magnetic resonance imaging of metastatic bone ...

Bone Metastases

Cancer Metastasis – Biology and Treatment VOLUME 12 Series Editors Richard J. Ablin, Ph.D., University of Arizona, College of Medicine and The Arizona Cancer Center, AZ, U.S.A. Wen G. Jiang, M.D., Wales College of Medicine, Cardiff University, Cardiff, U.K. Advisory Editorial Board Harold F. Dvorak, M.D. Phil Gold, M.D., Ph.D. Danny Welch, Ph.D. Hiroshi Kobayashi, M.D., Ph.D. Robert E. Mansel, M.S., FRCS. Klaus Pantel, Ph.D.

For further volumes: http://www.springer.com/series/5761

Bone Metastases A Translational and Clinical Approach Edited by

Dimitrios Kardamakis, M D, P hD, DM R T Department of Radiation Oncology, University of Patras Medical School, Patras, Greece

Vassilios Vassiliou, M D, P hD Department of Radiation Oncology, University of Patras Medical School, Patras, Greece and

Edward Chow, M B B S, P hD, F R CP C Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, Ontario, Canada

Editors Dr. Dimitrios Kardamakis, MD, PhD, DMRT University of Patras Medical School Dept. of Radiation Oncology 265 04 Patras Greece [email protected]

Dr. Vassilios Vassiliou, MD, PhD University of Patras Medical School Dept. of Radiation Oncology 265 04 Patras Greece [email protected]

Dr. Edward Chow, MBBS, PhD, FRCPC University of Toronto Sunnybrook Health Sciences Centre Dept. of Radiation Oncology 2075 Bayview Avenue Toronto, Ontario M4N 3M5 Canada [email protected]

ISBN 978-1-4020-9818-5

e-ISBN 978-1-4020-9819-2

DOI 10.1007/978-1-4020-9819-2 Library of Congress Control Number: 2009920108 c Springer Science+Business Media B.V. 2009 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

To Katerina and Anastasis, whose support and patience make it possible Dimitrios Kardamakis To my family for the invaluable help they have offered and to cancer patients and researchers who struggle against cancer in their everyday life Vassilios Vassiliou To my colleagues and my patients who teach and guide me Edward Chow

Preface by Editors

Bone metastases are common in the event of malignancy and after liver and lungs, bone is the third most common site of distant metastases. Metastatic bone disease is inevitably associated with severe complications such as pain, impaired mobility, pathological fractures, spinal cord or root compression and hypercalcemia. These complications deteriorate severely the clinical and physical status of patients, affect negatively their quality of life and can also be life threatening. The understanding of the pathophysiology of bone metastases and the investigation and application of newer diagnostic and therapeutic modalities is therefore of uttermost importance. “Bone metastases: A translational and clinical approach” is intended to serve both as an introductory and reference book that focuses on the field of metastatic bone disease. All invited contributing authors are expert researchers who have prominent publications in the field of bone metastases or related topics. More specifically, the book describes thoroughly the molecular and cellular mechanisms involved in the formation of bone metastases, comments on the role of angiogenesis, presents the newer advances made in the understanding of the clinical picture and symptoms of patients, analyses the role of bone markers in research and clinical practice and deals with all aspects of imaging modalities applied for the detection and evaluation of bone metastases. Furthermore, it covers extensively the use of radiotherapy, surgery and systemic treatments for the management of metastatic bone disease, giving special attention to the role and indications of each therapeutic mode. New therapeutic approaches such as the combination of radiotherapy or radiopharmaceuticals with bisphosphonates are also commented upon. Finally, two chapters are dedicated to the assessment of the therapeutic response by applying clinical, radiological and biochemical parameters or methods. Overall the textbook presents thoroughly all aspects of metastatic bone disease, providing comprehensive and concise information that serves as a reference for researchers, oncologists, orthopaedic surgeons, clinicians and medical students alike. Moreover it can serve as a guide for the clinical and therapeutic management of patients with metastatic bone disease. The editors would like to thank all contributing authors for their scholarly efforts and complement them for their outstanding work. We also thank Dr Richard Ablin and Wen Jiang, editors of the book series on Cancer Metastasis: Biology vii

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and Treatment, for inviting us to edit a volume for the aforementioned book series. Finally, the assistance from the Publishing Editor Dr. Christina Miranda Alves dos Santos and her assistant, Ms. Melania Ruiz Esparza was more than valuable. Patras Patras Toronto, ON

Dimitrios Kardamakis Vassilios Vassiliou Edward Chow

Contents

Part I Fundamental Concepts of Bone Metastases 1 BONE ANATOMY, PHYSIOLOGY AND FUNCTION . . . . . . . . . . . . . Vassiliki Tzelepi, Athanassios C. Tsamandas, Vassiliki Zolota and Chrisoula D. Scopa

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2 PATHOPHYSIOLOGY OF BONE METASTASES . . . . . . . . . . . . . . . . 31 G. David Roodman 3 ANGIOGENESIS AND BONE METASTASIS: IMPLICATIONS FOR DIAGNOSIS, PREVENTION AND TREATMENT . . . . . . . . . . . 51 Pelagia G. Tsoutsou and Michael I. Koukourakis 4 NATURAL HISTORY, PROGNOSIS, CLINICAL FEATURES AND COMPLICATIONS OF METASTATIC BONE DISEASE . . . . . 77 Vassilios Vassiliou, Edward Chow and Dimitrios Kardamakis 5 BONE BIOMARKERS IN RESEARCH AND CLINICAL PRACTICE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 Janet E. Brown and Edward Chow Part II Imaging Modalities 6 RADIOLOGIC EVALUATION OF SKELETAL METASTASES: ROLE OF PLAIN RADIOGRAPHS AND COMPUTED TOMOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Christina Kalogeropoulou, Anna Karachaliou and Peter Zampakis

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7 THE CONTRIBUTION OF NUCLEAR MEDICINE IN THE DIAGNOSIS OF BONE METASTASES . . . . . . . . . . . . . . . . . . 137 Andor W.J.M. Glaudemans, Marnix G.E.H. Lam, Niels C. Veltman, Rudi A.J.O. Dierckx and Alberto Signore 8 MAGNETIC RESONANCE IMAGING OF METASTATIC BONE DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Ekaterini Solomou, Alexandra Kazantzi, Odysseas Romanos and Dimitrios Kardamakis Part III Therapeutic Strategies 9 RADIOTHERAPY AND BONE METASTASES . . . . . . . . . . . . . . . . . . . 185 Jan W.H. Leer and Yvette M. van der Linden 10 BIPHOSPHONATES IN THE MANAGEMENT OF METASTATIC BONE DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Fred Saad and Arif Hussain 11 COMBINED RADIOTHERAPY AND BISPHOSPHONATES: STATE OF ART . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233 Vassilios Vassiliou and Dimitrios Kardamakis 12 BIPHOSPHONATES IN THE TREATMENT OF BONE METASTASES – OSTEONECROSIS OF THE JAW . . . . . . . . . . . . . . . 251 Cesar Augusto Migliorati 13 SURGICAL MANAGEMENT OF BONE METASTASES . . . . . . . . . . 263 Markku Nousiainen, Cari M. Whyne, Albert J.M. Yee, Joel Finkelstein and Michael Ford 14 THE ROLE OF CHEMOTHERAPY IN THE TREATMENT OF BONE METASTASES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Thomas Makatsoris and Haralabos P. Kalofonos 15 HORMONOTHERAPY OF BONE METASTASES . . . . . . . . . . . . . . . . 299 Konstantinos Kamposioras and Evangelos Briasoulis 16 RADIONUCLIDE THERAPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321 Giovanni Storto

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Part IV Assessment of Therapeutic Response 17 ASSESSMENT OF THERAPEUTIC RESPONSE . . . . . . . . . . . . . . . . . 345 Orit Freedman, Mark Clemons, Vassilios Vassiliou, Dimitrios Kardamakis, Christine Simmons, Mateya Trinkaus and Edward Chow 18 OUTCOME MEASURES IN BONE METASTASES CLINICAL TRIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 Amanda Hird and Edward Chow Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

Contributors

E. Briasoulis Department of Medical Oncology, Ioannina University Medical School, Ioannina, Greece, [email protected]; [email protected] J.E. Brown Cancer Research UK Clinical Fellow/Senior Lecturer in Medical Oncology, Cancer Research UK Clinical Centre in Leeds, St James’s Hospital, University of Leeds, Leeds, UK, [email protected] E. Chow Department of Radiation Oncology, Sunnybrook Health Sciences Centre, University of Toronto, 2075 Bayview Ave, Toronto, Ontario, Canada M4N 3M5, [email protected] M. Clemons Department of Medical Oncology, Princess Margaret Hospital (5-205), 610 University Avenue, Toronto, Ontario, Canada M5G 2M9, [email protected] R.A.J.O. Dierckx Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen, 9713 GZ, The Netherlands, [email protected] O. Freedman University of Toronto, Toronto, Ontario, Canada, [email protected] J. Finkelstein Department of Surgery, Division of Orthopaedic Surgery, Sunnybrook Health Sciences Centre, 2075 Bayview Ave., MG-361, Toronto, Ontario, Canada M4N 3M5, [email protected] M. Ford Division of Orthopaedic Surgery, Department of Surgery, Sunnybrook Health Sciences Centre 2075 Bayview Ave., MG-375, Toronto, Ontario, Canada M4N 3M5, [email protected] A.W.J.M. Glaudemans Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen, 9713 GZ, The Netherland, [email protected] xiii

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A. Hird Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, University of Toronto, 2075 Bayview Ave, Toronto, Ontario, Canada M4N 3M5, [email protected] A. Hussain Pathology and Biochemistry, Director, Medical Genito-Urinary Oncology, University of Maryland Greenebaum Cancer Center, 22 S. Greene St., Baltimore, MD 21201, USA, [email protected] H.P. Kalofonos Division of Oncology, University of Patras Medical School, 26504 Patras, Greece, [email protected] C. Kalogeropoulou Department of Radiology, University Hospital of Patras, 26500 Patras, Greece, [email protected] K. Kamposioras University General Hospital “Attikon”, Athens, Greece, [email protected] A. Karachaliou Department of Medical Physics, University of Patras Medical School, 26500 Patras, Greece, [email protected] D. Kardamakis Department of Radiation Oncology, University of Patras Medical School, 26500 Patras, Greece, [email protected] A. Kazantzi Department of Clinical Radiology, Magnetic Resonance Unit, University of Patras Medical School, 26500 Patras, Greece, [email protected] M.I. Koukourakis Radiation Oncology Department, Democritus University of Thrace, Alexandroupolis, Greece, [email protected] M.G.E.H. Lam Department of Radiology and Nuclear Medicine, University Medical Center Utrecht, Utrecht, 3584 CX, The Netherlands, [email protected] J.W.H. Leer Department of Radiotherapy, UMC St Radboud, Nijmegen, The Netherlands, [email protected] T. Makatsoris Division of Oncology, University of Patras Medical School, 26504 Patras, Greece, [email protected] C.A. Migliorati NSU College of Dental Medicine, 3200 S. University Drive, Fort Lauderdale, Florida 33328, USA, [email protected] M.T. Nousiainen Department of Surgery, University of Toronto, Sunnybrook Health Sciences Centre, Holland Orthopaedic and Arthritic Centre, Toronto, Ontario, Canada M4Y 1H1, [email protected]

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O. Romanos Department of Clinical Radiology, Magnetic Resonance Unit, University of Patras Medical School, 26500 Patras, Greece, [email protected] G.D. Roodman Veterans Affairs Pittsburgh Healthcare System, Department of Medicine/Hematology-Oncology, Pittsburgh, Pennsylvania; and University of Pittsburgh, Department of Medicine/Hematology-Oncology, Pittsburgh, Pennsylvania, USA, [email protected] F. Saad Division of Urology, Director, Genito-Urinary Oncology, University of Montreal Hospital Centre, U of M Endowed Chair in Prostate Cancer, University of Montreal, 1560 Sherbrooke St east, Montreal, Quebec, Canada H2L 4M1, [email protected] C.D. Scopa Department of Pathology, University of Patras School of Medicine, Patras, Greece, [email protected] A. Signore Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen, 9713 GZ, The Netherlands; Medicina Nucleare, “Sapienza” University, 2nd Faculty of Medicine, 00189 Rome, Italy, [email protected] C. Simmons Department of Medical Oncology, Sunnybrook Health Sciences Centre, University of Toronto, 2075 Bayview Ave, Toronto, Ontario, Canada M4N 3M5, [email protected] E. Solomou Department of Clinical Radiology, Magnetic Resonance Unit, University of Patras Medical School, 26500 Patras, Greece, [email protected] G. Storto IRCCS, CROB, Rionero in Vulture; Institute of Biostructures and Bioimages, CNR, Naples; Department of Biomorphological and Functional Sciences, University “Federico II”, Naples, SDN Foundation, Institute of Diagnostic and Nuclear Development, Naples, Italy, [email protected] M. Trinkaus Department of Medical Oncology, Sunnybrook Health Sciences Centre, University of Toronto, 2075 Bayview Ave, Toronto, Ontario, Canada M4N 3M5, [email protected] A.C. Tsamandas Department of Pathology, University of Patras School of Medicine, Patras, Greece, [email protected] P.G. Tsoutsou Radiation Oncology Department, Democritus University of Thrace, Alexandroupolis, Greece, [email protected] V. Tzelepi Department of Pathology, University of Patras School of Medicine, Patras, Greece, [email protected]

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Contributors

Y.M. van der Linden Radiation Oncologist, Radiotherapeutic Institute Friesland, Leeuwarden, The Netherlands, [email protected] V. Vassiliou Department of Radiation Oncology, University of Patras Medical School, 26500 Patras, Greece, [email protected] N.C. Veltman Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, Groningen, 9713 GZ, The Netherlands, [email protected] C.M. Whyne Department of Surgery, University of Toronto, Orthopaedic Biomechanics Laboratory, Sunnybrook Health Sciences Centre, IBBME and IMS, Toronto, Ontario, Canada M4N 3M5, [email protected] A.J.M. Yee Department of Surgery, University of Toronto, Sunnybrook Health Sciences Centre, 2075 Bayview Ave., Rm MG 371-B, Toronto, Ontario, Canada M4N 3M5, [email protected] P. Zampakis Department of Radiology, University Hospital of Patras, 26500 Patras, Greece, [email protected] V. Zolota Department of Pathology, University of Patras School of Medicine, Patras, Greece, [email protected]

Part I

Fundamental Concepts of Bone Metastases

Chapter 1

BONE ANATOMY, PHYSIOLOGY AND FUNCTION Vassiliki Tzelepi, Athanassios C. Tsamandas, Vassiliki Zolota and Chrisoula D. Scopa Department of Pathology, Room F1-35, University of Patras Medical School, 26504 Patras, Greece, e-mail: [email protected]

Abstract:

Bone metastases depend on reciprocal interactions between malignant cells and bones that will determine the homing and growth of malignant cells in the bone microenvironment. Additionally, the final step of bone metastasis (bone destruction or production) that determines the clinical phenotype of the metastatic foci (osteolytic or osteoblastic metastasis, respectively) is actually mediated by the bone cells under the influence of various factors secreted by malignant cells. In fact, metastatic lesions are the result of disruption of the normal bone remodeling process. Thus, understanding of the normal histology and physiology of the bone is fundamental in the elucidation of bone metastasis mechanisms and the development of therapeutic interventions. This chapter presents a description of the normal structure, physiology and function of the bone, emphasizing the aspects that are most relevant to the metastatic process. It begins with a description of the anatomy and histology of normal bone and continues with a detailed discussion on the microscopic and functional characteristics of bone cells and non-cellular matrix. Finally, a discussion on embryological development of bones, comments on bone functions and a conclusion on how the different constituents of bones are involved in the highly coordinated processes of bone remodeling, mechanotransduction and mineral homeostasis, are presented. By reviewing the structure, physiology and function of the bone, the reader will be able to understand the mechanisms implicated in bone metastasis, the pathobiologic basis of the clinical phenotype and the mechanism of action of the therapeutic strategies used in clinical practice.

Key words: Osteoblast · Osteoclast · Osteocyte · Remodelling · Mechanotransduction · Mineral homeostasis · Intramembranous ossification · Enchondral ossification · Cancellous bone · Compact bone D. Kardamakis et al. (eds.), Bone Metastases, Cancer Metastasis – Biology and Treatment 12, DOI 10.1007/978-1-4020-9819-2 1, C Springer Science+Business Media B.V. 2009

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1.1 Introduction Bones are individual organs composed of multiple tissues including bone, cartilage, fat, connective tissue, hematopoietic tissue, nerves and vessels. The human skeleton is composed of 206 bones and is divided into the axial skeleton that includes the skull, hyoid, sternum, ribs and vertebrae and the peripheral skeleton that includes the bones of the limps and the pelvis. The acral skeleton is part of the peripheral skeleton and consists of the bones of the hands and feet [1]. Bone formation and function involves a complex coordination among multiple cell types. Additionally, bones are dynamic structures that are constantly remodeled during life in response to various mechanic and hormonic stimuli. This process requires a tightly regulated interplay among the various cell types of bone. Bones are classified according to their shape and size in cuboid bones (i.e., carpal and tarsal bones), flat bones (bones of the skull, ilium) and tubular bones. The latter are further subdivided into long (i.e., humerus, radius, ulna, femur, tibia, fibula) and short (i.e., metacarpal and metatarsal bones) tubular bones [1]. Additionally, bones are classified according to the manner of embryological development. Thus, membranous bones are formed de novo from undifferentiated connective tissue (intramembranous ossification) whereas enchondral bones are formed by enchondral ossification in which undifferentiated mesenchymal cells differentiate into chondrocytes and form a cartilaginous anlage that will subsequently be replaced by bone [1, 2]. Enchondral ossification of long bones forms the growth plate which divides bone into distinct anatomic regions. The epiphysis is the bone region located from the growth plate to the joint surface. The region on the other side of the growth plate is called metahysis, whereas the bone in the central region in between the two metaphyses is the diaphysis. Metaphysis is distinguished from diaphysis due to its higher vascularization and higher proportion of cancellous bone [2]. However, despite their differences in location, size, shape, and embryological development, bones are composed of the same cell types and in many cases they are histologically indistinguishable [1].

