Table Of ContentTherapeutic Aerobic Exercise in a Mouse Model of Breast Cancer: Effects on Tumor
Progression, Angiogenesis, and Response to Chemotherapy
by
Allison Shayna Betof
Department of Pathology
Duke University
Date:_______________________
Approved:
___________________________
Mark W. Dewhirst, Supervisor
___________________________
Lee W. Jones
___________________________
Salvatore V. Pizzo
___________________________
Christopher D. Kontos
___________________________
Zeljko Vujaskovic
Dissertation submitted in partial fulfillment of
the requirements for the degree of Doctor of Philosophy in the Department of
Pathology in the Graduate School
of Duke University
2012
ABSTRACT
Therapeutic Aerobic Exercise in a Mouse Model of Breast Cancer: Effects on Tumor
Progression, Angiogenesis, and Response to Chemotherapy
by
Allison Shayna Betof
Department of Pathology
Duke University
Date:_______________________
Approved:
___________________________
Mark W. Dewhirst, Supervisor
___________________________
Lee W. Jones
___________________________
Salvatore V. Pizzo
___________________________
Christopher D. Kontos
___________________________
Zeljko Vujaskovic
An abstract of a dissertation submitted in partial
fulfillment of the requirements for the degree
of Doctor of Philosophy in the Department of
Pathology in the Graduate School
of Duke University
2012
Copyright by
Allison Shayna Betof
2012
Abstract
Over the past decade, exercise has gained increasing attention from both
clinicians and patients as a beneficial adjunct therapy to maintain or enhance quality of
life in breast cancer patients. Recent epidemiological studies indicate that aerobic
exercise following a diagnosis of breast cancer may be associated with reductions in
cancer-‐‑specific and all-‐‑cause mortality. However, the mechanisms by which physical
activity affects tumor physiology are poorly understood. Accordingly, clinicians lack
critical information to properly advise breast cancer patients on the use of exercise as an
adjunct to more conventional therapies.
The beneficial effects of exercise on systemic vasculature are well known,
including improved endothelial function and increased perfusion. In this work, we
evaluate the hypothesis that exercise slows tumor growth and improves the structure
and function of tumor blood vessels, resulting in decreased hypoxia and increased
effectiveness of cyclophosphamide chemotherapy.
To investigate the effects of exercise on tumor microenvironment, we injected
syngeneic 4T1 breast tumor cells into the mammary fat pad of immunocompetent
BALB/c mice. The exercise intervention (voluntary wheel running) was designed to
mimic four clinically relevant scenarios: (1) patients who are sedentary before and after
diagnosis, (2) previously sedentary patients who begin exercising after diagnosis, (3)
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previously active patients who stop exercising after diagnosis, and (4) previously active
patients who continue to exercise after diagnosis. Animals in Groups 3 and 4 exercised
prior to tumor cell transplant, whereas Groups 1 and 2 were sedentary during that time.
Immediately after transplant, Groups 2 and 4 were running, and Groups 1 and 3 were
sedentary. Tumor growth was monitored for 18 days, and then perfusion was mapped
using MRI and tumors were removed for analysis. In a follow-‐‑up experiment, BALB/c
mice were immediately implanted with tumor cells and then randomized to running or
sedentary conditions with or without cyclophosphamide chemotherapy given one week
after tumor cell transplant (three 100 mg/kg doses, every other day). Tumors were again
allowed to progress for three weeks, and MRI was performed prior to tumor removal.
Animals voluntarily ran 5 to 6 km per day prior to transplant and 4 to 5 km per
day after transplant. Body weight was unaffected by exercise. Voluntary wheel running
reduced tumor growth rate nearly twofold and significantly increased apoptosis.
Additionally, running after tumor implantation caused significant increases in
microvessel density (CD31) and vessel maturity (colocalization of CD31 with NG2 and
desmin). Hypoxia (EF5) was significantly reduced in the exercising animals, and MRI
showed that tumors were more uniformly perfused in the running groups. Furthermore,
exercise significantly enhanced the effectiveness of cyclophosphamide in slowing tumor
growth. Taken together, these results suggest that aerobic exercise slows breast tumor
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growth, improves tumor vessel structure and function, and augments the effectiveness
of cyclophosphamide chemotherapy.
These findings have important implications for the use of exercise in cancer
treatment. Using a clinically relevant animal model, we provide the first conclusive
evidence that exercise may do more than decrease symptoms and improve quality of life
for cancer patients. Our data suggest that exercise may, in fact, be an effective antitumor
intervention both alone and in combination with other cytotoxic therapies.
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Dedication
This dissertation is dedicated to:
My parents. You paved the path, then walked with me hand-‐‑in-‐‑hand.
Ari and Shauna. You have supported and guided me through the lowest of lows
to the highest of highs.
Rachel Brems. Not many people are lucky enough to have more than one family.
You made me part of yours, and I am so grateful that you are forever part of
mine.
Anya and Kayla. You have filled my life with light and joy. I work with the hope
that you will grow up to live in a world that no longer fears the word cancer.
