Development of a Metabolic Syndrome Mouse Model of Breast Cancer

131 Pages·2015·3.88 MB·English

by  Anishah Mandani

Development of a Metabolic
Syndrome Mouse Model of
Breast Cancer

ANISHAH MANDANI
ID# 2223777

Thesis submitted for
Bachelor of Biomedical Science with Honours 2015
University of Otago, Christchurch
Abstract

Metabolic syndrome is a cluster of disorders, including obesity, atherosclerosis,
inflammation and insulin resistance. It is associated with increased risk of various types of
cancers including breast cancer. Obesity in particular is a risk factor for an aggressive
tumour phenotype and reduced survival of patients with breast cancer. To understand
the underlying mechanisms I aimed to develop and characterise a metabolic syndrome
mouse with an orthotopic model of breast cancer.

Apolipoprotein E (ApoE) is involved in the catabolism of triglycerides and cholesterol, and
the ApoE knockout mouse model is prone to obesity and development of atherosclerosis.
The double knockout ApoE/ArKO mouse displays all features of metabolic syndrome. At 6
months of age, wild type, ApoE and ApoE/ArKO C57BL/6 mice were inoculated with the
murine breast cancer cell line E0771. Growth of tumours in the mammary fat pad and
mouse weight were measured until tumours reached ethical endpoint. The hypoxia
marker, pimonidazole, was injected 90min prior to euthanasia, and plasma, organs and
tumours were harvested and weighed. Half of each tumour was formalin fixed and
paraffin embedded for Immunohistochemical (IHC) analysis of cancer associated
adipocytes (perilipin), proliferation (phosphohistone-H3), estrogen receptor status (ERα)
and hypoxia (pimonidazole adducts). The other half was frozen and processed for tumour
lysates, which were used to measure hypoxia inducible factor 1 (HIF-1α) by Western
blotting, and adipokines, using an antibody array. Vascular endothelial growth factor
(VEGF) and Insulin-like growth factor binding protein 5 (IGFBP5) concentrations were
further analysed by an ELISA assay. HIF-1α levels in EO771 cells were analysed by
subjecting the cells to hypoxic conditions.

ApoE mice weighed more than wild type and ApoE/ArKO mice, and showed increased
cellular proliferation. ApoE/ArKO mice had the least omental fat and the smallest
tumours. IHC analysis showed that EO771 tumours in ApoE mice had the highest number
of intratumoral, perilipin positive adipocytes (p<0.01), and tumours from ApoE/ArKO had
the highest percentage of phosphohistone-H3 positive cells (p<0.05). All tumours stained

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negative for ER-α. Pimonidazole staining was prominent in all tumours, with secondary
tumours staining strongly. From Western blot analysis, tumours grown in ApoE/ArKO
mice had the highest level of HIF-1α, and HIF-1α levels were higher in secondary tumours
compared to primary tumours (p<0.05). A range of adipokines was differentially
expressed in tumours grown in the three different genetic backgrounds. VEGF
concentrations were higher in primary tumours from ApoE/ArKO mice than ApoE and
wild type mice. Primary tumours from ApoE/ArKO mice had significantly lower
concentrations of IGFBP5 (p<0.01).

My findings show that breast tumours grown in ApoE/ArKO mice have an aggressive
tumour phenotype, with increased proliferation, tumour hypoxia and VEGF
concentration. These models represent valuable tools for research that will bridge the
gap between cell culture models and breast cancer patients.

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Acknowledgments

I would like to express my gratitude to my supervisors Associate Professor Gabi
Dachs, Dr. Margaret Currie and Dr. Elisabeth Phillips for their guidance and support
throughout the entire honours project. I would like to thank Associate Professor Gabi
Dachs profusely for her leadership and guidance throughout the duration of this
project and sharing immense skills and knowledge that made this journey run
smoothly and enjoyable especially handling the mice. I would also like to thank her
for critically reading the manuscripts and giving me great presentation and writing
skills.

Thanks to Dr. Katie Saunders and Arron Dyer for helping me and building my
confidence in handling mice. Especially Arron for assuring me that the mice will not
bite and if they did it won’t hurt as it was very intimidating at the start.

I am beholden to Dr Elisabeth Phillips for giving an insight to the practical matters of
the everyday research work. I have learnt enormous laboratory skills that will be
valuable in my scientific career. I would like to thank Christina and Lizzie for teaching
me how to carry out western blotting and ELISA assay.

Thanks to all the members of the department of pathology for feedback and
encouragement and a special thanks goes to the members of the Mackenzie Cancer
Research Group (MCRG) for their support throughout the year. I would also like to
thank my fellow honour students especially Morgan and Rebecca for their
inspiration, support and help during our projects in the lab. The have been great lab
partners.

I would acknowledge Dr Stephanie Bozonet for the mouse VEGF ELISA kit she kindly
offered. A special thanks to the Christchurch District Health Board Histology
department for allowing me to use their cover slip machine and the patience during
busy times.

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I would also like to thank New Zealand Breast Cancer Foundation and Mackenzie
Cancer Research Group for funding my project

Lastly, I would thank my family, for understanding and supporting me throughout
this process and the difficult times. It would not have been possible without their
encouragement.