1.2 Bone Histology and Structure Anatomically, bones are composed of the periosteum, the cortex and the medulla. Periosteum surrounds the external surfaces of bones except in the region of articular cartilage [1] and serves as a transitional region between the bone and the adjacent soft tissues [3]. The cortex lies beneath the periosteum and is thicker along surfaces that bear greater load such as the diaphysis of long bones. The medulla represents the inner layer of bones and contains blood vessels, nerves and the hemapoietic or fatty bone marrow [1]. Periosteum is composed of two distinct layers, an outer fibrous and an inner cellular layer. The outer layer is in continuity with the overlying tissues and consists of fibroblasts, collagen, elastin fibers and a network of nerves and vessels. The inner cellular layer contains osteoprogenitor cells, fibroblasts and osteoblasts,

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along with microvessels and sympathetic nerves. Cellularity of the inner layer decreases with age since both osteoprogenitor cells and osteoblasts become fewer in number with maturity. Additionally, fibroblasts and vascular density of periosteum decrease with age. These age-related changes possibly contribute to the decline of periosteal bone formation rate and responsiveness to hormones and cytokines noted with increased age of the organism [1, 3]. The periosteum contributes to the regulation of cortical thickness and the maintenance of bone size and shape. The cortex is composed of compact bone whereas the medulla is composed of cancellous bone. Compact bone is hard and tan-white macroscopically and denser than cancellous bone on X-rays. On the contrary, cancellous bone is fenestrated. The percentage of compact and cancellous (or trabecular) bone tissue in a bone depends on the biomechanical requirements. Bones that are exposed to large torsional forces, like long bones, are composed mainly (80%) of compact bone, whereas bones that transmit weight-bearing forces, such as the vertebral bones, are predominantly composed of cancellous bone. In long bones, compact bone tissue is thicker in the middiaphysis, an area that is exposed in large torsial and weight-bearing forses, and thinner adjacent the articular surfaces where cancellous bone predominates and is responsible for the transmission of weight-bearing forces. Histologically, compact and cancellous bone tissues are made of two types of bone: woven and lamellar bone (Fig. 1.1). This categorization depends on the organization of type I collagen fibers of bones’ extracellular matrix. Both types can be found in cancellous and compact bone, but represent different phases of bone development. Woven bone is formed during periods of rapid osteogenesis as in the case of embryological development, normal bone growth, fracture repair and neoplastic bone

Fig. 1.1 Tissue section adjacent a fracture. Woven bone is deposited above lamellar bone

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formation (bone forming neoplasms, Codman’s triangle). It is an immature, disorganized type of bone that is characterized by an irregular arrangement of collagen fibers and a random distribution of cells, whose long axis is parallel to the neighboring collagen fibers. Woven bone is more cellular and more mineralized than lamellar bone. A characteristic feature of woven bone is that it is rapidly formed and resorbed and due to its structural organization can resist forces equally in all directions. Additionally, even though it is weaker than lamellar bone, it is more flexible [1, 4]. Lamellar bone is an organized type of bone tissue that is slowly fabricated and eventually replaces woven bone (Fig. 1.1). In normal adults, the entire skeleton is made of lamellar bone [1, 4]. It is organized in lamellas, which are composed of up to five sublayers of mineralized collagen with intermingled osteocytes [5]. Collagen fibers are distributed in parallel arrays and osteocytes are deposited in an organized fashion, since their long axis is parallel to the regularly deposited collagen fibers. As compared to woven bone, lamellar bone displays a decreased cellularity. Additionally, in contrast to woven bone, its mineralization occurs slowly and continues long after the initial deposition of organic matrix. Minerals are deposited almost exclusively within collagen fibers. Thus, lamellar bone is stronger and more rigid, but has less elasticity than woven bone [1, 4]. Lamellae of cortical (compact) bone follow three architectural patterns: circumferential, concentric and interstitial. Circumferential lamellae are the most common architectural pattern in the developing skeleton. They are the first lamellae to be deposited, being gradually replaced by concentric lamellae that surround the Haversian canals (Fig. 1.2). The Haversian canals and the lamellae that surround it are known as secondary osteons or Haversian systems. A few circumferential lamellae remain in the inner and outer surface of compact bone (just beneath the periosteum and along the endosteum, respectively) and are associated with bone growth during adult life, whereas the rest compact bone in adult life is made of concentric lamel-

Fig. 1.2 (A) Demineralized section of adult cortical bone. Haversian canals are surrounded by concentric lamellae; (B) Haversian canals contain blood vessels and mesenchymal cells. Bone lining cells and osteoblasts line the surface of the canal (arrow). Osteocytes reside in their lacunae (arrowhead)

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lae. The remaining spaces between the concentric lamellae are filled by interstitial lamellae [1, 2, 4]. The Haversian canals are a part of a branching interconnecting network that courses the cortex and are viewed as circular or cylindrical structures on tissue sections depending on the plan of section. They are filled with loose connective tissue stroma that contains vessels, nerves and mesenhymal cells (including stem cells) and are created by bone resorption. The process usually begins in circumferential lamellae of the endosteal surface of compact bone and rarely in lamellae of the sub-periosteal surface. As resorption proceeds, a canal is formed in which osteoclastic activity is oriented in the leading edge (cutting cone) followed by osteoblastic activity and new bone formation. Osteoblasts deposit lamellae of bone in a targetlike fashion around the canal. This action gradually decreases the diameter of the initial canal and create the concentric lamellae of compact bone. In each lamellae, collagen fibers are oriented in parallel to each other and to the long axis of the cells, a fundamental characteristic of lamellar bone. However, adjacent lamellae display slightly different pitches which enhances the strength of the bone [1, 4]. Nutritional support of the cells embedded in bone matrix depends on diffusion of oxygen and metabolites from vessels of the Haversian canal. Each Haversian canal and the lamellae that surround it comprise a relatively self-contained metabolic unit. Additionally, osteocyte communication through a complex network of cell processes is usually limited within the boundaries of a Haversian system. Cement lines define the boundaries of each Haversian system. They are thin and appear basophilic on tissue sections. Cement lines are believed to represent the remnants of mineralized substance that is secreted at the initial phase of bone formation and are characterized by reduced mineralization, absence of collagen and high concentration of sulfated mucosubstances. It has been proposed that cement lines mark the site of bone resorption that precedes bone formation. In normal bone, cement lines delineate the boundaries of the Haversian system in an orderly arranged pattern. In Paget disease of the bone, cement lines display a mosaic arrangement, representing the disorganized bone formation, characteristic of the disease [1, 6]. The space between the Haversian systems is filled up by interstitial lamellae. In contrast to the circular, target-like arrangement of the concentric lamellae, interstitial lamellae are irregularly shaped and represent remnants of previous generations of circular lamellas. Osteocytes of interstitial lamella are located far away from Haversian canals and may have no access to nutritional supplies and oxygen. Consequently they undergo necrosis leaving their lacuna empty [1, 4]. Cancellous bone consists of an interconnected network of trabeculae of lamellar bone and is located within the medullary cavity of bones. Red hematopoietic or yellow fat marrow resides between the trabeculae (Fig. 1.3). Large trabeculae may contain Haversian systems, surrounded by concentric lamellae, whereas smaller ones are composed of lamellae of bone oriented in the same direction as the trabeculae [1, 4]. Trabeculae that do not contain blood vessels are vascularized from adjacent bone marrow [2]. The surfaces of the trabeculae are lined by quiescent osteoblasts, being the areas where bone resorption begins. The large surface provided by the trabeculae of cancellous bone enables the skeleton to rapidly respond to

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Fig. 1.3 (A) Demineralized section of cancellous bone. Note the trabeculae, surrounded by yellow bone marrow (adipose tissue); (B) The trabeculae are composed of lamellae of bone oriented in the same direction as the trabeculae. Quiescent osteoblasts and bone lining cells line the surface of the trabeculae (arrow)

metabolic demands. Trabeculae are deposited in relation to the lines of mechanical stress according to Wolff’s law and distribute weight-bearing forces along a variety of different pathways [1, 4].

1.3 Bone Cell Types: Histology and Physiology Bone tissue, regardless of its type contains the same cell types: osteoblasts, osteoclasts, osteocytes and bone-lining cells.

1.3.1 Osteoblasts Osteoblasts are responsible for the synthesis and calcification of bone extracellular matrix and also regulate osteoclast maturation and activation. They derive from spindle-shaped cells known as osteoprogenitor cells that lie in the periosteum, the Haversian system and medullary canals [1, 6]. The differentiation of osteoblasts from osteoprogenitor cells during bone development and growth is not fully understood. However, some transcription factors that control osteogenesis have been identified. Runt-related transcription factor 2 (Runx2) and sex determining region Y-box 9 (Sox9) are basic regulators of osteogenesis and chondrogenesis. Osterix (Osx) is a zinc finger-containing protein that is important for osteoblastic differentiation. It appears that Runx2 regulates early cartilagous and pre-osteoblastic differentiation and plays an important role in the formation of cartilage anlage during enchondral bone formation, whereas Osx is important for commitment of preosteoblasts to osteoblastic phenotype [6]. However, regulation of osteoblast development in adult life remains largely unknown. Even though Runx2 seems to participate in the regulation of the activity of mature osteoblasts, transgenic expression of Runx2 in immature osteoblasts results in osteopenia, due to impaired osteoblastic maturation and enhanced osteoclastic activity. Thus, Runx2, apart from its role in osteoblasts, may be implicated in the regulation of osteoclastic function. Moreover, Runx2 expression in osteoblasts has

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Fig. 1.4 (A) Active osteoblasts line the surface of a trabeculae (arrows) and produce osteoid (arrowhead) (B) Multinucleated osteoclasts in resorption pits (arrows)

different effects depending on the stage of osteoblastic maturation and temporal control of Runx2 is important for the regulation of osteoblastic activity [7]. Osteoblasts line all bone surfaces and are spindle-shaped when they are metabolically inactive (Fig. 1.3) [1]. Some authors designate the metabolically inactive, spindle shaped osteoblasts as preosteoblasts [8]. Preosteoblast retain the capacity to proliferate but do not deposit bone matrix. They can produce type I collagen precursor molecules and express osteonectin, alkaline phosphatase, insulin-like growth factor, PTH-receptor and several integrins. Preosteoblasts differentiate into active osteoblasts, which are polyedral cells with abundant amphophilic to basophilic cytoplasm, eccentric nucleus with one to three nucleoli and prominent perinuclear halo (Fig. 1.4A). These cells are actively producing bone, but do not divide [8]. Ultrastructuraly, active osteoblasts have a well-developed Golgi apparatus, which corresponds to the perinuclear halo often seen in tissue sections, extensive granular endoplasmic reticulum, numerous mitochondria and abundant lysosomes [1]. The ultrastructure characteristicts of osteoblasts impart their function which is to produce a variety of extracellular matrix components [2]. Bone sialoprotein, osteocalcin, vitamin D3 receptor, vitronectin, thrompospondin, decorin and several bone morhogenetic proteins (BMP) are expressed by active osteoblasts, but not preosteoblasts and help the discrimination between the two cell types [8]. Type I collagen and alkaline phosphatase are considered markers of early osteoblastic differentiation (preosteoblast), whereas osteocalcin is a molecular marker for the late stage of osteoblastic differentiation [6]. Osteoblasts display multiple cytoplasmic processes that connect the cells with adjacent osteoblasts and osteocytes via gap junctions and mediate the propagation of signals among these cells [1, 9, 10]. Gap junctions are intercellular junctional complexes, formed by two juxtaposed hemichannels located in adjacent cellular membranes. Each hemichannel, also called connexon, is a hexameric of transmembrane proteins (connexins). This formation creates a transcellular conduit that permits the diffusion of ions, metabolites and small signaling molecules between the two cells and forms a functional syncytium that permits cell communication. Gap junction permeability and selectivity is dictated by its specific connexin composition, which allows a tight regulation of the type of signals that can be diffused.

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Signaling through gap junctions plays a critical role in osteoblast and osteocyte response to mechanic and hormonic stimuli. Mutations of gap junction proteins in mouse models and humans genetic diseases (i.e., oculodentodigital dysplasia, Charcot-Marie-Toth disease) are associated with skeleton malformations, which highlight their role in bone development and growth [9, 10].

1.3.2 Osteocytes As soon as osteoblasts become entrapped in the matrix they produce, they are called osteocytes. Osteocytes are the most common cell type of the bone, representing the 95% of all bone cells. Osteocytes’ half life is 25 years, whereas osteoblasts survive for up to three months and osteoclasts have a lifespan of only a few weeks [1, 8]. However, a small proportion of osteoblasts will become osteocytes (up to 30%). Most osteoblasts undergo apoptosis whereas others transform into inactive surface lining osteoblasts [8]. As active osteoblasts produce bone matrix (osteoid), they become embedded into their product. In this early stage of osteoblast to osteocyte differentiation, osteocytes are called large osteocytes, young osteocytes or osteoid-osteocytes. These osteocytes are larger than older osteocytes, retain their ability to synthesize collagen and their cytoplasm is characterized by a well developed Golgi apparatus. As mineralization of osteoid proceeds, osteocytes become smaller due to reduction in the endoplasmic reticulum and Golgi apparatus. Thus, protein synthesis also diminishes and these cells no longer express some of the markers of early osteocyte phenotype (osteocalcin, bone sialoprotein, alkaline phosphatase). Finally mature osteocytes reside in a lacunar space in the bone extracellular matrix (Fig. 1.2B) [8]. On histologic examination, osteocytes have small amounts of cytoplasm surrounding their nuclei in the lacunal space, but possess numerous long and delicate cytoplasmic processes (dendrites) that transverse the bone matrix through small tunnels called canaliculi. Their nucleus is not always visible in routine sections due to its small size [1]. Gap junctions are formed between their processes and the processes of neighboring osteocytes and surface osteoblasts and enable communication between cells. Thus, osteocytes are by no means isolated due to their remote location and envelopment in rigid mineralized extracellular matrix. Their long processes provide a large surface for contact with the matrix and extracellular fluid along the canaliculi and create a complex and integrated cell network. These properties are fundamental for their response to the mechanical and metabolic demands of the organism, since they represent sensors of strain stimuli. Osteocytes are stimulated by mechanical stimuli and influence the activity of osteoprogenitor cell, osteoblasts and osteoclasts, which in turn respond by remodeling bone mass according to environmental requirements (Wolff’s law) [11, 12]. Furthermore, osteocytes are fundamental in mineral homeostasis since they respond to changes in ion concentrations and stimulate exchange of ions between bone matrix and extracellular fluid [13]. Another function of osteocytes that is not characteristic of human osteocytes but has been well described in other vertebrate species, is osteocytic osteolysis.

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Osteocytes have a limited ability of bone resorption, which may be important for mineral mobilization during periods of increased requirements, such as pregnancy or lactation [8].

1.3.3 Osteoclasts Osteoclasts are multinucleated giant cells that mediate the resorption of bone matrix (Fig. 1.4B). They originate from fusion of preosteoclasts that derive from mononuclear hematopoietic progenitor cells of the granulocytic-macrophage colony-forming unit (GM-CFU) and the macrophage colony-forming unit (M-CFU) [1, 4, 14–20]. Various cytokines (interleukin -1, -3, -6, -11, -13, -18, tumor necrosis factor) and growth factors (granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor) regulate the development, differentiation and activity of osteoclasts [21]. Identification of the transcription factors implicated in osteoclastogenesis was largely based on studies of mutant mice with osteopetrosis. In fact, the first evidence of the hematopoietic origin of osteoclasts came from mice with osteopetrosis that were cured after injection of normal spleen cells [18]. A few years later and based on the results obtained from mice, a 3.5 month old girl with osteopetrosis was cured with bone marrow transplantation from her brother. The Y chromosome was present in osteoclasts but not in osteoblasts or other cells of the bone, thus establishing the hematopoietic origin of osteoclasts in humans [22]. Macrophage differentiation from progenitor cells is dependent on the transcriptional factor PU-1. Mice deficient in this factor lack not only macrophages, but also osteoclasts, due to deficiency of osteoclast progenitors. c-Fos is also required for osteoclast differentiation but its effect is distal to the activity of PU-1. Mice that lack c-Fos have decreased numbers of osteoclasts and increased number of macrophages, since c-Fos is implicated in the commitment of hematopoietic precursors to osteoclastic differentiation. NF-kB is also critical for the development of osteoclasts from macrophage precursors [15]. Farther maturation of osteoclasts is tightly regulated by adjacent cells especially osteoblasts and bone marrow stromal cells. TNF family receptors and ligands mediate a molecular cross talk between osteoclasts and osteoblasts that ensures the tight co-ordination of bone formation and resorption, that is vital to successful bone remodeling. RANK (receptor activator for nuclear factor kappa B) is a receptor expressed on preosteoclasts. Binding to its ligand (RANKL), which is located on the cell membrane of osteoblasts and bone marrow stromal cells, stimulates osteoclastogenesis. Its expression is upregulated by PTH, vitamin D, and various cytokines (PGE2, IL1a, TNF-a) that stimulate bone resorption. RANK, after binding to its ligand, activates TNF receptor-associated factors (TRAFs) which have been shown to activate NF-κB, c-jun N-terminal kinase (JNK) and c-fos [14]. These factors seem to be important for osteoclast differentiation and activity, since mice harboring mutations in their genes display osteopetrosis either due to low number of osteoclasts or due to the presence of a normal number of inactive osteoclasts [15].

