Application of a human bone engineering platform to an in vitro and in vivo breast cancer metastasis model

Application of a Human Bone Engineering Platform to an In Vitro and In Vivo Breast Cancer Metastasis Model Verena Maria Charlotte Reichert, MD Faculty of Built Environment and Enginee

Application of a Human Bone Engineering

Platform to an In Vitro and In Vivo Breast

Cancer Metastasis Model

Verena Maria Charlotte Reichert, MD

Faculty of Built Environment and Engineering, School of Engineering

Systems, Queensland University of Technology

Thesis submitted for:

Doctor of Philosophy (PhD)

2011

Keywords

Breast cancer, bone metastasis, human osteoblast matrix, breast cancer related bone disease, adhesion, single cell force spectroscopy, migration, invasion, tissue engineering, scaffold, polycaprolactone, osteoblasts, human mesenchymal stem cells

Abstract

Breast cancer in its advanced stage has a high predilection to the skeleton Currently, treatment options of breast cancer-related bone metastasis are restricted to only palliative therapeutic modalities This is due to the fact that mechanisms regarding the breast cancer celI-bone colonisation as well as the interactions of breast cancer cells with the bone microenvironment are not fully understood, yet This might be explained through a lack of

appropriate in vitro and in vivo models that are currently addressing the

above mentioned issue

Hence the hypothesis that the translation of a bone tissue engineering

platform could lead to improved and more physiological in vitro and in vivo

model systems in order to investigate breast cancer related bone colonisation was embraced in this PhD thesis

Therefore the first objective was to develop an in vitro model system that

mimics human mineralised bone matrix to the highest possible extent to examine the specific biological question, how the human bone matrix influences breast cancer cell behaviour Thus, primary human osteoblasts were isolated from human bone and cultured under osteogenic conditions Upon ammonium hydroxide treatment, a cell-free intact mineralised human bone matrix was left behind Analyses revealed a similar protein and mineral composition of the decellularised osteoblast matrix to human bone Seeding

of a panel of breast cancer cells onto the bone mimicking matrix as well as reference substrates like standard tissue culture plastic and collagen coated tissue culture plastic revealed substrate specific differences of cellular behaviour Analyses of attachment, alignment, migration, proliferation, invasion, as well as downstream signalling pathways showed that these cellular properties were influenced through the osteoblast matrix

The second objective of this PhD project was the development of a human ectopic bone model in NOD/SCID mice using medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold Human osteoblasts and mesenchymal stem cells were seeded onto an mPCL-TCP

scaffold, fabricated using a fused deposition modelling technique After subcutaneous implantation in conjunction with the bone morphogenetic protein 7, limited bone formation was observed due to the mechanical properties of the applied scaffold and restricted integration into the soft tissue

of flank of NOD/SCID mice Thus, a different scaffold fabrication technique was chosen using the same polymer Electrospun tubular scaffolds were seeded with human osteoblasts, as they showed previously the highest amount of bone formation and implanted into the flanks of NOD/SCID mice Ectopic bone formation with sufficient vascularisation could be observed After implantation of breast cancer cells using a polyethylene glycol hydrogel

in close proximity to the newly formed bone, macroscopic communication between the newly formed bone and the tumour could be observed

Taken together, this PhD project showed that bone tissue engineering platforms could be used to develop an in vitro and in vivo model system to study cancer cell colonisation in the bone microenvironment

Table of contents

Keywords I Abstract III Table of contents V List of illustrations and diagrams XIII List of abbreviations XIX Statement of original authorship XXV Acknowledgements XXVII

Chapter 1 - Overview 1

1 Overview 3

Chapter 2 - Literature Review - Mechanisms of breast cancer-related bone metastasis – overview of currently used in vitro and in vivo models 5

