise training in a small group setting. There were 8-10 subjects per one-hour time slot. Aerobic exercise
training modality were self-selected, and options included: cycling, walking, jogging, elliptical, rowing, or seated stepper. For the first 2-weeks of participation exercise volume and intensities started at 10-15 minutes of low intensity exercise (50-60% of the participants heart rate reserve (HRR) and 8-11 on the Borg Rating of Perceived Exertion scale (RPE)). Exercise duration was reduced by 5-10 minutes for severely deconditioned participants. By the 8th week of the study, the goal was for participants to be exercising for 30 minutes at a moderate intensity (65-75% of the HRR and RPE of 12-14).
Resistance exercise training included six resistance exercises per session, combined with the aerobic exercises previously described. The resistance training targeted large muscle groups: upper body, lower body, and core. The exercises were instructed in a circuit style with each session lastly 30 minutes. Two sets of every exercise were performed for 10-15 repetitions. The intensity progressed from light in week 1-5 (RPE 7-13) to moderate after week-5 (RPE 14-15). After week- 5 every exercise was performed in 2 sets for 10 repetitions. Modifications were made for orthopedic considerations.
The exercise training sessions were recorded in a participant log and stored in a locked filing cabinet at the Get Real and Heel training facility. Information about mode, time, amount, and intensity were recorded in the logs. Participants were continuously monitored and supervised at all times by training staff while exercising to ensure the safety of the participants. Adherence was calculated as follows: # of days attended / # of days prescribed x 100. Compliance was calculated as follows: aerobic compliance: # days completed at prescribed time (min) and intensity (RPE) / # days prescribed. Resistance Compliance: # days completed at prescribed sets x reps and intensity (RPE) / # days prescribed.
Statistical Analysis
Data was collected and analyzed with SPSS Statistics version 25.0 (SPSS, IN., Durham, NC, USA) and with Jamovi Statistics Version 0.9 (Jamovi Project Computer Software, 2020). Figures were created in GraphPad Prism version 8 (La Jolla, CA, USA). The α level was set a priori for all statistical procedures at α=0.05. Descriptive statistics were used to summarize subject characteristics and values were reported as mean ± SD. Group and time differences for participant characteristics were assessed using a 2 x 3 (group x time) ANOVA with Bonferroni post-hoc analysis. For acute exercise testing, all immune analyses were examined using a linear mixed model. The fixed factors were group and time with subjects as a random effect. Group x time interactions were resolved using simple effects to examine group responses at each time point. Data are presented as mean ± SD with model estimates expressed relative to baseline (time effects) or HC (group effects) and included 95% confidence intervals. Percent change was calculated as 100 × [(final – original)/(original)] where baseline or HC were the original for comparison purposes.
For the chronic effect of exercise, immune analyses were examined by the calculation of a change scores into linear mixed modelling. Mobilization was defined by the change score calculated from baseline to 0h post-exercise. Egress was defined by the change score calculated from the 0h to 1h post-exercise. The fixed factors were group and time with subjects as a random effect. Group x time interactions were resolved using simple effects to examine group responses at each time point. Data are presented as mean ± SD with model estimates expressed relative to baseline or HC group including 95% confidence intervals. Additionally, effects sizes were calculated using Cohen’s D (d) such that 0.2, 0.5 and 0.8 represent small, medium, and large differences, respectively.
CHAPTER IV: RESULTS