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Osteoblasts and bone marrow stromal cells also produce a second factor that is fundamental for ostoclastogenesis. Macrophage colony stimulating factor (M-CSF) is produced by osteoblasts and bone marrow stromal cells and binds to its receptor on osteoclast precursors and stimulates their survival and proliferation. In fact, M-CSF enhances the proliferation of osteoclast progenitor cells which differentiate into osteoclasts under the influence of RANKL. Genetic modification of M-CSF gene in mouse results in osteopetrotic phenotype due to failure of osteoclast development. However, osteopetrosis resolves spontaneously over time as a result of enhanced expression of GM-CSF. Thus, M-CSF and GM-CSF are redundant in osteoclastogenesis [15]. Osteoprotegerin (also known as osteoclastogenesis inhibitory factor, OCIF) acts as a negative regulator of osteoclastogenesis. It is a soluble receptor of RANKL and is produced by various cells, including osteoblasts, hematopoietic bone marrow cells and immune cells. It acts as a decoy receptor that binds to RANKL and prevents activation of RANK and osteoclast development and activation [15, 23]. It is evident so far that adjacent cells regulate the differentiation and function of osteoclasts. Additionally, adjacent cells and not osteoclasts themselves are the targets of various stimuli that influence bone resorption. For example parathormone (PTH) is a hormone produced by parathyroid glands and is involved in the restoration of low calcium levels to normal. This is achieved by various mechanisms including mobilization of calcium from bone by increasing bone resorption. However, PTH does not act directly on osteoclasts and its receptors have been recognized on osteoblasts and stromal cells. Binding of PTH on its receptors, stimulates the expression of RANKL and blunts the expression of osteoprotegerin, thus enhancing osteoclastogenesis in a paracrine fashion. This mechanism possibly accounts for the osteoclastic activity of vitamin D as well [15]. However, the regulation of the development and activity of osteoclasts is far more complicated than described above and involves complex signal networks rather than linear signal transduction pathways [23]. Osteoclasts are found within resorption pits (Howship’s lacunae) which are produced by digestion of bone matrix by themselves. Histologically, they have 4–20 nuclei (usually less than 12 but occasionally up to 100) and abundant amphophilic cytoplasm. They are polarized with one side located in intimate apposition to the bone surface whereas the nuclei and Golgi apparatus tend to congregate on the other side of the cell (Fig. 1.4B). Integrins, and in particular integrin αν β3 , which has been shown to be the main type expressed by mature osteoclasts, play a key role in osteoclast activity [16]. Integrins are transmembrane heterodimeric proteins that consist of an α- and β- chain. The extracellular domain of integrins recognize specific motifs of aminoacid sequences in proteins of the extracellular matrix, the most common being the sequence Arg-Gly-Asp, known as RGB. Their intracellular domain, on the other hand, is associated with proteins of the cytoskeleton and is connected to intracellular signal transduction pathways. Thus integrins integrate (hence their name) the extracellular milieu with the cytoskeleton and eventually the cell nuclei. They have been designated as the sensory organ of the cell [24]. Mice deficient in αν β3

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display an osteosclerotic phenotype in vivo and their osteoclasts have diminished bone resorbing activity in vitro [25]. Additionally, apart from their binding properties, integrin complexes of the osteoclasts are associated with signaling molecules such as c-Src kinase and the FAK-like kinase Pyk2 [15]. c-Src kinase is essential for osteoclast function and its disruption in mouse models results in inactive osteoclasts [21]. Thus, blocking of integrin signaling by specific, high affinity ligands may be effective in reducing bone resorption in various pathologic situations such as osteoporosis [26]. Binding of integrin to RGD sequences of proteins of the extracellular bone matrix (vitronectin, osteopontin and bone sialoprotein) causes reorganization of cytoskeleton of osteoclasts and polarization of actin filaments to form a circular structure (actin ring) [16]. F-actin of the actin ring localizes in punctuate plasma membrane protrusions (podosomes) that are tightly attached to the bone matrix and form the sealing zone, which creates an extracellular area isolated from the rest microenvironment. The sealing zone ensures compartmentalization of plasma membrane in two distinct regions with distinct morphology and different protein composition. The membrane surrounded by the sealing zone displays numerous fingerlike extensions (brush border, ruffled zone) that increase its surface. The formation of the ruffled zone is regulated by the integrin αν β3 , since osteoclasts in αν β3 knock-out mice display a poorly formed ruffled zone [25]. The rest of the cell membrane is also subdivided into two distinct domains, even though no physical barrier has been found. These domains are known as the basolateral domain and the functional secretory domain. The functional secretory domain is thought to correspond to the apical membrane of epithelial cells. Even though their exact function is not known, functional secretoty domains are implicated in excretion of resorbed bone remnants [27]. The ruffled border contains proton pumps (H+ ATPase) coupled to a Cl− channel that excrete HCL into the leak proof area. As a result, the sealed area between the cell and the bone surface has a pH of 4.5. Additionally, a Cl− /HCO− 3 exchanger is located on the membrane surface that is opposite to the brush border and ensures maintenance of intracellular pH [27]. The ruffled border is linked with the nuclei by a network of interconnecting actin filaments. This actin network transmits signals produced by anchorage to the nuclei and orchestrates bone resorption [1]. The cytoplasm adjacent to the brush border contains numerous lysosomes that fuse with the membrane and release their contents into the sealing zone [27]. Additionally, osteoclasts contain numerous mitochondria in order to meet the high energy requirements of the resorption process. Osteoclasts express high levels of carbonic anhydrase that produces H+ from CO2 and H2 O and enables continuous release of protons to the resorption pit [1]. In the sealing zone, HCl dissolves the solid hydroxyapatite to Ca+2 , HPO2− 4 and H2 O, whereas proteolytic enzymes, whose activity is enhanced in the acidic microenvironment, mediate resorption of organic bone components. Cysteine proteinases (cathepsins K, B, D, L) and matrix metalloproteinases are the most important proteolytic enzymes in the resorption pit [27]. The sealing of the space between osteoclast and bone surface is very important not only because it assures the functional separation and different protein composition of the brush border membrane,

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but also due to the fact that it prevents the diffusion of the highly destructive solution to adjacent tissues [21]. Bone resorption begins with demineralization of collagen, followed by degradation of non-collagen proteins and catabolism of collagen fibers [1]. Organic and inorganic products are trancytosized to the free surface of osteoclasts (the functional secretory domain, which is located opposite to the brush border) where they fuse to the membrane and release their content to the extracellular fluid. This is very important for calcium mobilization to blood circulation [6]. Degradation of proteins seems to continue in transcytotic vesicles, even though the proportion of extracellular to intracellular degradation is not known. The widely used osteoclastic marker tartrane-resistant acid phosphatase (TRAP), is associated with the production of reactive oxygen species in transcytotic vesicles and may contribute to the degradation of organic ingredients of bones [27]. Organic remnants are subsequently removed by macrophages [1]. Osteoclasts are detached from bone surface and either target a new site or undergo apoptosis. However, the signals that arrest the process of bone resorption are not well understood. It has been hypothesized that high calcium levels in the resorption site are sensed by plasma membrane receptors of the osteoclasts and mediate their detachment from this site [21]. A negative feedback mechanism has also been proposed. RANK-induced expression of c-fos enhances activity and secretion of interferon β, which in turn inhibits activation of adjacent osteoclast precursor cells and decreases bone resorption [6]. Additionally, increasing evidence suggests that various stimuli, such as estrogen and bisphonates, stimulate intracellular pathways that result in osteoclast death (apoptosis) [21]. Deregulated function of osteoclasts results in pathologic situations, such as osteopetrosis, Paget disease of the bones and osteoporosis. Osteopetrosis is attributed to decreased resorption of the bone by osteoclasts. Mutations in the proton pump of the ruffled membrane or in the carbonic anydrase have been identified in cases of congenital human osteopetrosis [28]. Paget disease is initiated by increased bone resorption by osteoclasts followed by compensatory increase in bone formation by osteoblasts, resulting in a disorganized bone tissue. Osteoporosis results from enhanced bone resorption that cannot be compensated by bone formation. Mutations in RANK gene cause a familial type of osteoporosis, known as familial expansible osteoporosis or familial Paget disease of the bone that is characterized by focal areas of bone remodelling with osteolytic regions [6].

1.3.4 Bone Lining Cells The surface of the bone matrix not undergoing remodelling is lined by specialized cells called bone lining cells. They represent the inactive form of osteoblasts or may be derived from other mesenchymal cells. They are flat spindle-shaped cells that extend along bone surfaces and are connected to each other with gap junctions. Their roles include partitioning of bone fluid compartment (lacunae and canalliculi) from interstitial fluids, formation of bone-marrow barrier and regulation of other bone cells function [2].

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1.4 Bone Non-cellular Matrix Bone tissue is characterized by abundant extracellular matrix which can be subdivided into organic and inorganic (mineral) matrix. Cellular constituents represent 8% of bone weight, whereas organic matrix makes up for 25% and minerals for 67% of bone weight [4]. Organic matrix is mainly composed of collagen type I, which is secreted by osteoblasts and is organized in layers (lamellae) [29]. Other types of collagen that can be found in bone extracellular matrix include collagen type V, III, XI and XIII [1]. Orientation of lamellae depends on stage of development and type of bone. Adult skeleton is composed of lamellar bone. In adult compact bone, collagen fibers (and lamellae) are mainly organized in concentric layers around Haversian systems, whereas in adult cancellous bone, lamellae are organized parallel to the long axis of trabecullae. In contrast to the fine organization of lamellae in lamellar bone, newly formed bone, either during development and growth or due to pathologic stimuli, is characterized by disorganized orientation of lamellae [1, 4]. Collagen type I is composed of two a1 chains and one a2 chain. After secretion, collagen chains assemble into a triple helix by a highly orchestrated process. Defects in collagen assembly due to mutation in genes encoding collagen chains result in bone genetic disease known as osteogenesis imperfecta. Recent data supports a link of gene mutations or genetic polymorphisms in genes encoding collagen chains with abnormal mineral density, low body mass and possibly osteoporosis. However, apart from its structural function, collagen regulates the biologic functions of bone cells, including cell apoptosis, proliferation and differentiation [29]. Non-collagen proteins (glycoproteins and proteoglycans) comprise a small proportion of bone organic matrix and include osteocalcin, fibronectin, osteonectin, thrombospondin-2 and osteopontin. They are implicated in mineralization of bone matrix [2] and are produced by osteoblasts or are derived and concentrated from the serum [1]. The latter is the result of the excellent adsorbent properties of bone mineral which binds circulating growth factors and various serum proteins such as 2-HS-glycoprotein [30]. The most abundant non-collagenous protein of the bone is osteocalcin, which is produced by osteoblasts and functions as a regulator of mineralization. Its levels in blood serum are a useful clinical marker of bone formation [1]. In addition to bone mineralization, non-collagenous proteins have important functions in the regulation of bone mass and fracture healing [29, 31]. For example, fibronectin contains the RGD aminoacid sequence that binds to integrin receptors of cell surface and activates intracellular signal transduction pathways. Osteonectin, also known as Secreted Protein Acidic and Rich in Cysteine (SPARC) is an important mediator of tissue remodeling and its loss is associated with osteopenia due to impaired bone turnover [29]. Osteopontin belongs to the Small IntegrinBinding LIgand N-linked Glycoprotein (SIBLING) family of proteins. Members of this family bind to integrin receptors through RGD sequences. Osteopontin regulates bone resorption and is expressed in both osteoclasts and osteoblasts, possibly mediating coupling of osteoblastic and osteoclastic activity during bone remodelling [29, 32, 33]. SIBLING proteins are also expressed by malignant epithelial

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cells, such as cells of the highly osteotropic breast and prostate cancer [34,35]. They are thought to enhance colonization of bone by malignant cells and protect cancer cells from host defense [36]. Their role in bone metastasis is further supported by the finding that OPN-deficient mice exhibit fewer metastatic foci after injection of the melanoma cell line B16 [37]. Two transcription factors that are critical for osteoblastic differentiation seem to mediate transcription of bone matrix proteins as well. These factors are Runx2 and osterix. However, the precise mechanism of transcriptional regulation and response to extracellular systemic stimuli such as hormones and mechanic forces remains elusive [29]. The inorganic phase constitutes the largest proportion of bone matrix and accounts for the rigity and strength of the skeleton [1]. Hydroxyapatite [Ca10 (PO4)6 (OH)2 ] is the most common bone mineral. Carbonate and magnesium are also contained inside the crystals of hydroxyapatite and facilitate release of ions from bone mineral phase when needed [2]. During bone development, mineralization occurs by the production of several small vesicles that contain crystals of hydroxyapatite and amorphous calcium phosphate. Large mineral aggregates are formed and deposited in and around collagen fibers. In adult bone, mineralization is a complex process involving the osteoblasts and numerous growth factors, cytokines and extracellular matrix proteins. Mineral is deposited between the ends of adjacent collagen molecules and eventually crystals of apatite are situated within and outside collagen fibers [1]. Bone mineralization is slower than bone matrix deposition. Thus, a region of the bone matrix close to the site of active bone formation (i.e., adjacent the layer of osteblasts in trabeculae or Haversian canals) is unmineralized. Unmineralized bone matrix is called osteoid (Fig. 1.4A). The width of osteoid layer depends on the rate of bone formation. In inactive regions, osteoid layer is thin, whereas in foci of active bone formation the layer is several times thicker [1]. Defects in bone mineralization result in the deposition of excessive amounts of osteoid, a pathologic situation called osteomalacia [2].

1.5 Vascularization of Bones Blood supply of bones is provided by three main sources: (a) the principle nutrient artery, (b) the metaphyseal arteries and (c) the periosteal arterioles. The nutrient artery is usually one and occasionally, i.e., in the femur, two in number. They enter long bones in the diaphysis, transverse the cortex and divide into ascending and descending branches within the medullary cavity [1]. They are also accompanied by one or two nutrient veins [38]. Each artery gives rise to smaller branch arteries, arterioles and capillaries that nourish the fatty and hematopoietic bone marrow. Arterioles of the Haversian canals originate from the nutrient artery and supply the inner two thirds of the cortex, whereas the outer third of the cortex is nourished by the small periosteal arterioles [1]. In medullary cavities containing hematopoietic bone marrow, nutrient arteries end up in a plexus of sinusoids which drain through a

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system of venules into larger venous channels and finally through a central longitudinal veins into the nutrient veins [38]. Metaphyseal arteries are more numerous than nutrient arteries, enter the metaphyseal region of bones and provide blood supply to the metaphysis and epiphysis of bones [1]. At birth all bones contain hematopoietic bone marrow in their medullary cavities. In adult skeleton, most hematopoietic bone marrow is replaced by fat marrow except from the bone marrow of vertebrae, ribs, sternum, pelvis and skull [2]. The sinusoids of the hematopoietic bone marrow are lined by an endothelial layer and also contain an incomplete basement membrane and an incomplete layer of adventitial cells [38]. Endothelial cells of bone marrow sinusoids allow migration of maturing blood cells and present increased permeability as demonstrated after injection of Indian ink. This may have implications for the formation of metastases to bones, since metastatic cells enter bones mainly through anastomoses of the nutrient artery to the sinusoidal network of bone marrow and most metastatic foci are found at red marrow containing medullary cavities [2]. In the contrary, yellow bone marrow is less vascular. Vessels in fat marrow are located between adipose cells and are composed of a continuous endothelium which is surrounded by a well developed basement membrane. Vascular density of bone marrow seems to affect response to growth signals and bone growth. Thus, bone turnover is higher adjacent to red marrow and bone response to PTH has been shown to be greater in red marrowassociated bones than bones containing fat marrow [2].

1.6 Histogenesis of Bone Embryological development of bone begins prior to day 40 of gestation by migration of primitive mesenchymal cells from the cranial neural crest for the craniofacial skeleton, the paraxial mesoderm for the axial skeleton and the lateral plate mesoderm for the appendicular skeleton. Activation of homeobox genes results in the development of localized cellular condensations at the sites of future bones [1]. By the seventh week of gestation, mesenchymal cells of condensations mature either to chondrocytes or osteoblasts. There are two biologically distinct pathways that will result in the development of grossly and microscopically indistinguishable bones; membranous and enchondral (also known as endochondral) ossification [1, 30].

1.6.1 Enchondral Ossification Most bones develop through a cartilagous intermediate anlage. These include bones of the vertebral column, pelvis and extremities [30]. Cartilage has the advantage of exhibiting both appositional and interstitial growth, whereas bone tissue can only enlarge through apposition of new bone on its surface. Appositional growth is insufficient for rapid increase in size, thus bones that require rapid growth such as tubular bones of the extremities, vertebrae and ribs are first formed as a cartilagous anlage [1]. Cartilage has an additional advantage compared to other soft tissues.

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It is rigid enough to withstand mechanical loads. In fact, cartilage combines both elasticity and rigity. Elasticity in cartilage is strain-rate dependent. Thus, cartilage is hard when forces are loaded rapidly, but soft when forces are applied slowly. This is attributed to the small pores formed between extracellular matrix components which enable movement of the water only when forces are applied for a few minutes or longer [39]. Growing cartilage will subsequently be replaced by bone in its entirety, apart from the epiphyseal growth plate that will be responsible for longitudinal growth of bones in prepubertal children. Epiphyseal growth plate will eventually be replaced by bone after puberty, a process known as closure of the growth plates [1]. In enchondral ossification, the cartilage anlage is initially avascular and has the shape of adult bone. Its growth results from the proliferation of chondrocytes and the accumulation of extracellular matrix, which is composed of proteoglycans and collagen. Cartilagous anlage is surrounded by the perichondrium, which will develop into periosteum once ossification begins. The first step of ossification is the differentiation of mesenchymal cells of perichondrium into osteoblasts. A layer of osteoblasts is produced along the middle region of the cartilagous shaft. Osteoblasts deposit a collar of woven mineralized bone in this region which indicates the transformation of perichondrium into periosteum. The periosteum, osteoblasts and the collar of woven mineralized bone form the primary center of ossification which is located in the midportion of the cartilagous shaft and surrounds in a collarlike way the middle region of the diaphysis of the developing bone. Primary centers of ossification develop in long bones by the third month of fetal life. The creation of the periosteal collar determines the cessation of interstitial growth of diaphysis and from thereafter further expansion of the diameter of the bone can be accomplished only by appositional growth. The chondrocytes which are surrounded by the periosteal collar enlarge due to cell hypertrophy and swelling. This is followed by perichondrocyte deposition of collagen type X, matrix mineralization and finally chondrocyte apoptosis and activation of chondroclastic resorption. As mineralized cartilage undergoes resorption, it is penetrated by a capillary network originating from periosteal vessels around the primary center of ossification. The capillaries are accompanied by primitive mesenchymal cells, including osteoblast- and osteoclast-progenitor cells. Resorption of cartilagous matrix results into the formation of longitudinal struts of cartilage with their long axis parallel to the long axis of the bone. Osteoblasts derived from the perivascular progenitor cells deposit layers of osteoid along the surface of cartilagous struts. Thus trabeculae composed of a central cartilagous core and a peripheral rim of bone are formed. These trabeculae are called primary trabeculae. The space developing in between the trabeculae as a result of cartilage resorption, is the medullary cavity and will be occupied by varying amounts of adipose tissue and hematopoietic bone marrow elements. Thus in enchondral ossification, the primary center of ossification is initially formed in the central region of the developing bone and progresses towards both ends of bone [1, 30]. In the majority of long bones, a similar process of ossification develops in the middle of the epiphysis and forms the secondary center of ossification. Secondary

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centers of ossification develop much later than primary centers, usually after birth. In secondary centers of ossification chondrocyte maturation, mineralization, apoptosis and primary trabeculae formation proceed in the same sequence as in the primary center of ossification and progresses centrifugally towards the periphery of the epiphysis. Thus a cylindrical segment of cartilage anlage is entrapped between the two enlarging ossification centers [1, 30]. When enchondral ossification originating from the primary center of ossification reaches the diaphyseal–epiphyseal junction, the epiphyseal growth plate is formed and ensures continual growth of the length of the bone. Here a sequence of bone formation identical to the one that was previously described for the primary center of ossification occurs. The different stages of chondrocyte maturation and bone formation are structured into merging regions that form the epiphyseal growth plate. These regions are: a zone of resting chondrocytes, a zone of proliferating chondrocytes that are arranged in spiral columns, a zone of chondrocyte hypertrophy and a zone of chondrocyte apoptotic necrosis and matrix mineralization. The zones of the epiphyseal growth plate merge with a zone of cartilage resorption that is brought about by osteoclasts (chondroclasts), resulting in the formation of primary trabeculae [1, 30]. The epiphyseal growth plates close at different times in different persons, in different bones of the same person, in different ends of the same bone and even in different regions of the same epiphyseal growth plates. For example, closure of growth plates begins earlier in girls than boys by 1.5 to 2 years and the lateral half of the growth plate of the distal tibia closes several months after the median half of the same growth plate [40]. Longitudinal bone growth is controlled at three different levels. Systemic control, local control and mechanic control are all responsible for adult bone length [39]. Systemic control is mediated by several hormones, including parathyroid hormone, growth hormone, thyroid hormone, androgens, estrogens, and adrenal cortical hormones. Local control is mediated by a variety of factors that include Parathyroid Hormone related Protein (PTHrP), Indian hedgehog, fibroblast growth factors, bone morphogenetic proteins and VEGF [39, 41]. The most important local regulators are Indian hedgehog gene and PTHrP [1]. They are involved in a feedback loop that controls the width of the proliferating zone [39]. Indian hedgehog is produced by prehypetrophic chondrocytes and promotes production of PTHrP [41]. This is accomplished by increasing the expression of TGF-β by perichondrial cells [42]. PTHrP is produced by perichondrial cells and chondrocytes of the periarticular perichondrium in response to TGF-β and diffuses to the prehypertrophic zone of the growth plate, where concentration of PTHrP receptors is high. PTHrP inhibits differentiation of proliferating chondrocytes and ensures a continuous supply of proliferating chondrocytes which is important for bone growth. Fibroblast growth factors (FGF) inhibit chondrocyte proliferation and decrease chondrocyte hypertrophy and matrix synthesis [40]. Several chondrodysplasia phenotypes, including achondroplasia, the most common form of dwarfism in humans, result from aberrations in FGF activity due to mutations in the gene coding the FGF receptor [42].

