EXERCISE AND THE IMMUNE RESPONSE IN LUNG CANCER

FACS analysis of PBMCs

The FACS panel comprises: CD4pos Helper T Cells, CD8pos Cytotoxic T Cells, Regulatory T Cells, Effector Memory T Cells, Naive/Memory Subsets, Activated/Exhausted T Cells, gamma-delta T Cells, Naive B Cells, Memory B Cells, Transitional B Cells, Classical Monocytes, Intermediate Monocytes, Non Classical Monocytes, NK CD16pos, NK CD16neg, Neutrophils, Basophils, Mast Cells, Viable vs. Dead Cells, Immune Checkpoint Subsets, Activation/Proliferation Markers. The total numbers and percentages of cells positive for the above listed markers are the primary outcome measures. The surface marker profile of PBMCs as a whole will define the outcome, showing differences between exercising and sedentary patients, e.g.: "In patients from the exercise group, XX% of naive B cells were found, and XX% of CD4pos Helper T Cells, and XX% CD8pos Cytotoxic T Cells were found. In patients from the control group, the proportion of naive B cells was XX...".

Cytokine analysis from peripheral blood sampling

IFN gamma, IL-13, GM-CSF, IL-1β, IL-5, sCD25 (IL-2Ra), IL-4, VEGF, IL-17A, IL-18, CCL4 (MIP-1β), IL-15, IL-12p70. The count of the above listed cytokines in pg/ml will be recorded in venous blood samples, with higher values after exercise indicating a more pronounced inflammatory response. Blood plasma samples from the patients will be collected at the beginning and after the 12 weeks exercise intervention for cytokine testing. Additionally, cytokine profiling will be done before and after one exercise bout to illustrate acute changes. Multiplex detecton kits (Legendplex) will be used for cytokine profiling. For every cytokine, differences will be recorded in one patient at enrollment and after completion of the 12 week training therapy. Differences between the exercising and non-exercising patients will be recorded as well. E.g. "IFNgamma levels were on average XX pg/ml before the intervention. After the intervention period of 12 weeks, IFNgamma levels were on average XX pg/ml."

Number of participants with changes in mitochondrial function (Seahorse analysis)

Mitochondrial function parameters are combined to assess how well mitochondria work in a given patient. Oxygen Consumption Rate (OCR), Extracellular Acidification Rate (ECAR), Glycolytic Rate, Glycolytic Capacity, Basal Oxygen Consumption Rate, Maximal Respiration, and Spare Respiratory Rapacity are assessed by means of a Seahorse assay using a Seahorse XF Analyzer from Agilent, that measures the real-time metabolic activity of live cells by simultaneously assessing mitochondrial respiration and glycolysis. The number of patients with changes in oxygen consumption rate (OCR), which indicates mitochondrial respiration, and the extracellular acidification rate (ECAR), which is linked to glycolysis, is analysed. Aberrations in mitochondrial function as well as changes from before to after the intervention in % of patients / total number of patients is assessed.

Mass spectrometry - Lipidomics

Marker Name (or m/z value), Retention Time and Intensity/Abundance of a given metabolite will be analysed in plasma samples. Markers comprise H4CH, H4A1, H4A2, TPA1, HDA1, IDPL, H4PL, Glutamine, L5CH, H3CH, TPA2, HDA2, L5PL, HDCH, H3A2, TPCH, L5PN, L5AB, V1FC, H3FC, L5FC, SPC, IDTG, V5FC, Glutamic acid, H4FC, Alanine, HDPL, V2PL, Glucose, LDCH, L4CH, H3A1, L4FC, V2TG. Lipidomics is used to describe the complete lipid profile within the patients' immune cells. It is a subset of the "metabolome". As opposed to classic analysis of carbohydrate metabolism, lipids are more stable, allowing for an analysis of frozen PBMCs. Our hypothesis is that upon the exercise program, lipidomics of the immune cells significantly change, allowing for a more favorable antitumor immune response. Total counts of each above-listed metabolite in the blood will be determined, e.g. in milligrams per deciliter and for every metabolite, changes between exercising and sedentary patients are recorded.

Aerobic capacity

With spiroergometry testing at the beginning of the exercise program and after completion of the program at 12 weeks, the investigators will assess potential changes in the patients' aerobic capacity. The normal range for womens' VO2max starts at 30 ml/kg/min and for men at 35 ml/kg/min with lower ranges expected in the usually elderly tumor patients. Improvements over the 12 week intervention period will be recorded.