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List of Abbreviations

AMPK AMP activated protein kinase
ApoE Apo lipoprotein E
ApoE/ArKO Apo lipoprotein E and Aromatase double knockout
ArKO Aromatase knockout
ATP Adenosine triphosphate
BMI Body mass index
BSA Bovine serum albumin
CAA Cancer associated adipocytes
CAF Cancer associated fibroblasts
CoCl₂ Cobalt Chloride
COL VI Collagen VI
COX-2 Cyclo-oxygenase 2
CRP C-reactive protein
CT X-ray computed tomography
CVD Cardiovascular diseases
DDT Dithiothreitol
DMEM Dulbecco’s modified eagles medium
DMSO Dimethyl sulfoxide
DPPIV Dipeptidyl peptidase 4
ECM Extracellular matrix
EGF Epithelial growth factor
ELISA Enzyme Linked Immunosorbent Assay

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EMT Epithelial to mesenchymal transition
ER Estrogen receptor
ERα Estrogen receptor alpha
FACS Fatty acyl CoA synthetase
FBS Fetal bovine serum
FCS Fetal calf serum
FCF Acidic Fibroblast growth factor
FFA Free fatty acids
FFPE Formalin Fixed Paraffin Embedded
HDL High density lipoprotein
HER2 Human epidermal growth factor 2
HFD High fat diet
HGF Hepatocyte growth factor
HIF-1α Hypoxia Inducible Factor 1 alpha
IBMX 3-Isolbutyl-1-methylxanthine
ICAM1 Intracellular adhesion molecule 1
IGF Insulin like growth factor
IGFBP 5 Insulin like growth factor binging protein 5
IL-6, 8, 10 Interleukin 6, 8, 10
JAK2 Janus kinase 2
LDL Low density lipoproteins
Lep Leptin
LFD Low fat diet
M-CSF Macrophage colony stimulating factor
MAPK Mitogen activated protein kinase

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MCP1 Monocyte chemo attractant protein1
MFP Mammary fat pad
MMP Matrix metalloproteinase
MRI Magnetic resonance imaging
MSG Metastases suppressor genes
Ob Obese gene
PBS Phosphate buffered saline
PGE2 Prostaglandin E2
pHH3 Phosphohistone H3
PI3K Phosphoinositide 3 kinase
PR Progesterone receptor
PVDF Polyvinylidene fluoride
RANKL Receptor activator of nuclear factor kappa-B ligand
Regulated on activation, normal T cell expressed and
RANTES
secreted
RBP4 Retinal binding protein 4
RIPA Radio Immunoprecipitation Assay Buffer
SDS Sodium dodecyl sulphate
STAT3 Signal transducer and activator transcription 3
SVF Stromal vascular function
TAM Tumour associated macrophages
TMB Tetramethylbenzidine
TBS-T Tris buffered saline
TGF- Transforming growth factor beta
TIMP1 Tissue inhibitor of metalloproteinases

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TNF- Tumour necrosis factor alpha
VEGF Vascular endothelial growth factor
WHO World Health Organization

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Table of Contents

1 Introduction ........................................................................................................... 1
1.1 Metabolic Syndrome ........................................................................................ 2
1.1.1 Metabolic Syndrome and Breast Cancer ........................................................... 3
1.1.1.1 Obesity as a Risk Factor ................................................................................ 3
1.1.1.2 Triglyceride as a Risk factor .......................................................................... 5
1.1.1.3 Other Disorders of Metabolic Syndrome as a Risk factor ............................ 5
1.2 Breast Tumour Microenvironment ................................................................... 7
1.2.1 Cancer Associated Fibroblasts, Tumour Associated Macrophages, Immune
Cells and Endothelial Cells in Breast Tumour Microenvironment ................................ 8
1.2.2 Adipocytes in Tumour Microenvironment ........................................................ 9
1.2.2.1 Cancer Associated Adipocytes ................................................................... 10
1.2.3 Tumour Microenvironment and Hypoxia ........................................................ 10
1.3 Role of Different Factors of Adipose Tissue in Breast Tumour Progression ...... 12
1.3.1 Hormones and Growth Factors ........................................................................ 12
1.3.2 Pro-inflammatory Cytokines ............................................................................ 12
1.3.3 Adipokines ........................................................................................................ 12
1.3.3.1 Leptin. ......................................................................................................... 13
1.3.3.2 Adiponectin ................................................................................................ 14
1.4 Animal Models in Obesity Related Breast Cancer Research ............................. 15
1.4.1 Types of Mouse Models Used In Cancer Research .......................................... 15
1.4.2 Obese Mouse Models in Breast Cancer Research ........................................... 16
1.5 Research Objectives ....................................................................................... 20
2 Materials and Methods ........................................................................................ 21
2.1 Mouse Model ................................................................................................. 21
2.2 Cells ............................................................................................................... 21
2.2.1 Tumour Model ................................................................................................. 21
2.2.2 Organ Harvest from Mice ................................................................................ 22
2.3 Cell Culture .................................................................................................... 22

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