2.1 Epidemiology – facts about breast cancer 7

2.2 Clinical presentation of bone metastases and treatment options 8

2.3 Tumour cell metastasis – a multistep process – from the primary site to the skeleton 11

2.4 Characteristics of the “the congenial soil” – bone 13

2.5 How the “seeds” find their “congenial” soil 18

2.6 Attachment at the metastatic site - interaction of integrins with bone matrix constituents 19

2.7 More factors involved in the promotion of bone metastasis 23

2.8 Osteomimicry of breast cancer cells 26

2.9 Osteolytic bone metastasis 27

2.10 Models investigating bone-cancer cell interaction in vitro 29

2.11 Models investigating bone-cancer cell interaction in vivo 37

2.12 Summary 46

Chapter 3 - Establishment of a human primary osteoblast matrix model 49

3.1 Introduction 51

3.2 Material and Methods 53

3.2.1 Characterisation of type I collagen coated tissue culture plastic

53

3.2.1.1 Collagen type I coating of tissue culture polystyrene or thermanox coverslips 53

3.2.1.2 Anti-col I staining of col I coated coverlips 53

3.2.1.3 Scanning electron microscopy analysis of col I coated thermanox coverslips 54

3.2.2 Generation and characterisation of primary human osteoblast matrices 54

3.2.2.1 Isolation of human primary osteoblasts and matrix production 54

3.2.2.2 Confocal laser microscopy to confirm completeness of decellularisation 55

3.2.2.3 Morphology - SEM 56

3.2.2.4 Morphology - Transmission Electron microscopy 56

3.2.2.5 Mineralisation - Alizarin red S 56

3.2.2.6 Mineralisation - Wako HRII calcium assay 56

3.2.2.7 Mineralisation - X-ray photoelectron spectroscopy (XPS) 57

3.2.2.8 Mineralisation - RAMAN analysis 57

3.2.2.9 Composition - Immunohistochemistry 58

3.2.2.10 Composition - Growth factor analysis 59

3.2.2.11 Composition - 2D PAGE with MALDI or LC/MS/MS 59

3.2.2.12 Composition - Western blot analysis 59

3.2.3 Image analysis 60

3.2.4 Statistical analysis 61

3.3 Results 62

3.3.1 Presence and Morphology of collagen I on coated thermanox

coverslips 62

3.3.2 Characterisation of the composition and morphology of the human decellularised osteoblast matrix 63

3.4 Discussion 75

3.5 Conclusion 79

Chapter 4 - Interactions of human breast cancer cells with the OBM 81

4.1 Introduction 83

4.2 Materials and Methods 85

4.2.1 Characteristics and growth conditions for cell lines used in this study 85

4.2.1.1 MCF10A 85

4.2.1.2 T47D 86

4.2.1.3 SUM1315 86

4.2.1.4 MDA-MB-231 86

4.2.1.5 MDA-MB-231SA 87

4.2.1.6 Gene expression profile 88

4.2.2 Morphology 89

4.2.2.1 Flow cytometry 89

4.2.2.2 Morphology - Transmitted light microscopy 90

4.2.2.3 Morphology – Fluorescent staining 90

4.2.2.4 Morphology - SEM 91

4.2.3 Cell proliferation 92

4.2.4 Cell adhesion 92

4.2.5 Cell migration 95

4.2.6 Cell Invasion 96

4.2.6.1 SEM image analysis 96

4.2.6.2 Confocal microscopy image analysis 98

4.2.7 Cell signalling pathways 98

4.2.8 Gene expression 100

4.2.8.1 RNA isolation 100

4.2.8.2 cDNA synthesis 100

4.2.8.3 qRT-PCR 101

4.2.9 Statistical analysis 102

4.3 Results 103

4.3.1 Characteristics of utilised cell lines 103

4.3.1.1 MCF10A 103

4.3.1.2 T47D 104

4.3.1.3 SUM1315 105

4.3.1.4 MDA-MB-231 106

4.3.1.5 MDA-MB-231-SA 107

4.3.2 Cell morphology of different cell lines on TCP, col I and OBM 113 4.3.2.1 Morphology on TCP, col I and OBM assessed with transmitted light and confocal laser scanning microscopy 113

4.3.2.2 Image analysis 116

4.3.3 Proliferation of MCF10A, T47D, SUM1315, MDA-MB231 and MDA-MB-231SA on TCP, col I and OBM 120

4.3.4 Assessment of established detachment forces of each cell line to disconnect from col I and OBM 121

4.3.5 Migratory features of the 5 BC cell lines on the three investigated substrates 123

4.3.6 Invasive potential of MCF10A, T47D, SUM1315, MDA-MB-231 and MDA-MB-231SA cells 127

4.3.6.1 SEM image analysis 127

4.3.6.2 Confocal microscopy image analysis 127

4.3.7 Western blot analysis of five cell lines on different substrates to

examine various cell signalling pathways 131

4.3.7.1 FAK 132

4.3.7.2 MAPK 133

4.3.8 mRNA expression profile of MCF10A, T47D, SUM1315, MDA-MB231 and MDA-MB-231SA cells on TCP, col I and OBM 135

