The effect of physical activity and energy restriction on tumor vessel architecture and hypoxia in the 4T1.2 murine breast cancer model
Open Access
- Author:
- Cain, Abriana
- Area of Honors:
- Nutritional Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Connie Jo Rogers, Thesis Supervisor
Alison D Gernand, Thesis Honors Advisor - Keywords:
- calorie restriction
physical activity
triple negative breast cancer
breast cancer
hypoxia
vessel architecture - Abstract:
- The American Cancer Society estimates that there will be 287,850 new cases of invasive breast cancer diagnosed and 43,250 breast cancer deaths in the United States in 2022. The average five-year survival rate for women with invasive breast cancer localized in the breast is 98.9% but is only 28.1% for women diagnosed with metastatic breast cancer. Thus, developing strategies to prevent metastatic disease is key to reducing mortality from breast cancer. Triple-negative breast cancer (TNBC) is a highly metastatic and aggressive form of breast cancer with limited treatment options due to being negative for all three hormone receptors that are traditionally used as therapy targets. The normalization of tumor vasculature is emerging as a new target for cancer treatment. One mechanism proposed for tumor vessel normalization is the normalization of hypoxia within the tumor microenvironment. The protein hypoxia inducible factor-1 alpha (HIF-1α) becomes activated in hypoxic cells and induces the production of over 200 proteins including vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). VEGF reduces hypoxia by stimulating the formation of endothelial cells and capillaries that enable the tumor to receive nutrients and oxygen rich blood. Endothelial cells release PDGF which enables pericytes, cells that wrap around endothelial cells to provide structural stability, to concomitantly release VEGF. Pharmacologic anti-angiogenic therapies inhibit VEGF resulting in decreased tumor growth, normalization of the vasculature and sensitization of tumors to chemotherapy by improving oxygenation and delivery of chemotherapy. However, these results are often short-lived, and long-term clinical benefit has not been observed. Thus, there is a need to develop novel interventions. Both physical activity and energy restriction have well established antitumorigenic effects in human and animal models. Emerging data suggest that physical activity and energy restriction not only reduce tumor growth but may improve tumor vascularization and hypoxia in preclinical tumor models, however no studies have assessed the combined effect of these interventions on tumor architecture and hypoxia. This study was a pilot study undertaken to understand the role of physical activity, energy restriction, and combination, in resulting tumor growth, blood vessel architecture, and markers of hypoxia and angiogenesis in the 4T1 murine mammary tumor model. The objectives of this project were to 1) characterize the effects of physical activity, energy restriction, or combination, on blood vessel architecture within the tumor microenvironment 2) characterize the effects of physical activity, energy restriction, or combination, on levels of hypoxia and angiogenesis within the tumor microenvironment. Mice (n=10-12/group) were randomized to one of four interventions: sedentary and ad libitum fed (SED+AL), physical activity and ad libitum fed (PA+AL), sedentary and 10% energy restricted (SED+ER), and physical activity and 10% energy restricted (PA+ER) for 34 weeks. All mice were injected orthotopically with 5 X104 4T1.2luc cells in the 4th mammary fat pad and were sacrificed at day 27-28 post tumor implantation. Half of the mice were assigned to assess the effects of physical activity and energy restriction on the microbiome and metabolome, the other half to this dissertation study. For the vessel architecture and immunohistochemistry cohort, whole tumors were removed from each mouse at sacrifice. Half of the tumor was used to assess tumor vessel architecture using immunohistochemistry (3-5 mice/group) and the other half was used to assess markers of hypoxia and angiogenesis using qPCR (3-6 mice/group). This was the first study to characterize the effects of the combination of physical activity and energy restriction on vessel architecture and tumor hypoxia in the 4T1.2 murine mammary tumor model. We found that sedentary and energy restricted mice as well as physical activity and energy restricted mice had significantly lowered tumor volumes at sacrifice compared to control (p= 0.003, Dunnett’s multiple comparisons, p<0.05). All other outcomes were similar between groups (p>0.05). We observed decreased vessel density in the sedentary energy restricted group and increased vessel density in the physical activity ad libitum group. Vessel maturity was increased in sedentary energy restricted mice as well as physical activity energy restricted mice. All markers of hypoxia (HIF 1 α, VEGF-A, VEGF-C, IL-6) and angiogenesis (Ang, Pecam-1, Cdhr5) were higher for all groups in comparison to control, except for a downregulation of VEGF-C, in sedentary energy restricted mice. Overall, energy restriction, and the combination of physical activity and energy restriction, improved vessel maturity in mice, and our findings suggest this normalization was not due to the normalization of hypoxia.