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Mechanical control of bone growth was first proposed in the 19th century by the Hueter-Volkmann law and is extensively applied in orthopedic surgical procedures used to correct genu varum (hemi-epiphysiodesis). It is generally accepted that compression decreases bone formation whereas tension increases bone length, even though the mechanisms that mediate this effect are not known. However, mild compression has shown to increase bone growth and it must exceed a certain level in order to act negatively on bone development [39]. Bone growth in width is of paramount importance for bone strength. Enlargement of the diameter of the bone diaphysis is accomplished by subperiosteal bone deposition. Osteoblasts of the periosteum deposit cortical bone on the external surface of the bone. This process does not involve an intermediate step of cartilage anlage and is called intramembranous ossification. This is followed by endosteal bone resorption and results in enlargement of medullary cavity, while cortical thickness depends on the relative rate of bone formation to bone resorption. However the coordinated bone resorption on the inner cortical surface and bone formation on the outer cortical surface ensures preservation of bone shape and maintenance of the relative proportion of cortical thickness to marrow cavity diameter as bone enlarges [40]. The final shape of bones is dependent on a continuous adaptation process known as bone modelling. Bone modelling is active during growth and sculpts the shape of bones by adding bone in some places and removing it from other places. Bone modelling is less active after skeletal maturity [1, 30]. However when bone strains exceed a certain level, modelling can be re-activated in order to strengthen loadbearing bones. Additionally, bone modelling can be activated in pathologic settings and is involved in the shaping of the callus formed in regions of healing fractures and the periosteal new bone formation evoked by local processes including tumors and infections [43].

1.6.2 Intramembranous Ossification Most flat bones of the skull (frontal, parietal, occipital and temporal bones) develop directly from connective tissue without an intermediate cartilagous model. This process of bone development in which the tissue that will be replaced by bone is a fibrous-like membrane is called intramembranous ossification. Apart from the bones that are entirely formed by intramembranous ossification, this process participates in the development of all bones, since bone cortices are partly formed from subperiosteal osteoblast progenitor cells. Initially, condensed vascular connective tissue is deposited in the site of future bones. This tissue contains osteoblast progenitor cells that differentiate into osteoblasts and begin to synthesize bone matrix. After mineralization of the bone matrix, osteoblasts are entrapped in the growing bone and become osteocytes. Continuous deposition of bone forms primitive cortical bone, whereas if bone formation stops, primitive cancellous bone is created. The mesenchymal tissue of the fibrous membrane in between the trabeculae is gradually replaced by hematopoietic or

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adipose tissue. The primitive cortical and cancellous bone is composed of woven bone that will later be replaced by lamellar bone [1, 30].

1.7 Bone Functions Bones along with the muscles, tendons and ligands attached on them are responsible for movement and standing [2,6]. Bone development was critical for the evolution of our species, since it facilitated locomotion and bipedalism [28]. Additionally, bones surround cavities in which critical organs are protected from external mechanical forces [2, 6]. For example, skull bones form the cranial cavity where the brain resides. Pleura and thoracic vertebrae form the thoracic cage which is important for the protection of the heart and lungs. The spinal cord is located in the spinal tube. In between the trabeculae of cancellous bone resides the hematopoietic bone marrow. Apart from their protective role, bone cells have the ability to respond to signals that regulate hematopoiesis. Bone resorption increases diameter of medullary cavity when needed for example in high altitudes [28]. Apart from the obvious structural functions mentioned above, bones are more than a rigid inactive organ and exert various metabolic functions in the body, since they are critically involved in mineral homeostasis. Bones deposit and store minerals, especially calcium and phosphate, which are released when needed. Thus, bone tissue responds to changes in blood calcium and phosphate levels and is involved in restoration of their levels within normal limits [2, 6]. Additionally, bones’ large surface absorbs toxins and heavy metals, minimizing their deleterious effects on other tissues [44]. Additionally, bones play a critical role in the regulation of hematopoiesis. Bone microenvironment provides a supportive microenvironment for the development of mature blood cells from hematopoietic stem cells, known as the stem cell niche. Interactions between progenitor cells and their microenvironment determine the maturation process, providing both permissive and instructive signals for stem cell differentiation. The hematopoietic stem cell niche is composed of the osteoblasts located along the endosteal surface and the bone marrow blood vessels. It has been proposed that osteoblastic niche provides a quiescent microenvironment for stem cells whereas the vascular niche drives proliferation and further differentiation of hematopoietic stem cells [45].

1.8 Bone Physiology 1.8.1 Remodelling Bone remodelling is defined as the replacement of older bone by newer bone and takes place in all bone surfaces. These are the periosteal surface, the endosteal surface, the surfaces of Haversian canals of compact bones and the surfaces of trabeculae of cancellous bone. Bone remodelling begins in embryonal life and continues

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throughout life. In adult skeleton bone remodelling represents the 90% of bone turnover [30]. Bone remodelling is mediated by a temporary bone structure called basic multicellular unit (BMU). Osteoclasts and osteoblasts are, as expected, the major components of this unit [2]. Osteocytes and bone lining cells are also involved in bone remodelling. Changes in bone strain and release of various hormones and cytokines stimulate osteocytes or bone lining cells. These cell types are connected to each other and to osteoblasts through a complicated system of cell processes that run through the bone matrix. The bone lining cells contract and secrete collagenase that digests a protective-coating layer of matrix and combined with cell contraction, exposes the mineralized bone surface. Bone exposion, together with signals released from osteoblasts, stimulates osteoclast formation. Osteoclasts mediate bone resorption which lasts two to four weeks and is followed by the reversal phase, during which resorption remnants are cleaned up. Subsequently, new bone is formed by osteoblasts and is mineralized. Bone formation lasts about four months [28]. In cortical bone, bone remodelling forms the cutting cone, a cone-shaped structure that bores holes in the hard and compact matrix of compact bone and is the top of a cylinder. The osteoclasts are gathered in the leading edge of the cutting cone. Osteoclasts are followed by a capillary loop surrounded by mesenchymal osteoblast progenitor cells. The later differentiate into osteoblasts that mediate the formation of new bone matrix (osteoid) and its minelarization, thus refilling the gap made by osteoclastic resorption. The same sequence of events occurs during remodelling of cancellous bones. Remodelling begins at the trabecular surface, where osteoclasts are activated and start to erode the bone matrix forming a resoption cavity. After removal of remnants, osteoblasts form new bone at the same site [30]. Bone mass is completely renewed every 10 years, since each year about 10% of bone is remodelled [6]. The ratio between bone formation to bone resorption determines the total bone mass. Disruption of the balance between the two processes may lead to bone loss or gain. This disruption may be reversible, so that bone mass returns to normal when stimuli (i.e., hypocalcaemia) is eliminated or may lead to irreversible deregulation of bone mass, especially bone loss (osteopenia or even osteoporosis) [2,30]. Additionally, bone loss may occur when mechanical loads stay below a threshold range (the remodelling threshold rage) and is called disuse-mode remodelling [43]. Bone remodelling activity varies between different bones and different regions of the same bone. Activity of remodelling (measured by the frequency with which a given site on the bone surface undergoes remodelling, the activation frequency) is identical between cortical and cancellous bone. However, cancellous bone is more (5–10 times) actively remodelled than cortical bone, possibly due to its higher surface to total volume ratio [30]. Thus, when the balance between bone formation and bone resorption is negative (i.e., less bone is formed than reabsorbed), it usually takes place next or close to the bone marrow cavity. This explains why 90% of bone loss during ageing or impaired-mode remodelling comes from bone next to the marrow [46].

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In bone remodelling, resorption which is the main function of osteoclasts, is coordinated with bone formation by osteoblasts. The two processes are coupled in order to maintain bone mass and usually when one increases the other will follow and other way round. The exact mechanisms that mediate the coupling of the two processes are not fully understood. Several factors have been shown to stimulate both bone resorption and formation. These include (among others) PTH, PGE, FGF, TGF-β [7]. In fact, PTH has both anabolic and catabolic function in bone mass, depending on the pattern and duration of action [47]. Additionally, during matrix degradation from osteoclasts, factors that enhance bone formation are released, such as the insulin like growth factor (IGF) and the transforming growth factor-β (TGF-β) [7]. Estrogens are among the major regulators of bone remodelling and act by altering the production or activity of factors that regulate bone resorption and formation [44]. In addition to hormones and growth factors that determine the rate of remodelling, mechanical loads seem to determine the site of remodelling [28]. Bone remodelling serves two basic functions of human skeleton, mechanical support and mineral homeostasis [2]. The human skeleton has to adjust to alterations of mechanical stress and this is accomplished by degradation of old bone and formation of new aligned with the new environmental demands. The adaptation of bones to local mechanical forces is the basis for orthopaedic and orthodontic procedures [7]. Additionally, repair of fatigue damages and micro-fractures is largely dependent on bone remodelling. Alterations of calcium levels in blood serum elicit a response in bone cells in order to restore calcium levels to normal [30]. Thus, mechanical forces and calcium levels are the major regulators of bone remodelling. However, there is a hierarchy in bone functions with calcium homeostasis being the most important. Thus, calcium deficiency will eventually lead to bone loss in spite of the presence of mechanical demands [7]. The normal pathway of bone remodelling is exploited by inflammatory cells and tumor cells which explain the bone loss that accompanies chronic inflammatory diseases, i.e., rheumatoid arthritis, and bone loss or gain that is noted in osteolytic or osteoblastic bone metastasis [6, 21, 28, 48–50].

1.8.2 Mechanotransduction Mechanotransduction refers to the process of translating mechanical signals generated by physical forces such as tension and compression into a cellular response, via the activation of intracellular signaling pathways. In bone, mechanotransduction is a basic function of osteocytes which are capable of monitoring the physical forces and eliciting bone response by activating effector cells (osteoblasts and osteocytes). The net effect of the coordinated action of bone cells is the organization of bone tissue according to the direction of mechanical forces. This leads to mechanical adaptation of bone structure and ensures efficient bearing of load by a relatively thin and light structure [51]. The asteroid morphology of osteocytes, with their numerous dendritic processes is fundamental for mechanical adaptation and transduction of mechanical signals.

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Osteoblasts are buried in a rigid mineralized matrix, but a complex network of cell dendrites connects each cell with its neighbors and with cells of the endosteal and periosteal bone surface [11]. Dendrites are connected with gap junctions, which facilitate the integration of signals in effector cells and the activation of osteoblasts and bone-lining cells. Additionally, the bone matrix around cell bodies (around the lacunae) and cell processes (along the course of canaliculli) does not calcify and is therefore more easily penetrated by water and small molecules than the mineralized bone tissue of the lamellae. This complex structure of pores and channels, named the lacuno-canalicular porosity is the basis of mechanotransduction [51]. Since bone tissue is stiff and rigid, the strain imposed by mechanical loads is very low and 10 times less than the one needed for in vitro activation of osteocytes. Consequently, activation of osteocytes directly by the strain produced by mechanical forces seems improbable. However, squeezing of extracellular fluid present in the lacuno-canalicular system produces shear forces at the cell membrane of the osteocyte body and dendrites. Mechanical perturbation of cell membrane is the principal stimulus of the cell. Streaming potentials may be generated by fluid movements over the cell surface, but their role in cell activation remains elusive [51]. Regarding the molecular basis of mechanic activation of osteocytes, two types of cell surface receptors are involved: those that respond to changes in the solid matrix structure of the bone and those that are activated indirectly by shear stress imposed by fluid flow [11]. Integrin dimmers bind to extracellular matrix proteins and activate via adaptor proteins two intracellular pathways: the MAPK (mitogen activated protein kinase) pathway and the PLC/IP3 (phospholipase C/triphosphate inositol) pathway. Moreover, integrin intracellular domains bind through adaptor proteins to the cytoskeleton and modulate gene expression via connections of actin cytoskeleton to the nuclear envelope. The MAPK pathway results in ERK1/2 activation, which in turn enhances activation of the transcription factor AP-1. IP3 opens stores of intracellular calcium, whereas cytoskeleton modulation by integrin ensures cell stabilization against applied forces. Additionally, stretch-activated ion channels are present on the cell membrane and respond to stress stimuli, allowing extracellular calcium to flow in cells. Increases in intracellular calcium through influx from the extracellular fluid along with release from intracellular stores, activate calcium responsive proteins, such as protein kinase C, CAM kinase and calcineurin, which in turn activate various transcription factors. Integration of various pathways results in transcriptional activation of bone growth-promoting genes including c-fos, IGF-1, cycloxygenase and osteocalcin [12]. Nitric oxide (NO) and prostaglandins are other important mediators of the mechanically induced cell responses. Their role in dilatation of blood vessels is well known. Endothelial cells sense blood fluid shear stress, generated by increased blood pressure and respond by increasing the diameter of blood vessel. Several similarities between the response of endothelial cells to mechanic stimuli and that of osteocytes have been found, including up-regulation of prostaglandins, expression of ecNOS (endothelial Nitric Oxide Synthase) and release of NO. Additionally, mechanical activation of osteocytes enhances production of growth actors (i.e., IGF) and other paracrine anabolic factors (i.e., PGE2 ). These factors are transferred to the bone

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surface via the lacuno-canalicular system either through intracellular or extracellular pathways and stimulate the recruitment of osteoblasts. Furthermore, various data suggest the existence of active suppression of osteoclasts by osteocytes, even though the exact mechanisms remain unknown [51].

1.8.3 Mineral Homeostasis Bones play a major role in mineral homeostasis and are the major reservoir of most essential minerals. Almost all body’s calcium (99%) and 85% of body’s phosphorus is deposited in bone matrix. Apart from the calcium and phosphorus hemostasis that are traditionally known to be regulated by bone, other essential minerals are also harbored in bone matrix. Bones contain 95% of body’s sodium and 50% of body’s magnesium [1]. The mineralized skeleton seems to have evolved to fulfil the need for calcium homeostasis in terrestrial environments. For the marine organisms bones are not critical for calcium homeostasis since calcium is plentiful in the sea. In these organisms mineralized skeleton is probably crucial for phosphate storage. However, for mammalian species of the land, bones are crucial calcium reservoirs, providing a continuous source of calcium between meals. Serum calcium is regulated between very narrow limits (8.5 to 10.2 mg/dL, corrected for serum albumin concentration). The strict extracellular levels of calcium reflect its numerous intra- and extracellular roles in various vital processes including neuromuscular activity, blood clotting and intracellular signal transductions. The tight regulation of calcium homeostasis is achieved through a finely regulated interplay between PTH, calcitonin and vitamin D. Bone, kidney and intestine are the target organs of these hormones [28, 52]. PTH and calcitonin act primarily on bones. Additionally, even though vitamin’s D major target is the intestine, Vitamin D receptors exist in the nuclei of osteoblasts and possibly regulate indirectly osteoclastogenesis [53]. PTH is the major regulator of calcium homeostasis in humans, acting primarily on the bones and the kidneys [47]. PTH is produced by chief cells of parathyroid glands in response to low levels of calcium. On the contrary, elevation of calcium levels result in decrease of PTH production. Parathyroid cells sense extracellular levels of calcium by the calcium-sensing receptor located on their cell membrane [52]. PTH activates PTH receptor expressed in target organs. The receptor is also activated by PTHrP, a hormone implicated in regulation of bone growth during development. PTHrP is also ectopically expressed by some malignant neoplasms resulting in hypercalcaemia known as humoral hypercalcaemia of malignancy. In bone, PTH receptors are expressed by osteoblasts, osteocytes and bone-lining cells [47]. After binding to its receptor, PTH enhances the expression of RANKL which in turn binds to the RANK receptor of the osteoclast progenitor cells and enhances osteoclastogenesis [15]. Additionally, PTH stimulates the production of collagenase-3 and decreases production of type I collagen from osteoblasts [54]. Thus, PTH stimulates bone resorption and calcium release through transformation of osteoblasts from cells involved in bone formation, to cells driving bone

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resorption, both directly and indirectly [54]. Continuous exposure to elevated levels of PTH, as in the case of primary hyperparathyroidism or chronic renal failure, results in osteitis fibrosa cystica which is characterized by increased bone resorption [55]. On the contrary, intermittent increase in PTH levels are associated with anabolic effects regarding bone mass. PTH causes sub-periosteal bone formation, increases trabecular and cortical thickness and improves bone mineral density. PTH analogs have been used for the therapy of osteoporosis especially in patients not responding to other treatment modalities [47], since its use stimulates bone formation instead of simply inhibiting bone resorption [56]. Thus theoretically, PTH analogs can be used to reverse the osteoporotic phenotype. The anabolic effects of PTH are mediated by its ability to stimulate the proliferation of preosteoblasts, the promotion of the differentiation of preosteoblasts and osteoblasts and the inhibition of osteoblast apoptosis [54, 56]. The function of PTH in regard to the bone mass is regulated by several feedback loops and depends on the timing of PTH administration (continuous or pulsative) rather than the levels of PTH. It has been proposed that pulsative administration of PTH, which is characterized by a rapid increase in PTH blood levels followed by rapid decline to baseline, is associated with rapid recovery from feedback loops and gives the opportunity of new activation of downstream molecules. In that case, the balance between bone formation and bone resorption is in favor of the former. In contrast, continuous PTH administration results in a constant down regulation of the PTH receptors and other effector molecules (i.e., cAMP) due to feedback mechanisms and the lower ratio of bone formation to bone resorption [56]. Calcitonin is involved in restoration of high calcium levels. It is produced by parafollicular cells (C cells) of the thyroid gland. Calcitonin acts on bones and kidneys, decreasing calcium release and increasing calcium excretion, respectively. Calcitonin is a widely used drug for the treatment of osteoporosis due to its antiosteoclastic effects. Calcitonin acts directly on osteoclasts and inhibits bone resorption interfering with osteoclasts’ development, differentiation and motility [57]. Vitamin D is the third major regulator of calcium homeostasis. It is synthesized in the skin after conversion of 7-dehydrocholesterol to vitamin D by ultraviolet light of sunlight or can be of dietary origin. Vitamin D is subsequently hydroxylated by 25-hydroxylase of the liver and 1-hydroxylase of the kidney. These modifications produce the active form of the molecule [1,25(OH)2 D] [58]. Vitamin D regulates bone mineralization indirectly by enhancing intestinal absorption of calcium. Its deficiency results in impairment of bone mineralization. Bones are characterized by the presence of excessive amounts of unmineralized matrix. In children with vitamin D deficiency, bone growth is defective and the developing bones are not rigid resulting in severe skeletal deformation (rickets). Upon deficiency in adults, the shape of the bones is not affected, but due to impaired mineralization, bones have loose their rigity and are susceptible to fractures (osteomalakia) [42, 53]. However, vitamin D has dual functions concerning bone mass. In normal conditions it enhances bone formation indirectly, due to increased absorption of calcium from the intestine as described above. On the contrary, in supraphysiological

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doses, vitamin D induces bone resorption and releases calcium from the bone to the blood circulation. This is possibly accomplished through direct actions on bone cells especially osteoblasts which express osteoblastic vitamin D receptor in their nuclei. Activation of vitamin D receptors of osteoblasts enhances the expression of factors that stimulate osteoclastogenesis (RANK, M-CSF) and increases bone resorption [53].