4.3.8.1 Attachment 135

4.3.8.2 Invasion 138

4.4 Discussion 140

4.5 Conclusion 157

Chapter 5 - Human bone and marrow derived progenitor cells and their potential for human bone tissue engineering using a medical grade polycaprolactone-tricalcium phosphate scaffold in NOD/SCID mice 161

5.1 Introduction 163

5.2 Materials and Methods 166

5.2.1 Cells 166

5.2.1.1 Isolation of human osteoblasts 166

5.2.1.2 Human mesenchymal precursor cells 166

5.2.2 Characteristics of human osteoblasts and human mesenchymal precursor cells under osteogenic conditions on TCP (2D) 167

5.2.2.1 Cell culture 167

5.2.2.2 Mineralisation - Alizarin red S 167

5.2.2.3 Mineralisation - Wako HRII calcium assay 167

5.2.3 In vitro characterisation of hOBs and hMSCs on a medical grade polycaprolactone tricalcium phosphate (mPCL-TCP) scaffold 167

5.2.3.1 Scaffold fabrication using fused deposition modelling technique 167

5.2.3.2 Cell seeding 168

5.2.3.3 Seeding efficiency and proliferation 168

5.2.3.4 Cell viability 169

5.2.3.5 Cell morphology 169

5.2.3.6 RNA isolation, primer design and qRT-PCR 170

5.2.4 Ectopic bone assay 172

5.2.4.1 Implantation 172

5.2.4.2 Calcein injection and imaging of live animals 173

5.2.4.3 Micro-CT analysis 173

5.2.4.4 Biomechanical testing 174

5.2.5 Histology and immunohistochemistry 174

5.2.5.1 Paraffin Embedding 174

5.2.5.2 Histochemistry and immunohistochemistry on paraffin embedded samples 174

5.2.5.3 Tartrate resistant acid phosphatase (TRAP) staining of paraffin embedded samples 176

5.2.5.4 Resin Embedding 176

5.2.5.5 Histomorphometry 177

5.2.6 Statistical analysis 177

5.3 Results 178

5.3.1 Characteristics of human osteoblasts and human mesenchymal precursor cells under osteogenic conditions on TCP (2D) 178

5.3.1.1 Mineralisation 178

5.3.1.2 Proliferation 179

5.3.1.3 qRT-PCR 180

5.3.2 Characterisation of human marrow and bone derived cells on the scaffold 181

5.3.2.1 Morphology and viability 181

5.3.2.2 Proliferation 183

5.3.2.3 qRT-PCR 185

5.3.3 Ectopic bone assay 186

5.3.3.1 Live imaging 186

5.3.3.2 Micro-CT analysis 186

5.3.3.3 Biomechanical testing 187

5.3.3.4 Histological analysis 188

5.4 Discussion 192

Chapter 6 - Human bone derived progenitor cells and their potential for human bone tissue engineering using an electrospun polycaprolactone scaffold with calcium-phosphate coating in NOD/SCID mice 201