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Contents
Abstract .......................................................................................................................................... iv
List of Tables ................................................................................................................................ xii
List of Figures ............................................................................................................................. xiii
List of Abbreviations .................................................................................................................. xv
Acknowledgments ................................................................................................................... xviii
1. Introduction ............................................................................................................................... 1
1.1 Epidemiology of Breast Cancer ...................................................................................... 2
1.2 Pathogenesis of Breast Tumors ....................................................................................... 3
1.3 The Hallmarks of Cancer ................................................................................................. 4
1.4 The Tumor Microenvironment ....................................................................................... 5
1.4.1 Components of the Physical Microenvironment .................................................... 6
1.4.2 The Physiological Microenvironment: Hypoxia and Angiogenesis .................... 8
1.5 Antiangiogenesis and Vascular Normalization ......................................................... 15
1.6 Introduction to Exercise Oncology .............................................................................. 18
1.7 Exercise and Cancer Symptom Control ...................................................................... 19
1.8 Exercise and Breast Cancer Survivorship: Epidemiological Evidence ................... 21
1.9 Biological Mechanisms of Exercise Effects on Breast Cancer: Clinical Evidence .. 24
1.10 Exercise and Cancer Progression: Preclinical Studies ............................................. 30
1.11 Biological Mechanisms of Exercise’s Effects on Breast Cancer: Preclinical
Evidence ................................................................................................................................. 41
1.12 Conclusions ................................................................................................................... 44
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2. Materials and Methods ........................................................................................................... 46
2.1 4T1 Breast Cancer Model ............................................................................................... 46
2.2 Exercise Intervention ...................................................................................................... 46
2.3 Magnetic Resonance (MR) Tumor Perfusion Imaging .............................................. 49
2.4 In Vivo and Ex Vivo Bioluminescence Imaging For Metastasis Detection ............ 50
2.5 Exercise Plus Chemotherapy Model ............................................................................ 51
2.6 Immunohistochemistry .................................................................................................. 53
2.6.1 Ki67 and Cleaved Caspase-‐‑3 .................................................................................... 54
2.6.2 CD31, Desmin, and NG2 .......................................................................................... 55
2.6.3 8-‐‑oxo-‐‑2’-‐‑deoxyguanosine ......................................................................................... 55
2.6.4 EF5 ............................................................................................................................... 56
2.6.5 Microscopy ................................................................................................................. 56
2.6.6 Image Analysis ........................................................................................................... 57
2.7 Optical Spectroscopy Measurement of Total Hemoglobin and Hemoglobin
Saturation ............................................................................................................................... 58
2.8 Angiogenesis-‐‑Related Gene Expression ...................................................................... 58
2.9 Statistics ............................................................................................................................ 60
3. Effect of Voluntary Aerobic Exercise on Primary Breast Tumor Growth and
Progression ................................................................................................................................... 62
3.1 Introduction ..................................................................................................................... 62
3.2 Results .............................................................................................................................. 64
3.2.1 Voluntary Exercise Performance ............................................................................. 64
3.2.2 Effect of Aerobic Exercise on Body Weight ........................................................... 66
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3.2.3 Effects of Exercise on Primary Tumor Growth ..................................................... 68
3.2.4 Effects of Exercise on Breast Tumor Metastasis .................................................... 73
3.3 Discussion ........................................................................................................................ 76
4. Effects of Voluntary Aerobic Exercise on the Tumor Microenvironment ...................... 81
4.1 Introduction ..................................................................................................................... 81
4.2 Results .............................................................................................................................. 85
4.2.1 Effects of Exercise on Tumor Vascularity and Angiogenic Signaling ............... 85
4.2.2 Exercise-‐‑Mediated Changes in Tumor Vessel Maturity ...................................... 88
4.2.3 Effects of Exercise on Tumor Perfusion ................................................................. 91
4.2.4 Effects of Exercise on Tumor Hypoxia ................................................................... 93
4.2.5 Exercise-‐‑Induced Decreases in Oxidative Stress ................................................... 95
4.2.6 Effects of Exercise on the Physical Microenvironment: Innate Immunity ........ 96
4.3 Discussion ........................................................................................................................ 97
5. Efficacy of Combining Aerobic Exercise with Cyclophosphamide Chemotherapy for
Breast Cancer Treatment .......................................................................................................... 105
5.1 Introduction ................................................................................................................... 105
5.2 Results ............................................................................................................................ 107
5.2.1 Effects of Chemotherapy on Voluntary Exercise Performance ........................ 107
5.2.2 Effects of Aerobic Exercise and Cyclophosphamide on Body Weight ............ 109
5.2.3 Effects of Aerobic Exercise and Cyclophosphamide on Primary Tumor Growth
............................................................................................................................................. 110
5.2.4 Exercise-‐‑Mediated Changes in Tumor Blood Flow ............................................ 115
5.3 Discussion ...................................................................................................................... 118
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Description:of Breast Cancer: Effects on Tumor. Progression, Angiogenesis, and Response to Chemotherapy by. Allison Shayna Betof. Department of Pathology.