References 1. Rosenberg AE, Roth SI (2007) Bone. In: Mills SE (ed.) Histology for histopathologists, 3rd edn. Lippincot Williams, Philadelphia, PA 2. Shea JE, Miller SC (2005) Skeletal function and structure: implications for tissue-targeted therapeutics. Adv Drug Del Rev 57:945–957 3. Allen MR, Hock JM, Burr DB (2004) Periosteum: biology, regulation and response to osteoporosis therapies. Bone 35:1003–1012 4. Walsh WR, Walton M, Bruce W, et al. (2003) Cell structure and biology of bone and cartilage. In: An YH, Martin KL (eds.) Handbook of histology methods for bone and cartilage. Humana Press, Totowa, NJ 5. Weiner S, Traub W, Wagner HD (1999) Lamellar bone: structure-function relations. J Struct Biol 126:241–255 6. Cohen MM Jr. (2006) The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A 140A:2646–2706 7. Harada S-I, Rodan GA (2003) Control of osteoblasts function and regulation of bone mass. Nature 243:349–355 8. Franz-Odendaal TA, Hall BK, Witten PE (2006). Buried alive: how osteoblasts become osteocytes. Dev Dyn 235:176–190 9. Stains JP, Civitelli R (2005) Cell–cell interactions in regulating osteogenesis and osteoblast function. Birth Def Res C 75:72–80 10. Stains JP, Civitelli R (2005) Gap junctions in skeletal development and function. Biochem Biophys Acta 1719:69–81 11. Knothe Tate ML, Adamson JR, Tami AE, et al. (2004) The osteocyte. Int J Biochem Cell Biol 36:1–8 12. Iqbal J, Zaidi M (2005) Molecular regulation of mechanotransduction. Biochem Biophys Res Commun 328:751–755. 13. Cullinane DM (2002) The role of osteocytes in bone regulation: mineral homeostasis versus mechanoreception. J Musculoskelet Neuronal Interact 2:242–244 14. Chambers TJ (2000) Regulation of the differentiation and function of osteoclasts. J Pathol 192:4–13 15. Teitelbaum SL (2000) Bone resorption by osteoclasts. Science 289:1504–1508 16. Teitelbaum SL (2000) Osteoclasts, integrins, and osteoporosis. J Bone Miner Metab 18:344–349 17. Walker DG (1972) Congenital osteopetrosis in mice cured by parabiotic union with normal siblings. Endocrinology 91:916–920 18. Walker DG (1973) Osteopetrosis cured by temporary parabiosis. Science 180:875 19. Walker DG (1975) Bone resorption restored in osteopetrotic mice by transplants of normal bone marrow and spleen cells. Science 190:784–785 20. Walker DG (1975) Spleen cells transmit osteopetrosis in mice. Science 190:785–787 21. Bar-Shavit Z (2007) The osteoclast: a multinucleated, heatopoietic-origin bone-resorbing osteoimmune cell. J Cell Biochem 102:1130–1139 22. Coccia PF, Krivit W, Cervenka J, et al. (1980) Successful bone-marrow transplantation for infantile malignant osteopetrosis. N Engl J Med 302:701–708

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23. Blair HC, Robinson LJ, Zaidi M (2005) Osteoclast signalling pathways. Biochem Biophys Res Commun 328:728–738 24. Clark EA, Brugge JS (1995) Integrins and signal transduction pathways: the road taken. Science 268:233–239 25. McHugh KP, Hodivala-Dilke K, Zheng MH, et al. (2000) Mice lacking αν β3 integrins are osteosclerotic due to dysfunctional osteoclasts. J Clin Invest 104:433–440 26. Engleman VW, Nickols GA, Ross FP, et al. (1997) A peptidomimetic antagonist of the a´ vâ3 integrin inhibits bone resorption in vitro and prevents osteoporosis in vivo. J Clin Invest 99:2284–2292 27. Vaananen HK, Zhao H, Mulari M, et al. (2000) The cell biology of osteoclast function. J Cell Sci 113:377–381 28. Rodan GA (2003) The development and function of the skeleton and bone metastasis. Cancer 97:726–732 29. Young MF (2003). Bone matrix proteins: their function, regulation and relationship to osteoporosis. Osteoporosis Int 14:S35–S42 30. Steiniche T, Hauge EM (2003) Cell Normal structure and function of the bone. In: An YH, Martin KL (eds.) Handbook of histology methods for bone and cartilage. Humana Press, Totowa, NJ 31. Alford AI, Hankenson KD (2006) Matricellular proteins: extracellular modulators of bone development, remodelling and regeneration. Bone 38:749–757 32. Dodds RA, Connor JR, James IE, et al. (1995) Human osteoclasts, not osteoblasts, deposit osteopontin onto resorption surfaces: an in vitro and ex vivo study of remodelling bone. J Bone Miner Res 10:1666–1680 33. Asou Y, Rittling SR, Yoshitake H, et al. (2001) Osteopontin facilitates angiogenesis, accumulation of osteoclasts, and resorption in ectopic bone. Endocrinology 142:1325–1332 34. Bellahcene A, Castronovo V (1997) Expression of bone matrix proteins in human breast cancer: potential roles in microcalcification formation and in the genesis of bone metastases. Bull Cancer 84:17–24 35. Waltregny D, Bellahcene A, Van Riet I, et al. (1998) Prognostic value of bone sialoprotein expression in clinically localized human prostate cancer. J Natl Cancer Inst 90:1000–1008 36. Jain A, Karadag A, Fohr B, et al. (2002) Three SIBLINGs enhance factor H’s cofactor activity enabling MCP like cellular evasion of complement-mediated attack. J Biol Chem 277:13700–13708 37. Nemoto H, Rittling SR, Yoshitake H, et al. (2001) Osteopontin deficiency reduces experimental tumor cell metastasis to bone and soft tissues. J Bone Miner Res 16:652–659 38. Wickramasinghe SN (2007) Bone Marrow. In: Mills SE (ed.) Histology for histopathologists, 3rd edn. Lippincot Williams, Philadelphia, PA 39. Rauch F (2005) Bone growth in length and width: the yin and yang of bone stability. J Mucoskelet Neuronal Interact 5:194–201 40. Forriol F, Shapiro F (2005) Bone development. Interaction of molecular components and physical forces. Clin Orthop Relat Res 432:14–33 41. Adams SL, Cohen AJ, Lassova L (2007) Integration of signaling pathways regulating chondrocyte differentiation during endochondral bone formation. J Cell Physiol 213:635–641 42. Ballock RT, O’Keefe RJ (2003) The physiology and pathophysiology of the growth plate. Birth Def Res C 69:123–143 43. Frost H (2004) A 2003 update of bone physiology and Wolff’s law for clinicians. Angle Orthod 74:3–15 44. Raisz LG (1999) Physiology and pathophysiology of bone remodelling. Clin Chem 45:1353–1358 45. Kropp HG, Avecilla ST, Hooper AT, et al. (2005) The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda) 20:349–356 46. Frost H (2001) From Wolff’s law to the Utah paradigm: insights about bone physiology and its clinical applications. Anat Rec 262:398–319

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Chapter 2

PATHOPHYSIOLOGY OF BONE METASTASES G. David Roodman Department of Medicine/Hematology-Oncology, University of Pittsburgh School of Medicine, VA Pittsburgh Healthcare System, Research and Development 151-U, University Drive C, Rm. 2E113, Pittsburgh, PA 15240, USA, e-mail: [email protected]

Abstract:

Bone is a very common site for cancer metastasis and may be the only site of metastasis in patients with breast cancer or prostate cancer. The exact incidence of bone metastasis is unknown, but it has been estimated that approximately 300,000–400,000 people in the United States die from bone metastasis each year. Bone metastasis can involve any bone but has a predilection for areas of red bone marrow. Bone lesions are characterized as either osteolytic or osteoblastic, but this classification actually represents extremes of a continuum in which normal bone remodeling, where bone destruction and formation are balanced, is unbalanced. Increased bone destruction is characteristic of osteolytic metastasis and markedly increased bone formation results in osteoblastic metastasis. However, patients can have both osteolytic and osteoblastic metastasis as well as mixed lesions containing both elements. Bone is a frequent site of involvement in patients with prostate cancer, breast cancer and multiple myeloma (MM) because the propensity of these tumors to home to bone and the capacity of bone marrow to support the growth of the tumor. In patients with osteolytic and osteoblastic bone metastasis, both osteoclast formation and activity are increased. In osteoblastic bone metastasis osteoblast is also increased by tumor derived factors. However, the factors which enhance osteoclast or osteoblast activity differ among different tumor types. The identification and characterization of the pathophysiologic mechanisms and factors that underlying bone metastasis have provided important new therapeutic targets for treating these patients, who are currently incurable, and are reviewed in this chapter.

Key words: Metastasis · Bone · Pathogenesis of Bone Metastasis · Osteoclast · Osteoblast D. Kardamakis et al. (eds.), Bone Metastases, Cancer Metastasis – Biology and Treatment 12, DOI 10.1007/978-1-4020-9819-2 2, C Springer Science+Business Media B.V. 2009

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2.1 Introduction Bone is a very common site for cancer metastasis and may be the only site of metastasis in patients with breast cancer or prostate cancer. The exact incidence of bone metastasis is unknown, but it has been estimated that approximately 300,000– 400,000 people in the United States die from bone metastasis each year [1]. Bone metastasis is frequently seen in patients with breast cancer and prostate cancer with up to two-thirds to three-fourths of the patients developing bone metastasis [2], and 15–30% of patients with lung, bladder, uterine, rectal, thyroid or renal cancer developing bone metastasis during the course of their disease (Table 2.1). The development of bone disease has a major impact on the survival of patients with cancer. In a study by Saad et al. [3] in which 3,049 patients with MM, breast cancer, prostate cancer, lung cancer or other solid tumors were analyzed, patients with MM have the highest fracture incidence at 43%, followed by breast cancer and prostate cancer, with lung cancer and other solid tumors only having 17% (Table 2.2). Further, the occurrence of fractures has a major effect on survival for these patients. An increased risk of death was significantly associated with both nonvertebral and vertebral fractures for all tumor types. The risk of death in patients with breast cancer for example was increased 24% in patients with nonvertebral fractures and 19% in patients with vertebral fractures. Similarly, an increase risk of death was seen in patients with nonvertebral fractures who have prostate cancer. In Table 2.1 Bone metastasis is a frequent complication of cancer. Myeloma is the most frequent cancer to involve bone followed by breast cancer and prostate. Once cancer metastasizes to bone, it is incurable for the overwhelming majority of patients. The incidence of bone metastasis and median survival of patients with bone metastasis is depicted in this table

Myeloma Melanoma Bladder Thyroid Lung Breast Prostate

Incidence of Bone Metastases (Thousands)a,b

Median Survival (Months)b−d

70–95 20–25 14–45 40 60 30–40 65–75 65–75

6–54 12 6 6–9 48 6–7 19–25 12–53

a

Mathers, et al. [88]. Coleman [89]. c American Cancer Society [90]. d Zekri, et al. [91]. b

Table 2.2 Percentage of cancer patients presenting with fractures Multiple Myeloma Breast Cancer Prostate Cancer Lung Cancer and Other Solid Tumors Adapted from Saad, et al. [3].

n

Percent

513 1130 640 766

43 35 19 17

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Pathophysiology of Bone Metastases

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MM patients, there was a 20% increase risk of death in patients who sustained a fracture compared to patients who did not. Further, the economic burden of metastastic bone disease is tremendous [4]. The mean medical cost for patients with metastatic bone disease was $75,329 compared to $31,082 from patients without bone involvement and similar tumors. The incremental cost for all cancer types studied with bone involvement was $44,442 with the highest increment seen in patients with MM ($63,455). These authors estimated that the national cost burden for patients with metastatic bone disease was 12.6 billion dollars. These results clearly demonstrate that metastatic bone disease has a tremendous impact both on the well-being of patients, the survival of patients, and the cost of their care. In addition, the morbidity that is associated with bone metastasis can be devastating. Patients can have severe bone pain, pathologic fractures, nerve compression syndrome including spinal cord compression [5], and if they have high levels of bone resorption, life-threatening hypercalcemia.

2.2 Clinical Manifestations of Bone Metastasis Bone metastasis can involve any bone but has a predilection for areas of red bone marrow. Bone lesions are characterized as either osteolytic or osteoblastic (Fig. 2.1), but this classification actually represents extremes of a continuum in which normal bone remodeling, where bone destruction and formation are balanced, is unbalanced. Increased bone destruction is characteristic of osteolytic metastasis and markedly increased bone formation results in osteoblastic metastasis. However, patients can have both osteolytic and osteoblastic metastasis as well as mixed lesions containing both elements. For example, patients with MM have pure lytic lesions, while patients with prostate cancer have predominantly osteoblastic lesions. Patients with breast cancer also have osteolytic lesions, but at least 15–25% of these patients may have skeletal disease, which is predominantly osteoblastic in nature [6]. Because there still is reactive new bone formation in response to the bone destruction in patients with bone metastasis from solid tumor, bone scans can identify these sites of new bone formation. However, in MM where bone formation is markedly suppressed or even absent, bone scans can underestimate the extent of the bone disease [7]. Even in patients who have predominantly osteoblastic metastasis there is still ongoing bone destruction. This has been shown by studies of Coleman and others in prospective trials of bisphosphonates for the treatment of bone metastasis [8]. These studies clearly demonstrate that patients with prostate cancer can have very high levels of bone resorption markers in their urine and serum. Further, agents that block bone resorption can decrease bone pain and pathologic fractures in patients with prostate cancer [9].

2.3 Bone as a Preferential Site for Bone Metastasis Bone is a frequent site of involvement in patients with prostate cancer, breast cancer and MM because the propensity of these tumors to home to bone and the capacity of bone marrow to support the growth of the tumor. The metastatic process, which

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(A)

(B)

(C)

Fig. 2.1 Osteoclasts and osteoblasts in normal bone and bone metastasis. (A) Osteoclasts and osteoblasts in normal bone. The large osteoclast (arrow) is actively resorbing bone. Osteoblasts are small cuboidal shaped cells that actively lay down bone matrix. Magnification 100× (generously provided by Dr. Hua Zhang, Helen Hayes Hospital, New York). (B) Osteolytic bone metastasis. Renal carcinoma cells are seen invading the bone marrow, and osteoclasts (arrows) are actively resorbing bone adjacent to the tumor cells. Magnification 200× (courtesy of Dr. Brendan Boyce, University of Rochester, New York). (C) Osteoblastic metastasis. Thickened trabeculae and large numbers of osteoblasts are seen next to the bone surface. Tumor cells from adenocarcinoma of the lung are easily seen between the two large trabeculae. Magnification 200× (courtesy of Dr. Brendan Boyce, University of Rochester, New York)

involves bone destruction and/or formation, enhances both the growth of the tumor due to the release of activated growth factors from bone during the bone resorptive process and the production by bone marrow stromal cells and osteoblasts of growth factors and cytokines, which enhance the survival and growth of the tumors. Many of these stromal cell-derived growth factors and cytokines are upregulated due to adhesive interactions between tumor cells and bone marrow stromal cells (Fig. 2.1, Table 2.3) [10]. In addition, soluble factors produced by tumors themselves further enhance the production of cytokines by bone marrow stromal cells, which also

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Table 2.3 Adhesive Interactions Involved in Bone Metastases Tumor ∗

Breast CA Breast CA Myeloma ∗

Receptor

Integrin

Effect

Urokinase Bone sialoprotein VCAM1, fibronectin

β1 Integrin αv β5 α4 β1 , α5 β1

Tumor progression Adhesion and progression Growth factor production

CA: carcinoma. Table 2.4 Stromal/Osteoblast-Derived Factors that may Affect Bone Metastases PDGF

RANKL

VEGF FGFs IGFs TGF-β

IL-6 BMPs SDF-1 MCP-1

PDGF: platelet-derived growth factor; RANKL: ligand of the receptor activator for nuclear factor κB; VEGF: vascular endothelial growth factor; IL-6: interleukin 6; FGFs: fibroblast growth factors; BMPs: bone morphogenic proteins; IGFs: insulin-like growth factors; SDF-1: stromal cell-derived factor 1; TGF-β: transforming growth factor β; MCP-1: monocyte chemoattractant protein 1.

increase tumor survival and growth. Cytokines that have been shown to be active in this process are listed in Table 2.4 [10]. In addition, blood flow is high in areas of red marrow [11], which may account for the predilection of metastasis to these sites. Further, bone is the largest repository of immobilized growth factors in the body, including transforming growth factorβ (TGF-β), insulin-like growth factors (IGF)-1 and -2, fibroblast growth factors (FGF), platelet-derived growth factors (PDGF), bone morphogenetic proteins, and calcium [12], which make bone a perfect site for metastasis. These growth factors, which are released and activated during bone resorption [13], provide fertile ground in which tumor cells can grow. This “seed-and-soil hypothesis” of the mechanism of bone metastasis was first advanced by Stephan Paget in 1889 [14], and is supported by animal models of bone metastasis. Recently, the bone marrow has also been shown to play a major role in maintaining tumor cells in a dormant state, so they can be reactivated at later states. Tumor stem cells can home to bone via cytokines and chemokines, in particular, SDF-1 expressed by osteoblasts and stromal cells in the marrow microenvironment, and its cognate receptor, CXCR4, on tumor cells. SDF-1 is chemotactic for tumor cells and directs these cells toward the marrow. The marrow microenvironment maintains normal hematopoiesis through production of cytokines and chemokines that enhance the growth and differentiation of hematopoietic stem cells (HSCs) and directs their homing to the marrow. However, HSCs are not distributed uniformly throughout the bone marrow, but are highly localized in “niches” that allow the HSCs to remain in a dormant state. Upon release from the “niche,” stem cells enter the cell cycle and proliferate and differentiate to the formed elements of blood [15].