6.1 Introduction 203

6.2 Material and Methods 203

6.2.1 Cells 203

6.2.1.1 Isolation of human osteoblasts 203

6.2.2 In vitro characterisation of the electrospun PCL scaffold 203

6.2.2.1 Scaffold fabrication 203

6.2.2.2 Scaffold characteristics and testing in biological applications 203

6.2.2.3 Calcium phosphate coating 205

6.2.2.4 Cell seeding 206

6.2.2.5 Cell viability 206

6.2.2.6 Cell morphology 206

6.2.3 Ectopic bone assay 206

6.2.3.1 Implantation 206

6.2.3.2 Implantation of PEG gels incorporated with human breast cancer cells 207

6.2.3.3 Micro-CTanalysis 208

6.2.3.4 Histology 208

6.3 Results 209

6.3.1 In vitro characterisation of hOBs on the electrospun scaffold 209 6.3.1.1 Morphology and viability 209

6.3.2 Morphology assessment of BC cells in PEG gels cultured in vitro 209

6.3.3 Ectopic bone assay 210

6.3.3.1 Implantation 210

6.3.3.2 Micro-CT-analysis 211

6.3.3.1 Histological analysis 211

6.4 Discussion 215

6.5 Conclusion – Chapter 5 and 6 218

Chapter 7 - Future directions 221

Chapter 8– Bibliography 227

List of illustrations and diagrams

Chapter 2

visualized by imaging modalities

9

plasmin in the pericellular activation cascade for MMPs

26

mineralised rat tail collagen fibrils

67

OBM in addition to human native bone

71

Figure 23: Immunohistochemistry for human bone ECM 72

factors in the OBM

74

Chapter 4

study

98

SUM1315, MDA-MB-231 and MDA-MB-231SA cells cultured on TCP, col I and OBM

114

T47D,SUM1315, MDA-MB-231 and MDA-MB-231SA cells cultured on TCP, col I and OBM

115

T47D, SUM1315, MDA-MB-231 and MDA-MB-231SA cells cultured on TCP, col I and OBM

117

MCF10A, T47D, SUM1315, MB-231 and

MDA-118

MB-231SA cells cultured on TCP, col I and OBM

MCF10A, T47D, SUM1315, MB-231 and MB-231SA cells cultured on TCP, col I and OBM

MDA-119

MDA-MB-231 and MDA-MDA-MB-231SA cells cultured on TCP, col I and OBM

120

SUM1315, MDA-MB-231 and MDA-MB-231SA from col I and OBM

122

of MCF10A, T47D, SUM1315, MB-231 and MB-231SA on TCP, col I and OBM

MDA-125

and persistence time of MCF10A, T47D, SUM1315, MDA-MB-231 and MDA-MB-231SA on TCP, col I and OBM

126

MCF10A, T47D, SUM1315, MB-231 and MB-231SA on OBM

MDA-128

cells

129

SUM1315, MDA-MB-231 and MDA-MB-231SA cells on OBM at d 3

130

microscopy images to determine invasiveness

levels of integrin alpha 2, 3, 5, v and beta 1and 3

Chapter 5

and hOBs

179

in 3D

184

qRT-PCR analysis of 2D and 3D cultures

185

injections as well as scaffolds before and after implantation

186

results from biomechanical testing

188

mineralisation

189

Kossa/van Gieson and H&E stained sections

190

Chapter 6

meshes

205

hOBs on e-spun PCL-CaP scaffolds

209

gels

210

Figure 85: Images of PCL-CaP scaffold and PEG gels before and

after implantation

211

stained histology sections

213

human tissue engineered bone construct

bone metastasis and tumour growth in bone

20

breast cancer cells with the bone microenvironment

List of abbreviations

cells

DIC Differential interference contrast

IGF Insulin-like growth factor

phosphate

immunodeficient

PCL Polycaprolactone

Cbfa1

SPARC Secreted protein, acidic cysteine-rich, a.k.a

Statement of original authorship

“The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other higher education institution To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made.”

Wednesday, 6 July 2011

Acknowledgements

I would like to gratefully acknowledge Prof Dietmar W Hutmacher for his friendship and the supervision of my work I greatly thank Prof Judith Clements for stimulating critical discussions, her support and intellectual input and proofreading and also would like to thank Prof Colleen Nelson

I am very very grateful to Dr Anna Taubenberger, her friendship, her enthusiasm, dedication, patience and diligence, her guidance in the laboratory as well as her fantastic input, advice and support in writing as well

as proofreading I am grateful to Dr Maria Woodruff for her friendship and support in matters of histology I would also like to thank Dr Daniela Loessner for her advice and support in laboratory matters

I would like to acknowledge the DAAD (German academic exchange service) for the supporting scholarship towards my living allowances

Furthermore, I am very thankful to the members of the IHBI Regenerative Medicine group and the members of the Cancer Program as well as my fellow colleagues and research students for companionship, inspiration and advice

Lastly I would like to express my sincere gratefulness to my husband Johannes for his love, scientific advice, his patience, encouragement and unlimited support I am also grateful to my parents and my sister Annalena for their support, understanding, and patience over the years

Chapter 1 - Overview

1 Overview

and the impact on the quality of life of every woman suffering from this debilitating disease are still alarming

Breast cancer is a leading cause of cancer related death among women worldwide and numbers are still rising This might be due to the implementation of screening methods and more advanced imaging systems

to detect the primary tumour These are important diagnostic tools, as early diagnosis and treatment of this disease is crucial and vastly affecting the 5-year survival rate First line therapy of breast cancer has become more individualised and specialised with interdisciplinary medical teams deciding

on the therapeutic scheme based on the clinical situation and histological analysis of the tumour tissue The combination of anti-hormonal therapies with the application of chemotherapeutic drugs as well as radiation therapy and the introduction of the monoclonal antibody Trastuzumab (Herceptin®) dramatically improved the median survival rate of patients Hence, a lot of women are in complete clinical remission after the initial treatment without overt signs of metastatic cancer spread However, even years after disease free survival women might present with metastatic lesions The most common sites of metastatic breast cancer spread are the skeleton, liver, lung and brain While a lot of progress has been made in the treatment of initial breast cancer, only palliative treatment modalities are available once the cancer cells have spread throughout the body

