Research Proposal: Exercise as a Form of Lung Cancer Treatment
Morgan Allen
Department of Biology, Liberty University
BIOL 415L: Cell Biology Laboratory
Dr. William Moore
February 17, 2025
Background and Rationale: Lung cancer is the deadliest form of cancer, claiming
124,730 lives in the U.S. annually (American Cancer Society Medical and Editorial Team, 2025). Although
regular exercise has been known to reduce the effects of various types of cancer, recent studies have
shown exercise to be successful in treating lung cancer (cdc.gov, 2024). Exercise increases the
proliferation and activation of T-cells, which participate in the adaptive immune response to cancer,
reduce inflammation, and modulate expression of cytokines that stimulate various immune cells, causing
either growth or regression of cancer cells (Wang & Zhou, 2021.)
A 2022 study treated one group of mice with saline and one group with urethane, which
develops lung cancer. The lung cancer mice were separated into three groups, a control group (no
exercise), an endurance group, and a HIIT (high intensity interval training) group (Ge et al., 2022). After
12 weeks, cancer tissues were collected from the mice for RT-PCR, immunofluorescence staining, and
western blotting analysis. Endurance exercise was found to reduce the proportion of M1-type TAMs
(tumor-associated macrophages; these play a significant role in cancer progression) in lung cancer
tissues, while HIIT regulated M1 and M2 polarization of TAMs by increasing the levels of IL-10 and IL-12
(inflammatory cytokines) in
lung cancer tissues and circulating IFN-γ.
A second study in 2023 injected mice with lung cancer cells (LLC1.) These mice were separated
into three groups: a control group that participated in no exercise, a group that participated in voluntary
exercise (running 2-15 km per day on a wheel), and a group that was forced to run over 15 km per
day(Leimbacher et al., 2023). Mice were euthanized and lung tissue was examined. Reduced tumor
growth was apparent only in the group subject to intense exercise; the voluntary exercise group and
control group growth shared the same amount of tumor growth. This study showed that a consistent,
direct link between exercise and
reduced lung cancer tumor growth has not been proven.
Any method of treatment that shows promise as a therapeutic avenue for reducing tumor
growth, and thereby ameliorating symptoms, could be used to improve quality of life and prognosis for
people with lung cancer. There remains a gap in knowledge as to how exercise mimetics affect human
cells (most tests have been done on animals) and which conditions are directly responsible for reduced
cell proliferation. Locating the specific exercise-induced conditions that lead to tumor regression would
allow for the development of more effective treatments for those suffering from lung cancer. The
proposed research would examine the effects of exercise mimetics (such as increased oxygen and
glucose levels, decreased blood pH levels, and the presence of exercise-specific hormones) on human
lung cancer cells (A549 line) to determine if the tested exercise mimetic
result in decreased cell proliferation or increased apoptosis.
Specific Aim: The aim of the proposed research is to determine if a reduced pH (7.0), increased
(300%) oxygen and glucose levels, and the presence of epinephrine and exercise-induced metabolites
will reduce proliferation of A549
human lung cancer cells.
Hypothesis: I hypothesize that the cells exposed to a reduced pH (7.0), increased oxygen (300%)
and glucose levels, and the presence of epinephrine and exercise-induced metabolites will have a lower
rate of proliferation than that of the
control group.
Experimental Design: Human lung cancer cells (cell line A549) would be acquired from ATCC. One
group of the cells is the control; one will be subject to exercise mimetic testing. The control group will be
incubated in media in a 5% oxygen atmosphere at 98.6 degrees Fahrenheit, in a pH of 7.4.
To mimic the effects of exercise-induced acidosis, Sigma Aldrich lactate (0.5 mg/mL) and Aldon
glucose (0.5 mg/mL) will be added to the medium of the experimental group, simulating the slightly acidic
pH levels and high energy demands present in the body during intense exercise. 1 μM Caroline
epinephrine will also be added to the media to mimic the release of epinephrine during exercise (Miao et
al., 2024). To simulate the increased oxygen levels in the lung tissue during intense exercise, cells will be
incubated in a 15% oxygen atmosphere at 100.6 degrees Fahrenheit (to simulate the increase in body
temperature. Inflammatory cytokine IL-6 (Stem Cell Technologies) was added to the media, which
stimulates the IDO (indoleamine 2,3-dioxygenase) pathway, allowing for the production of metabolites
such as Kynurenine, β-Hydroxybutyrate, N-lactoyl-
phenylalanine, β-Aminoisobutyric acid, and Kynurenic acid.
After the cells have been allowed to rest for one week, proliferation will be assessed through
performing an MTT assay. First, an MTT solution will be dissolved in PBS at a concentration of 5 mg/mL
(Romar et al., 2016). This solution should be stored at –20 degrees Celsius. Control and experimental
cells will be transferred to a 96 well plate where 50 μL of serum-free media and 50 μL of MTT solution
will be added to each well. The cells will be incubated at 37 degrees Celsius for three hours. After
incubation, 150 μL of DMSO will be added to each well. The plate will be shaken gently to dissolve
crystals. After one hour, absorbance at 590 nM will be taken. Absorbance is proportional to the number
of viable cells present in the media; therefore, the control group is predicted to have a significantly
higher absorbance than that of the experimental group.
Potential Pitfalls and Alternative Approaches: Since the link between exercise and reduced tumor
growth seems to be linked to exercise intensity, it is possible that the mimetics for this experiment may
not resemble exercise that is “intense” enough to contribute to the tumor-reducing effect. Conversely,
the mimetics could be too strong, and not equivalent to the atmosphere one could attain through
exercise alone. If decreased cell proliferation is not evident after the first MTT assay, the experiment
could be repeated using higher oxygen levels and concentrations of hormones.
References
2024. Physical activity and cancer. Centers for Disease Control and Prevention.
https://www.cdc.gov/physical-activity-basics/health-benefits/lowers-risk-of-cancer.html (Accessed
February 17, 2025.)
American Cancer Society medical and editorial content team. 2025. “Key Statistics for Lung Cancer.”
American Cancer Society. https://www.cancer.org/cancer/types/lung-cancer/about/key-
statistics.html (Accessed February 17, 2025.)
Ge, Z., Wu, S., Qi, Z., and Ding, S. (2022). Exercise modulates polarization of TAMS and expression of
related immune checkpoints in mice with lung cancer. Journal of Cancer 13, 3297–3307.
Leimbacher, A., Villiger, P., Desboeufs, N., Aboouf, M., Armbruster, J., Ademi, H., Flüchter, P., Rütten, M.,
Gantenbein, F., Thomas, T., et al. (2023). Voluntary Exercise Does Not Always Suppress Lung
Cancer Progression.
Miao, S.-N., Chai, M.-Q., Liu, X.-Y., Wei, C.-Y., Zhang, C.-C., Sun, N.-N., Fei, Q.-Z., Peng, L.-L., and Qiu, H.
(2024). Exercise accelerates recruitment of CD8+ T cell to promotes antitumor immunity in lung
cancer via Epinephrine. BMC Cancer 24.
Romar, G.A., Kupper, T.S., and Divito, S.J. (2016). Research techniques made simple:
Techniques to assess cell proliferation. Journal of Investigative Dermatology 136.
Wang, Q., and Zhou, W. (2021). Roles and molecular mechanisms of physical exercise in cancer
prevention and treatment. Journal of Sport and Health Science 10, 201–210.
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