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Cancers that metastasize to bone appear to use a similar mechanism to either usurp the HSC niche or use a similar type of niche to grow within the marrow and to maintain “cancer stem cells” in a dormant state that are resistant to chemotherapeutic agents. Like normal HSCs, cancer stem cells can lay dormant for long periods of time until they are activated to differentiate and proliferate into malignant cells [16]. The cellular composition of the stem cell niche is an area of active investigation, and its components are just beginning to be identified. Osteoblasts are an important component of the stem cell niche, and form the “endosteal niche” [17]. Cytokines and chemokines produced by osteoblasts as well as adhesive interactions between osteoblasts and HSCs maintain HSCs in G0 and provide a signal for homing of HSCs to the bone marrow. Prostate cancer cells like HSCs utilize a similar mechanism to home to the bone marrow and lodge there [18]. As noted above, several chemokines play critical roles in homing of HSC and cancer cells to the marrow. SDF-1, which is expressed by osteoblasts and endothelial cells, acts as a chemoattractant for both HSCs and cancer cells including MM cells to home to the bone marrow [19]. Recently, annexin II (AXII) has been identified as important in HSC and cancer cell lodgment in the bone marrow and the mobilization of HSCs and cancer cells to the peripheral blood [18]. Recent evidence suggest that other cells in the bone microenvironment the osteoclasts (OCLs), which are responsible for the bone destructive process, may also be involved in tumor cell mobilization. Kollet and coworkers examined the contribution of OCL to the mobilization of immature hematopoietic progenitors, and showed that OCLs secrete MMP-9 and cathepsin K, which cleave SDF-1, OPN and stem cell factor in the surrounding extracellular matrix [20]. This in turn weakens HSC anchorage provided by the endosteal niche. These results show that OCLs are involved in stem cell mobilization. Whether OCLs are also involved in tumor stem cell mobilization to increase bone metastasis or permit metastasis to solid organs remains to be determined.

2.4 The Role of Adhesive Interactions to Bone Metastasis Interactions between specific cell surface molecules on bone cells, bone marrow cells, and tumors are critical to both tumor invasion and the metastatic process. The importance of these interactions has been demonstrated by studies in human breast carcinoma, MM, and prostate carcinoma. Van der Pluijm and coworkers [21] found that the urokinase receptor and β1 integrins formed functional adhesion complexes at distinct sites at the cell surface of metastatic human breast carcinoma cells and that the urokinase receptor is capable of regulating the adhesive function of integrins on breast carcinoma cells. They showed that the addition of a blocking peptide for the urokinase-integrin complex inhibits the attachment of breast carcinoma cells to vitronectin. Using a mouse model of breast carcinoma metastasis, those authors reported that transplantation of nude mice with MDA-231 breast carcinoma cells that overexpress this blocking peptide, results in a significant reduction in tumor progression in bone compared with empty vector-transfected cells. Furthermore, mice

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that were transplanted with MDA-231 cells and received continuous administration of the peptide for 28 days had significantly reduced tumor progression in bone compared with animals that were treated with a scrambled control peptide. These results show that breast carcinoma progression in bone requires adhesive interactions between molecules that are expressed in the bone and molecules that are expressed in tumor cells. Similarly, Sung and coworkers [22] have shown human breast carcinoma cells adhere, proliferate, and migrate to bone through the interactions between αv β3 and αv β5 integrins and bone sialoprotein. In patients with MM, adhesive interactions between the α4 β1 and α5 β1 integrins and vascular cell adhesion molecule 1 (VCAM-1) or fibronectin appear to play an important role in upregulating the expression of cytokines and growth factors by bone marrow stromal cells, further enhancing tumor growth and chemotherapy resistance of the tumor. Damiano and Dalton [23] have shown that these adhesive interactions play an especially important role in the capacity of MM cells to resist standard chemotherapeutic agents, including doxorubicin and melphalan. It is very likely that adhesive interactions between a variety of tumor cells and bone marrow stromal cells result in the release of growth factors by stromal cells and osteoblasts, which also further enhance tumor growth. Similarly, in MM blocking α4 β1 binding to marrow stromal cells affects both the bone destructive process and tumor growth. Adhesive interactions between MM cells, for example, and marrow stromal cells enhanced both tumor growth and production of the factors such as RANKL and TNF-α (discussed below), which enhance OCL formation and activity and further amplify the bone destructive process. Thus, adhesive interactions between tumor cells and stromal cells play a critical role in the homing of the tumor to the bone, the growth of the tumor in the bone, and the upregulation of growth factor production by stromal cells required for tumor cell survival.

2.5 Regulation of Normal Bone Remodeling The adult skeleton is continually being remodeled to replace defective bone as well as to release calcium for various metabolic processes. This occurs through coordinated activity between the osteoblast and the OCL. In the normal bone remodeling sequence, osteoclastic bone resorption is followed by new bone formation at the site of new bone formation and then the process is balanced. However, in bone metastasis this process is either severely imbalanced or even uncoupled, so that bone resorption may be followed by inadequate bone formation, or exuberant bone formation, or no bone formation at all. In patients with bone metastasis, bone destruction is mediated by the OCL, the normal bone resorbing cells, rather than by tumor cells. The RANKL signaling pathway plays a critical role in both normal and malignant bone remodeling by regulation of OCL activity. RANK is a transmembrane signaling receptor, which is a member of the tumor necrosis receptor superfamily. It is found on the surface of OCLs precursors [24, 25]. RANK ligand

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(RANKL) is expressed as a membrane-bound protein on marrow stromal cells and osteoblasts, and secreted by activated lymphocytes. Its expression is induced by cytokines that stimulate bone resorption [26] such as PTH, 1,25 OH Vitamin D3 and prostaglandins [27, 28]. RANKL binds to RANK receptor on OCL precursors and induces OCL formation. Rank signals through the NF-κB and JunN terminal kinase pathways and induces increased osteoclastic bone resorption and enhanced OCL survival [29]. The important role of RANKL in normal osteoclastogenesis has been clearly demonstrated in RANKL or RANK gene knockout mice. These animals lack OCLs and as result develop severe osteopetrosis [30, 31]. OPG is a soluble decoy receptor for RANKL and is a member of the TNF receptor superfamily [32]. It is produced by osteoblasts as well as other cell types and blocks the interactions of RANKL with RANK, thereby limiting osteoclastogenesis. In normal subjects, the ratio RANKL/OPG is very low. Studies using knockout mice for the OPG gene have shown the importance of OPG. OPG deficient mice develop severe osteopenia and osteoporosis [30, 32–35]. Osteoblasts are bone-forming cells that arise from mesenchymal stem cells [36]. The factors controlling osteoblast differentiation are less well understood than for OCLs. The one transcription factor that has been clearly linked to osteoblast differentiation is core binding factor alpha 1 (Cbfa1), also known as Runx-2. Cbfa1 is responsible for the expression of most genes associated with osteoblast differentiation [37]. In mice lacking the Cbfa1 gene bone does not develop [38, 39]. Many extracellular factors can enhance the growth and differentiation of osteoblasts, including PDGF, FGF, and TGF-β.

2.6 Factors Involved in Osteolytic Bone Metastasis In patients with osteolytic bone metastasis, both OCL formation and activity are increased. However, the factors which enhance OCL activity differ among different tumor types. A common mediator of many effects of these tumor-derived factors on OCL formation is RANKL, which is increased in the bone microenvironment in patients with MM and plays an important role in the bone destructive process in breast cancer and prostate cancer as well (Fig. 2.2).

2.6.1 RANKL RANKL expression is increased when MM cells or breast cancer cells bind to marrow stromal cells [40]. RANKL then induces osteoclastogenesis, which results in the release of growth factors that further enhance the growth and survival of tumor cells. In addition, factors produced by tumor cells directly enhance RANKL expression. Using the 5T2 model of MM, Oyajobi and co-workers [41] showed that MIP-1α can induce RANKL expression in bone marrow stromal cells. In addition, IL-6, which is induced when murine MM cells bind to bone marrow stromal cells, also enhances RANKL production. IL-6 also enhances the survival of MM cells.

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Fig. 2.2 The vicious cycle involved in osteolytic metastasis. Tumor cells, in particular breast cancer, secrete parathyroid hormone related peptide as the primary stimulator of osteoclastogenesis. In addition, tumor cells produce other factors that increase osteoclast formation including IL-6, PGE2, tumor necrosis factor, and M-CSF. These factors increase RANK ligand expression, which directly act on osteoclast precursors to induce osteoclast formation and bone resorption. The bone resorption process releases factors such as TGF-β, which increase parathyroid hormone related peptide production by tumor cells as well as growth factors that increase tumor growth. This symbiotic relationship between bone destruction and tumor growth further increases bone destruction and tumor growth Source: Adapted from Roodman NEngl J Med.2004,350.1655.

RANKL has no direct effects on the growth of tumor cells, although its increased production enhances bone destruction, which further enhances the bone metastatic process. A recent study has reported that RANKL can also act as a chemoattractant for tumor cells expressing RANK [42]. A human antibody to RANKL, Denosumab, has been developed, which is in clinical trial for osteoporosis, arthritis and bone metastasis. The antibody has a similar action to OPG and lowers bone receptor markers in patients and is in phase III trials either being compared to placebo or to zoledronic acid, the most potent bisphosphonate used to treat patients with bone metastasis [43]. In MM, several other OCL-inducing factors have been identified in addition to RANKL, which appear to play an important role in the bone destructive process in these patients. These include IL-6, MIP-1α and IL-3.

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2.6.2 MIP-1α MIP-1α is a chemokine that is produced by MM cells in 70% of MM patients, and is a potent inducer of human OCL formation. MIP-1α can increase OCL formation independently of RANKL and can potentiate both RANKL and IL-6 stimulated OCL formation [44]. Bataille et al. reported that gene expression profiling of MM cells from patients demonstrated that MIP-1α is the gene most highly correlated with bone destruction in MM [45]. Further, Abe and coworkers have shown that elevated levels of MIP-1α also correlate with an extremely poor prognosis in MM [46]. In vivo models of MM have demonstrated that MIP-1α can induce OCL formation and bone destruction. Blocking MIP-1α expression in MM cells injected into SCID mice or treating the animals with a neutralizing antibody to MIP-1α results in decreased tumor burden and bone destruction [47,48]. MIP-1α also plays an important role in homing of MM cells to the bone marrow. MIP-1α increases adhesive interactions between MM cells and marrow stromal cells by increasing expression of β1 integrins. This results in production of RANKL, IL-6, VEGF and TNFα by marrow stromal cells, which further enhances MM cell growth, angiogenesis and bone destruction. Masih-Khan et al. reported that the t4:14 translocation in MM results in a constitutive expression of the FGFR3 receptor, which in turn results in high levels of MIP-1α [49]. Patients with the t4:14 translocation have a very poor prognosis, which may reflect the increased MIP-1α production in this patient population.

2.6.3 Interleukin-3 Interleukin-3 (IL-3) in addition to RANKL and MIP-1α, is also significantly elevated in bone marrow plasma of MM patients as compared to normal controls [50]. IL-3 can induce OCL formation in human bone marrow cultures at levels similar to those measured in MM patient samples, and OCL formation induced by marrow plasma from MM patients can be inhibited by using a blocking antibody to IL-3 [50]. IL-3 also indirectly influences osteoclastogenesis by enhancing the effects of RANKL and MIP-1α on the growth and development of OCLs. It also stimulates MM cell growth directly [50]. IL-3 also inhibits osteoblast formation through a factor produced by macrophages in the marrow microenvironment [51].

2.6.4 Interleukin-6 Interleukin-6 (IL-6) has been long recognized as a proliferative factor for plasma cells, but is unclear if IL-6 levels correlate with disease status [52]. IL-6 is a potent inducer of human OCL formation [53]. Levels of IL-6 are elevated in MM patients with osteolytic bone disease when compared to MM patients without bone disease, as well as in patients with monoclonal gammopathy of unknown significance (MGUS) [54]. Most studies support the finding that IL-6 is produced by

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cells in the bone marrow microenvironment through direct contact with MM cells rather than MM cells themselves. These cells most likely are OCLs and stromal cells. Increased osteoblast production of IL-6 has been also reported in cocultures of human osteoblasts with MM cells [55]. Although the precise role of IL-6 in MM bone disease is yet to be determined, IL-6 production by OCLs can increase tumor burden leading to enhanced bone destruction as well as act as an autocrine/paracrine factor to increase OCL formation [56].

2.6.5 PTHrP and TGF-β In breast cancer, other factors which can enhance RANKL production appear to be involved. One of the major factors produced by breast cancer cells is parathyroid hormone related peptide (PTHrP), which induces osteoclastic bone resorption through induction of RANKL by marrow stromal cells. Breast cancer cells also produce IL-6, IL-8, prostaglandin E2, M-CSF, IL-1 and tumor necrosis factor-α, which may increase OCL formation in bone metastasis [57–59]. The increased levels of PTHrP produced by breast cancer cells appear to be due to release of TGF-β in the bone microenvironment. Several laboratories have shown that, when breast carcinoma cells metastasize to bone and induce bone resorption, TGF-β is released in active form from bone. In particular, Chirgwin and Guise [57] have reported that breast carcinoma cells produce parathyroid hormone-related peptide (PTHrP), which induces osteoclastic bone resorption and releases TGF-β from the bone matrix. TGF-β then increases PTHrP production further, creating a vicious cycle in which tumor cells induce bone destruction and, through this process, release growth factors that enhance the growth of the tumor (Fig. 2.2). TGF-β is a potent, multifunctional cytokine that is produced by many cells, including osteoblasts and bone marrow stromal cells that can regulate cell growth and stimulate matrix production. TGF-β is a major factor in bone remodeling, and tumor-derived agents that enhance TGF-β production have been associated with increased bone formation [60]. TGF-β normally functions as a suppressor of tumor growth. Lang and coworkers [61] have shown that mice lacking TGF-β due to haploinsufficiency are more susceptible to tumors. Furthermore, TGF-β is immunosuppressive, which can increase tumor survival further by suppressing the immune system. TGF-β also can stimulate normal stromal cells and osteoblasts to secrete growth factors that enhance tumor growth. In patients with MM, Brown and coworkers [62] reported that TGF-β can depress dendritic cell function in these patients, further enhancing the growth of these tumors and protecting them from immune surveillance. Guise and coworkers [63] reported that the administration of a neutralizing monoclonal antibody to PTHrP inhibited the development of breast carcinoma bone metastasis by MDA-MB-231 cells in nude mice. It is noteworthy that those authors also showed that inhibiting TGF-β responsiveness of the tumor by using a dominant negative TGF-β receptor also reduced bone metastasis.

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Because of the importance of TGF-β in osteolytic bone metastasis, inhibitors of TGF-β type 1 receptor kinase have been explored in preclinical models of skeletal metastasis. Bandyopadhyay and coworker [64] have shown that systemic administration of a TGF-β receptor 1 kinase inhibitor reduced the number and the size of lung metastasis and bone metastasis in a model of metastatic breast cancer. Similarly, Ehata and coworkers [65] have also shown that a novel TGF-β type 1 receptor kinase inhibitor blocked bone metastasis by human breast cancer cells in preclinical mouse models. The inhibitor decreased the invasion of the breast cancer cells induced by the TGF-β in vitro and suppressed transcription of PTHrP, and IL-11 mRNA in the breast cancer cells. Thus, blocking TGF-β signaling is an important area of investigation for inhibiting bone metastasis in patients with breast cancer. Kang and coworkers [66] have shown that TGF-β induced signaling by means of the Smad transcription factor and plays an important role in breast cancer bone metastasis. These investigators demonstrated active Smad signaling in both human and mouse bone metastatic lesions. Further, Smad4 contributed to the formation of osteolytic bone metastasis and was essential for the induction of IL-11, a gene implicated in bone metastasis in mouse systems. Javelaud and coworkers [67] have also demonstrated the importance of TGF-β in melanoma bone metastasis. These investigators reported in a mouse model of melanoma metastasis to bone that overexpression of the inhibitory Smad7, which blocked TGF-β signaling, impaired bone metastasis. Thus, TGF-β plays a major role in the bone metastatic process for many solid tumors. Kang and coworker [68] have used gene expression profiling to identify the genes required for breast cancer bone metastasis. They found that IL-11, osteopontin (OPN), CXCR4 and CTGF were the genes required for bone metastasis, and that overexpression of IL-11 and OPN with either CTGF or CXCR4 was sufficient for bone metastasis. The results suggest that for a tumor to be metastatic to bone, it must express a set of genes that includes genes for homing (CXCR4), angiogenesis (CTGF) and osteolysis (IL-11 and OPN). CTGF and IL-11 are TFG-β responsive genes, which once the tumor metastasizes to bone and increases TGF-β release from bone, further amplify the bone destructive process.

2.6.6 Platelet-Derived Growth Factor Platelet-derived growth factor (PDGF) is a polypeptide produced by osteoblasts in the bone microenvironment that shares extensive sequence homology with the oncogene c-Cis. PDGF increases cell replication, bone resorption, collagen degradation, and collagenase expression as well as inhibiting osteoblast function. The mitogenic activity of PDGF increases the growth of tumor cells as well as enhances OCL activity. Yi and coworkers used MCF-7 cells in a model of breast carcinoma metastasis to bone in nude mice [69] and reported that, breast carcinoma cells that overexpressed HER-2-NEU produced large amounts of PDGF and showed an enhanced propensity

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of these cells to metastasize to bone. Furthermore, they suggested that PDGF played a causative role in the development of osteosclerotic bone metastasis in this model. Thus, upregulation of PDGF may enhance osteoblast formation and activity in bone metastasis and may enhance the growth of tumor cells through its mitogenic effects on tumors.