Bone is the most common site of cancer metastasis for breast as well prostate cancer Chapter 2 will provide more detailed epidemiological information about the occurrence of breast cancer related bone metastasis

as well as the pitfalls of the current diagnostic tools to detect these bone metastatic lesions and available treatment options

One reason for the poor treatment options of breast cancer related bone disease is that the underlying molecular mechanisms are not fully deciphered

yet This relates back from the lack of appropriate in vitro and in vivo models

that are able to mimic the pathophysiology of human bone metastasis Chapter 2 will also focus on the existing knowledge of the interactions

between breast cancer cells and the bone microenvironment Furthermore, it

will provide an overview of currently used in vitro and in vivo models

As a result, more recently, cancer biologists are turning to more complex and realistic 2.5-3D culture methods to provide cells with a more physiological environment Hence, Chapter 3 will introduce a new model system established by the Hutmacher group, namely a decellularised human osteoblast matrix as representative of the human bone matrix, which will be

subsequently applied in a detailed in vitro study in Chapter 4 Here, the

influence of the human decellularised bone matrix on key characteristics (adhesion, migration, proliferation and differentiation) of a panel of breast cell lines was investigated

Breast cancer related bone metastases are of complex nature and currently

available in vivo models that are trying to mimic this intricate process are

presenting considerable shortfalls Chapter 5 will present a human bone tissue engineering strategy, which is currently applied and investigated in bone regeneration and the treatment of critical sized bone defects in its translation to an ectopic human bone model in NOD/SCID mice This study

will be concluded in Chapter 6, demonstrating results of preliminary in vivo

experiments

The last Chapter will present future directions on how the in vitro and in vivo

models presented in this PhD thesis will be further used to dissect the complexity of breast cancer related bone disease

Chapter 2 - Literature Review - Mechanisms of

overview of currently used in vitro and in vivo

models

2.1 Epidemiology – facts about breast cancer

Breast cancer (BC) is a leading cause of cancer related death among women worldwide [1] According to the Australian Institute of Health and Welfare (AIHW), 35 Australian women were diagnosed and 7 women died from this disease each day in 2006 Over the last 20 years the outcome of women diagnosed with BC has significantly improved The overall 5-year survival rate was 88% for women diagnosed with BC between 2000 and 2006 in comparison to 73% for women diagnosed in 1982 to 1987 This might be due

to earlier diagnosis with more advanced diagnostic tools, screening methods

as well as the implementation of systemic adjuvant therapy [2] However, despite the increase in 5-year survival rate, it still remains a major health burden and has a significant impact on the quality of life of every patient

When first diagnosed, most patients do not show any clinicopathological signs of overt secondary cancer lesions (metastases) [3] and women are able to achieve a state of complete clinical remission after surgical treatment

in conjunction with individualized first line therapy such as chemo-, radio- and anti-hormonal therapy [3] An increasing risk of metastasis occurs with a larger-sized primary tumour, loss of histopathological grade and the presence of lymph-node metastasis [4] These criteria also serve as prognostic markers in BC patients

Localised metastatic spread firstly occurs through the lymphatic vessels through which cancer cells are able to spread to the contralateral breast as well as to the axillary lymph nodes Affected lymph nodes in the axilla can be detected and removed during first line surgery yet still lead to a worse prognosis and a decreased survival time in comparison to patients with tumour-negative axillary lymph nodes [5] Despite this, roughly one–third of women suffering from BC that has not spread to the lymph nodes develop distant metastasis On the other hand, some patients that have affected lymph nodes are able to remain free of distant metastases 10 years after local therapy [6, 7]

Development of distant metastases usually occurs in 10 to 15% of all BC patients within three years of detection of the primary tumour However, BC

has the unusual ability amongst malignancies in its trend for late metastatic recurrence even 10 years or more after initial diagnosis and disease free survival [8]

The skeleton, liver, lung and brain account for the most common sites affected by BC cell colonisation It was shown that the highest frequency of secondary cancer lesions is located in bone (83%) whereas liver and lung are usually affected to a lesser extend (27%) [3, 9, 10] This demonstrates, that the skeleton is the most common site of the first distant relapse of BC [3,

9, 10] The median survival after bone metastases have been diagnosed is relatively long with 24-40 months In contrast, the median survival of soft tissue metastases like the liver is only three months after detection [3, 11] However, the involvement of the skeleton is a major cause of morbidity and hospitalization in addition to the tremendous impact in the quality of life of each woman

2.2 Clinical presentation of bone metastases and treatment options

Once women show overt sings of bone metastases, they have entered a so far incurable stage of the disease Symptoms associated with metastatic bone disease are highly debilitating Due to extensive bone loss and tumour expansion serious morbidity is caused by symptoms like severe bone pain, pathological fractures, spinal cord compression, bone marrow aplasia and hypercalcaemia [9]

Bone metastases are classified due to their radiographic appearance as either osteolytic or osteoblastic Both types of lesions result from alterations

in the balance of the normal bone remodelling This results from a pathological activity of bone resorbing cells, the osteoclasts and their counterparts, the osteoblasts which are responsible for new bone formation Osteolytic lesions show an increase in bone resorption, but compensatory bone formation is impaired In contrast, osteoblastic lesions are characterised by disorganised new bone formation and insufficient bone

resorption [9] The majority of BC related bone metastases are characterised

as osteolytic, only approximately 15% are of osteoblastic or mixed entity [12] The frequency of bone metastases can be difficult to estimate due to limited diagnostic tools Skeletal scintigraphy and X-ray are the most common imaging modalities Skeletal scintigraphy detects enhanced osteoblastic activity and skeletal vascularity However, it can occasionally result in false negative findings when pure osteolytic metastases are growing rapidly, when bone turnover is slow, or if the site is avascular [13] Uncovering osteolytic metastases by X-ray requires a loss of 35-70% of normal bone mineral content [13] Therefore lesions may only be detected in an advanced stage

of bone destruction Computed tomography (CT), magnetic resonance imaging (MRI), positron emission computed tomography (PET) are other modalities to detect bone metastases They are used less frequently because of expense, availability and specificity of these methods (Fig 1) [13]

Fig 1: Schematic of bone structure and types of metastases visualized by various imaging modalities (Left) Each modality visualizes different aspects of tissues, plain radiography (XR) and computed tomography (CT) visualise bone structure, CT and magnetic resonance imaging (MRI) visualize tumours and bone marrow, skeletal scintigraphy (SS) and single photon emission computed tomography (SPECT) reveal bone osteoblastic metabolism and positron emission tomography (FDG-PET) visualise tumour metabolism CT, MRI and FDG- PET can potentially detect small bone marrow metastases early, before any structural changes in cortical bone are visible [13] (Right) Example of discordant PET/CT and SS results A 45-year-old woman with biopsy-proven left iliac osseous metastasis detected on (A) axial PET/CT image (gold arrow), but not on (B) SS A 58-year-old woman with biopsy- proven metastatic BC to (C) L4 on coronal PET image (blue arrow), but (D) not on bone scintigraphy Reproduced from [14]

Additionally to the imaging techniques mentioned above, biochemical markers can be used to detect skeletal pathologies Those markers are for example urine calcium levels, urine levels of N-terminal telopeptide of collagen type I, free deoxypyridinoline or pyridinoline, hydroxyproline and serum levels of alkaline phosphatase [15] However, those markers are not exclusive to bone metabolism Available assays for detection of these biochemical markers lack sufficient sensitivity or specificity for diagnosis of bone metastases, but can be used to monitor responses to treatment [16] Levels of deoxypyridinoline and pyridinoline, for example, decreased after treatment with anti-resorptive drugs such as bisphosphonates [15]

Current treatment options for cancer related bone disease are rarely curative and advanced pain management is often the major treatment avenue Palliative treatment with anti-resorptive drugs has been found to reduce the frequency of skeletal related events Additionally, bone metastasis associated complications of bone destruction such as pain and morbidity could be diminished, thus improving the quality of life but without prolonging survival [17]

One approach in treating bone destruction as result of metastatic BC disease

is the application of bisphosphonates Bisphosphonates are stable analogues of pyrophosphates that bind to hydroxyapatite bone mineral surfaces with high affinity They are selectively internalised by osteoclasts where they subsequently inhibit their activity [18] However, although experiments in rodents with metastatic bone disease showed that bone resorption was significantly reduced after administration of bisphosphonates there was no evidence of new bone deposition or bone repair [19] Hence, this might stop the bone resorption but the damage that has already been caused remains untreated

It is also hypothesised that bisphosphonates may be used as anti-tumour drugs in very early stages of BC, due to their anti-proliferative effect on

cancer cells in vitro and anti-angiogenic effect at the primary tumour site in