2.6.7 Vascular Endothelial Growth Factor Vascular endothelial growth factor (VEGF) induces the growth of vascular endothelial cells as well as enhances OCL formation and activity. Investigators in Japan have shown that VEGF can rescue the osteopetrotic phenotype of the op/op mouse, suggesting that VEGF is an OCL stimulatory factor [70]. VEGF is produced by marrow stromal cells rather than by the vascular endothelium of bone. FGF, TGF-β, and the insulin growth factors increase VEGF production by several cell types, and VEGF production is upregulated by MM cells when they bind to bone marrow stromal cells [71]. This upregulation of VEGF results in increased angiogenesis that enhances the proliferation of MM cells. Thus, VEGF appears to be an important cytokine regulating the growth of MM cells and probably the growth of other tumor cells that bind to bone marrow stromal cells. Gupta and coworkers [71] have shown in cocultures of MM cells with bone marrow stromal cells that VEGF significantly increased interleukin 6 (IL-6) secretion by bone marrow stromal cells and that stromal cells from MM patients and normal donors secreted VEGF, FGF, and IL-6. Thus, VEGF produced by bone marrow stromal cells has multiple effects on tumor growth, including increased angiogenesis, increased growth of tumor cells, upregulation of IL-6 (another growth and survival factor for MM cells), as well as increased OCL formation.

2.6.8 Insulin-Like Growth Factors Insulin-like growth factors (IGFs) are produced by osteoblastic cells and are regulated by a number of factors produced by bone marrow stromal cells in the bone marrow microenvironment. These include TGF-β, FGF, PDGF, and prostaglandins. IGFs induce proliferation of osteoblasts and play a major role in stimulating differentiation of osteoblasts [72]. In addition, IGFs increase bone resorption by stimulating the formation of OCLs and activating preexisting OCLs. IGFs can also regulate OCL activity through their regulation of RANKL and RANK levels in bone [73]. IGFs are potent mitogens for tumor cells. For example, Ferlin and coworkers [74] showed that IGFs induce the survival and proliferation of MM cells independent of IL-6 production by bone marrow stromal cells. These authors found that IGF acts as a potent survival/proliferation factor for MM cells, and strongly suggested a role for IGFs in the pathophysiology of MM.

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2.7 Factors Increasing Osteoblast Activity in Bone Metastasis In patients with osteoblastic bone metastasis, osteoblast formation and activity are increased although there is still underlying bone destruction. Prostate cancer is the cancer which has predominantly osteoblastic bone metastasis with increased numbers of irregular bone trabeculae [69]. Risk factors responsible for increased osteoblast activity in prostate cancer are just beginning to be defined.

2.7.1 Endothelin-1 Endothelin-1 has been identified as a possible factor involved in the development of osteoblastic metastases [75]. In vitro studies of samples from patients with breast cancer showed that endothelin-1 can stimulate bone formation and osteoblast proliferation [76]. Recent studies suggest that endothelin-1 may increase osteoblast activity by inhibiting expression of DKK1 by marrow stromal cells [77]. In addition, serum endothelin-1 levels are increased in patients with osteoblastic metastases from prostate cancer [78]. Use of a selective endothelin-1 receptor antagonist decreased both osteoblastic metastases and tumor burden in an animal model, although it had no effect on tumor growth at orthotopic sites [75]. These observations suggest that blocking osteoblastinducing activity by tumors may decrease tumor growth in bone. Consistent with these observations are recent studies using an endothelin-1 receptor antagonist, Atrasentan, in clinical trials in patients with prostate cancer and bone metastasis. The basis for this trial was that endothelin-1 levels are increased in advanced prostate cancer, endothelin-1 is produced by prostate cancer cells and that blocking endothelin-1 activity in preclinical models of osteoblastic bone metastasis had beneficial effects on tumor burden. However, in clinical trials with Atrasentan, Caraduchi and coworkers [79] showed no effect of Atrasentan on progression-free survival or overall survival of patients. However, there was a significant decrease in cancer-induced bone pain and bone alkaline phosphatase, a marker of bone formation. Further, progression of tumor was decreased in patients who only had bone metastasis but no other sites of metastasis. Hall and coworkers [80] have shown that the Wnt signaling pathway plays an important role in the osteoblastic metastasis in prostate cancer. The Wnt signaling pathway promotes the proliferation, expansion and survival of pre and immature osteoblastic cells [81]. Osteoblasts produce several soluble inhibitors of the canonical Wnt pathway; including Dickkopf (DKK1), secreted frizzled related proteins (sFRP), and Wnt inhibitor factor (Wif-1). Hall and coworkers showed that blocking expression of the Wnt signaling pathway antagonist DKK1 in an osteolytic prostate cancer cell line resulted in increased osteoblast activity in the metastasis. In contrast, expressing DKK1 in a prostate cancer cell line that induced both osteoblastic and osteolytic metastasis when injected into tibia of mice, resulted in conversion of the tumor to a highly osteolytic tumor. Recently, Li and coworkers [82] showed that

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normal prostate cells do not express the Wnt stimulator Wnt-7b, but high grade prostate cancer cells do. Further, 16 of 38 bone metastases from patients with breast cancer also expressed Wnt-7b by gene array. DKK1 was not expressed in normal prostate cancer cells but was expressed in 2 of 3 specimens from osteolytic bone metastasis from patients. They interpreted these results to show that DKK1 expression is low in most prostate cancers, and thus allows enhanced Wnt signaling and results in increased osteoblast activity. In contrast to prostate cancer, osteoblast activity is suppressed in MM. Tian and co-workers reported the production of the Wnt antagonist, DKK1, by primary CD138+ MM cells but not by plasma cells from MGUS patients. They demonstrated that levels of DKK1 mRNA correlated with focal bone lesions in patients with MM [83, 84]. In contrast, patients with advanced disease as well as some human MM cell lines did not express DKK1, suggesting that these inhibitors may mediate bone destruction only in the early phases of disease [83]. Anti-DKK1 antibody administration to SCID-hu mice injected with 1◦ MM cells, inhibited MM cell growth and increased bone formation in the implanted fetal bone [85]. MM cells also produce sFR2 [58], another Wnt antagonist, which suppresses osteoblast differentiation in MM. In addition to inhibition of osteoblastogenesis, elevated DKK1 levels can also enhance osteoclastogenesis. Wnt signaling in osteoblasts increases expression of OPG [86] and downregulates the expression of RANKL [87], suggesting a possible mechanism by which inhibition of Wnt signaling in osteoblasts would indirectly increase osteoclastogenesis. Taken together, these studies indicate that DKK1 is a key regulator of bone remodeling in both physiological and pathological conditions and that blocking Wnt signaling may contribute to both stimulation of osteoclastogenesis and inhibition of osteoblasts in myelomatous bones. Thus, multiple stimulators of OCL activity and suppressors of osteoblast differentiation are present in patients with bone metastasis and together result in the devastating bone disease present in these patients.

2.8 Summary The identification and characterization of the pathophysiologic mechanisms underlying bone metastasis have provided important new therapeutic targets for treating these patients, who are currently incurable. Bone metastasis takes a tremendous toll on patients both physically, financially and impacts their survival. Current therapies using intravenous bisphosphonates have greatly improved the outlook of the patients with bone metastasis, but only slow the progression of the disease rather than completely eradicating it [8]. The identification of RANKL as a major mediator of the bone destructive process in bone metastasis for multiple tumor types has led to phase III clinical trials of a human antibody to RANKL, Denosumab, which prevents skeletal-related events in patients with bone metastasis from a variety of tumors. Similarly, endothelin-1 receptor antagonists, TGF-β, receptor kinase antagonists and antibodies to DKK1 are in preclinical or clinical trials. Thus, the future

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appears much brighter for patients with bone metastasis, and the availability of these new agents along with bisphosphonates to prevent development of metastasis or eradicate metastasis and decrease the tumor burden in bone is an exciting possibility.

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20. Kollet O, Dar A, Lapidot T (2007) The multiple roles of osteoclasts in host defense: bone remodeling and hematopoietic stem cell mobilization. Annu Rev Immunol 25:51–69 21. van der Pluijm G, Sijmons B, Vloedgraven H, et al. (2001) Urokinase-receptor/integrin complexes are functionally involved in adhesion and progression of human breast cancer in vivo. Am J Pathol 159:971–982 22. Sung V, Stubbs JT III, Fisher L, et al. (1998) Bone sialoprotein supports breast cancer cell adhesion proliferation and migration through differential usage of the alpha(v)beta3 and alpha(v)beta5 integrins. J Cell Physiol 176:482–494 23. Damiano JS, Dalton WS (2000) Integrin-mediated drug resistance in multiple myeloma. Leuk Lymphoma 38:71–81 24. Hsu H, Lacey DL, Dunstan CR, et al. (1999) Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A 96:3540–3545 25. Nakagawa N, Kinosaki M, Yamaguchi K, et al. (1998) RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res Commun 253:395–400 26. Boyle WJ, Simonet WS, Lacey DL (2003) Osteoclast differentiation and activation. Nature 423:337–342 27. Yasuda H, Shima N, Nakagawa N, et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 95:3597–3602 28. Hofbauer LC, Heufelder AE (1998) Osteoprotegerin and its cognate ligand: a new paradigm of osteoclastogenesis. Eur J Endocrinol 139:152–154 29. Roodman GD (2007) Treatment strategies for bone disease. Bone Marrow Transplant 40:1139–1146 30. Dougall WC, Glaccum M, Charrier K, et al. (1999) RANK is essential for osteoclast and lymph node development. Genes Dev 13:2412–2424 31. Tsukii K, Shima N, Mochizuki S, et al. (1998) Osteoclast differentiation factor mediates an essential signal for bone resorption induced by 1 alpha, 25-dihydroxyvitamin D3, prostaglandin E2, or parathyroid hormone in the microenvironment of bone. Biochem Biophys Res Commun 246:337–341 32. Lacey DL, Timms E, Tan HL, et al. (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176 33. Bucay N, Sarosi I, Dunstan CR, et al. (1998) osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12:1260–1268 34. Li J, Sarosi I, Yan XQ, et al. (2000) RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Natl Acad Sci U S A 97:1566–1571 35. Simonet WS, Lacey DL, Dunstan CR, et al. (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309–319 36. Aubin JE (1998) Bone stem cells. J Cell Biochem Suppl 30–31:73–82 37. Yang X, Karsenty G (2002) Transcription factors in bone: developmental and pathological aspects. Trends Mol Med 8:340–345 38. Komori T, Yagi H, Nomura S, et al. (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764 39. Otto F, Thornell AP, Crompton T, et al. (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765–771 40. Mancino AT, Klimberg VS, Yamamoto M, et al. (2001) Breast cancer increases osteoclastogenesis by secreting M-CSF and upregulating RANKL in stromal cells. J Surg Res 100:18–24 41. Oyajobi BO, Williams PJ, Story B, et al. (2001) Myeloma bone disease and tumor burden reversed by a neutralizing antibody to macrophage inflammatory protein (MIP 1-α/CCL3) in vivo. J Bone Miner Res 16:S192

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42. Jones DH, Nakashima T, Sanchez OH, et al. (2006) Regulation of cancer cell migration and bone metastasis by RANKL. Nature 440:692–696 43. Roodman GD, Dougall WC (2008) RANK ligand as a therapeutic target for bone metastases and multiple myeloma. Cancer Treat Rev 34:92–101 44. Han JH, Choi SJ, Kurihara N, et al. (2001) Macrophage inflammatory protein-1alpha is an osteoclastogenic factor in myeloma that is independent of receptor activator of nuclear factor kappaB ligand. Blood 97:3349–3353 45. Magrangeas F, Nasser V, Avet-Loiseau H, et al. (2003) Gene expression profiling of multiple myeloma reveals molecular portraits in relation to the pathogenesis of the disease. Blood 101:4998–5006 46. Hashimoto T, Abe M, Oshima T, et al. (2004) Ability of myeloma cells to secrete macrophage inflammatory protein (MIP)-1alpha and MIP-1beta correlates with lytic bone lesions in patients with multiple myeloma. Br J Haematol 125:38–41 47. Alsina M, Boyce B, Devlin RD, et al. (1996) Development of an in vivo model of human multiple myeloma bone disease. Blood 87:1495–1501 48. Choi SJ, Oba Y, Gazitt Y, et al. (2001) Antisense inhibition of macrophage inflammatory protein 1-alpha blocks bone destruction in a model of myeloma bone disease. J Clin Invest 108:1833–1841 49. Masih-Khan E, Trudel S, Heise C, et al. (2006) MIP-1alpha (CCL3) is a downstream target of FGFR3 and RAS-MAPK signaling in multiple myeloma. Blood 108:3465–3471 50. Lee JW, Chung HY, Ehrlich LA, et al. (2004) IL-3 expression by myeloma cells increases both osteoclast formation and growth of myeloma cells. Blood 103:2308–2315 51. Ehrlich LA, Chung HY, Ghobrial I, et al. (2005) IL-3 is a potential inhibitor of osteoblast differentiation in multiple myeloma. Blood 106:1407–1414 52. Solary E, Guiguet M, Zeller V, et al. (1992) Radioimmunoassay for the measurement of serum IL-6 and its correlation with tumour cell mass parameters in multiple myeloma. Am J Hematol 39:163–171 53. Roodman GD, Kurihara N, Ohsaki Y, et al. (1992) Interleukin 6. A potential autocrine/paracrine factor in Paget’s disease of bone. J Clin Invest 89:46–52 54. Sati HI, Apperley JF, Greaves M, et al. (1998) Interleukin-6 is expressed by plasma cells from patients with multiple myeloma and monoclonal gammopathy of undetermined significance. Br J Haematol 101:287–295 55. Karadag A, Oyajobi BO, Apperley JF, et al. (2000) Human myeloma cells promote the production of interleukin 6 by primary human osteoblasts. Br J Haematol 108:383–390 56. Abe M, Hiura K, Wilde J, et al. (2004) Osteoclasts enhance myeloma cell growth and survival via cell-cell contact: a vicious cycle between bone destruction and myeloma expansion. Blood 104:2484–2491 57. Chirgwin JM, Guise TA (2000) Molecular mechanisms of tumor-bone interactions in osteolytic metastases. Crit Rev Eukaryot Gene Expr 10:159–178 58. Oshima T, Abe M, Asano J, et al. (2005) Myeloma cells suppress bone formation by secreting a soluble Wnt inhibitor, sFRP-2. Blood 106:3160–3165 59. Park BK, Zhang H, Zeng Q, et al. (2007) NF-kappaB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GM-CSF. Nat Med 13:62–69 60. Festuccia C, Bologna M, Gravina GL, et al. (1999) Osteoblast conditioned media contain TGF-beta1 and modulate the migration of prostate tumor cells and their interactions with extracellular matrix components. Int J Cancer 81:395–403 61. Lang SH, Clarke NW, George NJ, et al. (1999) Scatter factor influences the formation of prostate epithelial cell colonies on bone marrow stroma in vitro. Clin Exp Metastasis 17:333–340 62. Brown RD, Pope B, Murray A, et al. (2001) Dendritic cells from patients with myeloma are numerically normal but functionally defective as they fail to up-regulate CD80 (B7-1) expression after huCD40LT stimulation because of inhibition by transforming growth factor-beta1 and interleukin-10. Blood 98:2992–2998

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63. Guise TA, Yin JJ, Taylor SD, et al. (1996) Evidence for a causal role of parathyroid hormonerelated protein in the pathogenesis of human breast cancer-mediated osteolysis. J Clin Invest 98:1544–1549 64. Bandyopadhyay A, Agyin JK, Wang L, et al. (2006) Inhibition of pulmonary and skeletal metastasis by a transforming growth factor-beta type I receptor kinase inhibitor. Cancer Res 66:6714–6721 65. Ehata S, Hanyu A, Fujime M, et al. (2007) Ki26894, a novel transforming growth factor-beta type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line. Cancer Sci 98:127–133 66. Kang Y, He W, Tulley S, et al. (2005) Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway. Proc Natl Acad Sci U S A 102:13909–13914 67. Javelaud D, Mohammad KS, McKenna CR, et al. (2007) Stable overexpression of Smad7 in human melanoma cells impairs bone metastasis. Cancer Res 67: 2317–2324 68. Kang Y, Siegel PM, Shu W, et al. (2003) A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3:537–549 69. Yi B, Williams PJ, Niewolna M, et al. (2002) Tumor-derived platelet-derived growth factor-BB plays a critical role in osteosclerotic bone metastasis in an animal model of human breast cancer. Cancer Res 62:917–923 70. Niida S, Kaku M, Amano H, et al. (1999) Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med 190:293–298 71. Gupta D, Treon SP, Shima Y, et al. (2001) Adherence of multiple myeloma cells to bone marrow stromal cells upregulates vascular endothelial growth factor secretion: therapeutic applications. Leukemia 15:1950–1961 72. Shinar DM, Endo N, Halperin D, et al. (1993) Differential expression of insulin-like growth factor-I (IGF-I) and IGF-II messenger ribonucleic acid in growing rat bone. Endocrinology 132:1158–1167 73. Wang Y, Nishida S, Elalieh HZ, et al. (2006) Role of IGF-I signaling in regulating osteoclastogenesis. J Bone Miner Res 21:1350–1358 74. Ferlin M, Noraz N, Hertogh C, et al. (2000) Insulin-like growth factor induces the survival and proliferation of myeloma cells through an interleukin-6-independent transduction pathway. Br J Haematol 111:626–634 75. Guise TA, Yin JJ, Mohammad KS (2003) Role of endothelin-1 in osteoblastic bone metastases. Cancer 97:779–784 76. Kasperk CH, Borcsok I, Schairer HU, et al. (1997) Endothelin-1 is a potent regulator of human bone cell metabolism in vitro. Calcif Tissue Int 60:368–374 77. Clines GA, Mohammad KS, Bao Y, et al. (2007) Dickkopf homolog 1 mediates endothelin-1stimulated new bone formation. Mol Endocrinol 21:486–498 78. Nelson JB, Hedican SP, George DJ, et al. (1995) Identification of endothelin-1 in the pathophysiology of metastatic adenocarcinoma of the prostate. Nat Med 1:944–949 79. Carducci MA, Saad F, Abrahamsson PA, et al. (2007) A phase 3 randomized controlled trial of the efficacy and safety of atrasentan in men with metastatic hormone-refractory prostate cancer. Cancer 110:1959–1966 80. Hall CL, Bafico A, Dai J, et al. (2005) Prostate cancer cells promote osteoblastic bone metastases through Wnts. Cancer Res 65:7554–7560 81. Westendorf JJ, Kahler RA, Schroeder TM (2004) Wnt signaling in osteoblasts and bone diseases. Gene 341:19–39 82. Li ZG, Yang J, Vazquez ES, et al. (2008) Low-density lipoprotein receptor-related protein 5 (LRP5) mediates the prostate cancer-induced formation of new bone. Oncogene 27:596–603 83. Tian E, Zhan F, Walker R, et al. (2003) The role of the Wnt-signaling antagonist DKK1 in the development of osteolytic lesions in multiple myeloma. N Engl J Med 349: 2483–2494

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84. Politou MC, Heath DJ, Rahemtulla A, et al. (2006) Serum concentrations of Dickkopf-1 protein are increased in patients with multiple myeloma and reduced after autologous stem cell transplantation. Int J Cancer 119:1728–1731 85. Yaccoby S, Ling W, Zhan F, et al. (2007) Antibody-based inhibition of DKK1 suppresses tumor-induced bone resorption and multiple myeloma growth in vivo. Blood 109:2106–2111 86. Glass DA II, Bialek P, Ahn JD, et al. (2005) Canonical Wnt signaling in differentiated osteoblasts controls osteoclast differentiation. Dev Cell 8:751–764 87. Spencer GJ, Utting JC, Etheridge SL, et al. (2006) Wnt signalling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand and inhibits osteoclastogenesis in vitro. J Cell Sci 119:1283–1296 88. Mathers, et al. (2000) IARC Globocon. http://wwwlpubmedcedntral.nih.gov. Accessed March 2006 89. Coleman (2001) Cancer Treat Rev. 27:165 90. American Cancer Society (2005) Cancer Facts and Figures. At: http://www.cancer.org/ docroot/STT/content/STT 1x Cancer Facts Figures 2005.asp. Accessed March 2006 91. Zekri, et al. (2001) Int J Oncol 19–379

Chapter 3

ANGIOGENESIS AND BONE METASTASIS: IMPLICATIONS FOR DIAGNOSIS, PREVENTION AND TREATMENT Pelagia G. Tsoutsou and Michael I. Koukourakis Radiation Oncology Department, Democritus University of Thrace, Medical School, Dragana 68 100, Alexandroupolis, Greece, e-mail: [email protected]

Abstract:

Angiogenesis consists of the mechanism by which new blood vessels are formed within the tumor to sustain its development and growth. This “angiogenic switch” is mediated by various molecules deriving from the cancer cell and/or the tumor-associated stroma and is promoted either by oncogene activation or by the physiological response of any cell to the hypoxic stress. The angiogenic switch is governed by the balance between angiogenic inducers and inhibitors. There is a certain affinity between tumors sites and metastatic sites; that is the theory of “seed and soil”: certain tumor cells (the “seed”) have a specific affinity for the milieu of certain organs (the “soil”). Metastases result only when the seed and soil are compatible. The pathophysiology of bone metastasis is complex, involving different cell populations and regulatory proteins. The traditional idea that bone metastases are either osteoblastic or osteolytic represents in fact a continuum. There is a multiplicity of reasons for a tumors’ propensity to metastasize to bone: Increased blood flow in red marrow and the presence of adhesive molecules on tumor cells are the most important. Adhesion triggers the secretion of angiogenic factors and bone resorbing factors from tumor cells that enable cancer cell survival and growth. The unique bone environment characterized by the continuous remodelling through osteoclast and osteoblast activity on trabecular surfaces provides cancer cells a soil rich in growth factors, such as transforming growth factor –β (TGF-β), fibroblast growth factor (FGF), plateletderived growth factor (PDGF)), and insulin growth factors (IGFs). Physical factors within the bone microenvironment, including low oxygen levels, acidic pH, and high extracellular calcium concentrations, may also enhance tumor growth. Bone-derived chemokines such as osteopontin, bone sialoprotein, and stromal-derived factor also act as chemoattractants for cancer cells.

D. Kardamakis et al. (eds.), Bone Metastases, Cancer Metastasis – Biology and Treatment 12, DOI 10.1007/978-1-4020-9819-2 3, C Springer Science+Business Media B.V. 2009

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Therefore, the most important angiogenic molecules in bone metastasis are VEGF, heparenase, TGF-β, IGF, PDGF, Interleukin- 8 (IL-8) and IL-6. Angiogenesis opens up a whole era of new treatment modalities for bone metastases: biphosphonates can act as anti-angiogenic agents and a field of targeted anti-angiogenic therapies for bone metastases is also emerging. Key words: Angiogenesis · Angiogenic factors · Bone metastasis · Cancer · Therapy

3.1 Introduction Angiogenesis has emerged as an important chapter in our understanding of tumor biology. New blood vessels formation within the tumor is essential to sustain its survival and growth. The pioneer work of the recently deceased Judah Folkmann and subsequent laborious work from various research groups all over the world during the past 20 years, revealed the role of angiogenesis as an essential part of tumor pathogenesis. Angiogenesis also became a major target for therapy and, indeed, the development of a large number of antiangiogenic agents in the recent years has opened a new era of therapeutics. Angiogenesis is fundamental to reproduction, development, and repair; it mainly consists of growth of blood vessels that can “turn on” and “turn off” within a brief period. Although a fundamental physiological procedure, angiogenesis can become pathologic and lead to the progression of many neoplastic and non-neoplastic diseases [1]. This “angiogenic switch” within a previously non- angiogenetic tumor is mediated by various molecules deriving from the cancer cell itself and/or the tumor-associated stroma fibroblasts and infiltrating macrophages and lymphocytes. Once a cancer cell becomes refractory to the normal regulatory mechanisms of division and differentiation, its progression and survival depend on its proximity to a vascular supply [2]. In fact, data of human lung cancer brain metastases indicate that tumor cell division takes place within 75 μm of the nearest blood vessel, whereas tumor cells residing beyond 150 μm from a vessel undergo programmed cell death [2]. These data are in accordance with the diffusion coefficient of oxygen in tumor tissue, which is approximately 120 μm [3]. Therefore, this explains the notion supported by Folkmann, that a tumor without any newly formed vessels can be no bigger than 1 mm in diameter. This early theory [4] suggested that tumors can persist in situ for months to years without neovascularization, when they are no more than 2–3 mm large but then become vascularized when a subgroup of cells in the tumor “switches” to an angiogenic phenotype. In the prevascular phase, cells, or even dormant micrometastases may replicate rapidly as well, but without the angiogenetic procedure their rate of proliferation reaches equilibrium with their rate of death [5]. To grow larger than that, the tumor needs new vessel supplies, therefore the angiogenetic process needs to be “switched on”. This is promoted either by oncogene activation or by the physiological response of any cell to the

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Angiogenesis and Bone Metastasis

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hypoxic stress by up-regulating hypoxia-dependent transcription factors that trigger the overexpression of angiogenic factors, such as VEGF [6–8]. The angiogenic switch is governed by the balance between angiogenic inducers and inhibitors [9]. The essential evidence for this assumption comes from in vitro bioassays, where both bFGF and VEGF can elicit a positive response from capillary endothelial cells, but if an inhibitor such as TSP-1 (thrombospondine 1) is added, the response is blocked [9]. As Hanahan points out: “a net balance of inhibitors over activators would maintain the switch in the off position, whereas a shift to an excess of activating stimuli would turn on angiogenesis” [9]. This raises the question how are all these regulators combined to produce a certain angiogenic result in a given tissue. Manipulation of this balance would find important therapeutic applications in oncology. The whole concept of the “angiogenic switch” along with the interplaying cells is described in Fig. 3.1.

osteoblasts

new bone

osteoclast

BONE development of metastasis macrophage Angiogenesis switch on: inducers

Angiogenesis switch off: inhibitors

Angiogenesis: induction of new blood vessel to support tumor growth

fibroblast endothelial cell

vessel Inducers of angiogenesis: bFGF, VEGF Inhibitors of angiogenesis: TSP-1

cancer cell

fibroblast

osteoblasts

osteoclast

switch on

lymphocyte

new bone

BONE switch off

Fig. 3.1 Interplay of tumor cells to support tumor growth through induction of angiogenesis. These are the basic cells interplaying in the angiogenetic procedure to support metastatic growth. Cancer cells travel through normal blood vessels. Those surviving this “trip” finally exavasate through a rupture of the basement membrane of the vessel and interact with the host environment (stroma, cells) in order to promote angiogenetic loops that will allow them to survive and proliferate. A cluster of tumor cells cannot grow beyond 2–3 mm, if not sustained by the growth of a newly formed vessel, a process called neovascularization. This newly formed blood vessel is formed by endothelial cells, that interact with the tumor cells and promote, both of them, the secretion of angiogenesis inducers (basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β) and others). This is the angiogenesis “switch on” that is in balance with an angiogenesis “switch off”, promoted by angiogenesis inhibitors, also secreted by these cells. Though the endothelial-cancer cell interplay is the basic feature of the angiogenetic process, other cells, such as the fibroblast, the lymphocytes, the osteoblasts and the osteoclasts interplay as well through the angiogenetic swith on/off, as previously described

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The neovascularization supports tumor growth through a “perfusion” effect, a “paracrine” effect [1] and an “autocrine” effect. Perfusion allows nutrients and oxygen to enter and catabolites to exit. The paracrine effect results from the production of growth factors by cells employed in the tumor microenvironment, mainly fibroblasts and infiltrating macrophages and lymphocytes [1]. The autocrine effect refers to the role of angiogenic proteins produced by cancer cells to stimulate the survival and proliferation of the cancer cell by binding to proper receptors [10–17]. Quantification of angiogenesis in a biopsy specimen may have a prognostic as well as a predictive value. There is a common positive association between tumor angiogenesis and the risk of metastasis, recurrence, or death with regard to many tumor types. High microvessel density might be a predictor of metastatic risk because high density increases the area of the vascular surface, thus increasing the possibility for a metastatic cell to escape into the circulation [1]. On the other hand, the angiogenesis “potential” of the tumor might preclude its response to radiotherapy or chemotherapy, while anti-angiogenic agents may reverse this effect, as seen in clinical studies with agents such as bevacizumab [Avastin] [18, 19].

3.2 The Process of Angiogenesis The basic cell that forms a blood vessel is the endothelial cell. The endothelial cells are among the cells with the longest life in the body (apart from the central nervous system), that is in a normal adult vessel, only 1 in every 10,000 endothelial cells (0.01%) happens to be in the cell division cycle at any given time [20]. However, in special circumstances intense endothelial cell proliferation, thus angiogenesis occurs. Figure 3.2 describes the process of neovascularisation through the interplay of endothelial-tumor cell. Inducers of angiogenesis – interplay of cancer and endothelial cell

cancer cell

• • • • • • • •

Neovascularization: Perfusion (O2) Paracrine effect (tumor growth) Autocrine effect: growth factors (fibroblasts, macrophages, lymphocytes) • Autocrine (proteins from tumor cell to itself for survival and proliferation)

blood vessel

endothelial cell

endothelial cell

Fig. 3.2 Interplay between endothelial and cancer cell

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The process of angiogenesis is complex. The basement membrane that surrounds endothelial cells is locally degraded, and they in turn change shape and invade into surrounding stroma and start proliferating [9]. A region of differentiation then appears, where the endothelial cells connect to each other and form a lumen of a new capillary tube. These tubes fuse and coalesce into loops leading to blood circulation. The classical proof of angiogenesis came from experiments whereby tumor fragments or cultured tumor cells were placed in an avascular site, the cornea of a rabbit eye [21]. The implants attracted new capillaries that grew to vascularize the expanding tumor mass. If the capillary formation was blocked tumor growth was dramatically impaired. Further experiments confirmed this result and showed that in the absence of an adequate vasculature, tumor cells either become necrotic and/or apoptotic [5, 22], restraining the increase in tumor volume that should result from continuous cell proliferation, the hallmark of cancer [9]. The blood vessels of tumors are known to be chaotic, leaky and inefficient [23]. The difference between immature tumor vessels and mature normal ones found in healthy tissues is the topic of current research. Vascular morphogenesis requires that endothelial cells undergo morphological changes such as activation, migration, alignment, proliferation, tube formation, branching, anastomosis, and maturation of intercellular junctions and basement membrane [24]. Each of these stages is either known or suspected to depend on VEGF and TGF-β morphogenetic protein signaling pathways [24]. VEGF is essential for initiation of angiogenic sprouting, and also regulates migration of capillary tip cells, proliferation of trunk cells, and gene expression in both. TGF-β regulates cell migration and proliferation, as well as matrix synthesis. These pathways develop an essential interplay in vascular morphogenesis [24]. Moreover, the inflammatory cytokine interleukin-1β has been recently identified as an essential trigger of VEGFdependent angiogenesis and is thought to play a considerable role in tumor vessels maturation [23].

3.3 Angiogenesis and Metastasis The process of cancer cell metastasis is by large obscure but it seems that there are key steps in its pathogenesis. After the initial event that confers the malignant phenotype, cancer cells continue to grow with nutrients supplied by simple diffusion from adjacent normal vessels. As soon as the tumor mass reaches 1–2 mm in diameter, angiogenesis is needed to sustain an adequate supply of nutrient and oxygen to the inner sectors of the growing mass. The onset of this process depends on the synthesis and secretion of proangiogenic factors by tumor cells. As already underlined, the newly growing vessels are immature, with thin often discontinuous walls so that tumor cells, having activated genes controlling cell-cell adhesion disruption and cellular motility, may penetrate in the lumen and reach the systemic circulation. The majority of circulating tumor cells are rapidly destroyed. Some of

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them will be trapped in the capillary beds of organs. Surviving cancer cells can start an intra-capillary growth or extravasate into the parenchyma through disruption of the capillary walls forming a micrometastasis. This micrometastases must in its turn survive and grow through development of a vascular network (angiogenesis). This provides the micrometastasis the ability to invade, penetrate newly formed blood vessels, and enter the circulation, therefore producing “metastasis from metastasis” [25]. It has been made evident early in clinical studies that there is a certain affinity between tumors sites and metastatic sites, i.e., certain tumors metastasize to specific organs [26]. Historically, it was Stephen Paget in 1889 that first developed the theory of “seed and soil” [27]. He proved that certain tumor cells (the “seed”) had a specific affinity for the milieu of certain organs (the “soil”). Metastases result only when the seed and soil are compatible [27]. In 1928, Ewing challenged this theory by implying that metastasis is the result of purely mechanical factors deriving from anatomical re-arrangements of the vascular system within the tumor; a theory that, however, has not gained uniform acceptance [28]. Later on, Zeidman and Buss demonstrated that tumor cells from different tumors interact differently with the capillary bed of a given organ [29]. Sugarbaker proved that each type of tumor provokes a specific pattern of metastases even when injected to the same site [30], while Fisher and Fisher demonstrated that tumor cells can traverse different organs at different rates [31]. Cancer metastasis depends on the interplay of tumor cells with various host factors. The cancer cells consist of multiple genetically unstable cell populations with diverse karyotypes, growth rates, cell-surface properties, antigenicities, immunogenicities, sensitivity to various cytotoxic drugs, and abilities to invade and produce metastasis [25, 26, 32, 33]. It has been shown that the expression of proangiogenic cytokines by malignant cells is under the regulation of the tissue microenvironment [25]. Microenvironmental factors influence expression levels of basic fibroblast growth factor (bFGF), a growth factor that controls the angiogenic switch of some tumors [34]. All this evidence suggests an important role of the microenvironment to a tissues’ ability to promote or inhibit angiogenesis, therefore regulating its metastatic potential. Figure 3.3 describes the interplay between tumor cell and host factors (the seed and soil hypothesis). On the other hand, a tumor can be “hostile” to the implantation of a certain metastasis, due to the function of the tissue microenvironment as a negative regulator of tumor cell growth [25]. Recently, the molecular basis of tumor dormancy has become the topic of research interest [25]. This is a very important aspect of cancer pathogenesis, especially in certain tumor types, such as breast cancer that can recur or metastasize after a long lag period. Cell cycle arrest and immune surveillance have been implicated in tumor dormancy [35, 36]. Interestingly, this dormant cell can become metastatically active if removed from the inhibitory influences of the organ microenvironment [37]. This can be attributed to the inhibition of angiogenesis taking place at this negatively regulatory environment [21].

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Seed and soil hypothesisOsteotropism Bone colonizing tumor cells express: • MMP-1 (proteolysis) • IL-11 (osteoclastogenesis) • Osteopontin (osteoclastogenesis) • Connective tissue growth factor (angiogenesis) • Chemokine receptor CXCR4 (homing to bone) • Bone chemokines, • O2 pressure, • Acidic pH, • Calcium tumor growth • Osteopontin, sialoprotein, stromal-derived factors serve as chemoattractants for cancer cells

cancer Cancer cell

Tumor cells interplay with host factors through: • proangiogenic cytokines • bFGF

osteoblasts osteoclast

• blood flow in the bone marrow • Adhesive molecules on tumor cells to stromal cells and matrix • Secretion of angiogenic and bone resorbing factors cancer survival and growth

Bone remodelling through osteoclast and osteoblast FGF PDGF IGF Bone chemokines

Fig. 3.3 The seed and soil hypothesis-osteotropism

3.4 Bone Metastases – Osteotropism In the United States alone, more than 350,000 individuals die each year with evidence of skeletal metastasis, mainly arising from breast or prostate tumors and to a lesser extent from lung and kidney cancers [38, 39]. The pathophysiology of bone metastasis is complex, involving different cell populations and regulatory proteins [25]. Bone metastases have been classified as either osteolytic or osteoblastic, depending on which cell types are involved, however, in clinical practice, most patients hospit both types. Mainly breast cancer provokes osteolytic metastases, whereas most prostate tumors form osteoblastic lesions [38]. This traditional idea that bone metastases are either osteoblastic or osteolytic represents in fact a continuum [40]. Although bone scanning detects only osteoblastic metastases, in most bone scans of patients even harboring mainly osteolytic metastases, some osteoblastic element is present and therefore is imaged. The advent of new molecular techniques, namely DNA microarray platforms has enlightened the genetic determinants that are critical for tumor cell survival in bone. Kang et al. has studied an underlying gene expression signature in bone-colonizing variants that explained the organ tropism to bone [41]. Compared to the parental tumor population, the bone-colonizing tumor cells expressed significantly more matrix metalloproteinases-1 (MMP-1), interleukin-11 (IL-11), osteopontin, connective

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tissue growth factor, and the chemokine receptor CXCR-4, which accounted for homing to bone (CXCR-4), proteolysis (MMP-1), angiogenesis (connective tissue growth factor), and osteoclastogenesis (IL-11 and osteopontin) [25, 41]. There is a multiplicity of reasons for a tumors’ propensity to metastasize to bone: Increased blood flow in red marrow and the presence of adhesive molecules on tumor cells recognizing the marrow stromal cells and matrix enhance cancer cell entrance and installation in the bones. Adhesion per se triggers the secretion of angiogenic factors and bone resorbing factors from tumor cells that enable cancer cell survival and growth. The unique bone environment characterized by the continuous remodelling through osteoclast and osteoblast activity on trabecular surfaces provides cancer cells a soil rich in growth factors, such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and IGFs. Physical factors within the bone microenvironment, including low oxygen levels, acidic pH, and high extracellular calcium concentrations, may also enhance tumor growth [42]. Bone-derived chemokines such as osteopontin, bone sialoprotein, and stromal-derived factor also act as chemoattractants for cancer cells [43–46]. Such interactions are described in Figs. 3.3 and 3.4. The predilection of some tumors to metastasize to the bones, for example breast cancer, was described more than 50 years ago by Walther in 1948 [47]. He found in an autopsy study (when adjuvant chemotherapy did not exist) that 64% of 186 patients who died of breast cancer had metastases to bone. Two more recent studies Osteotropism •

•

Avβ3 integrin: adhesion molecule of cancer cell binds to bone matrix protein

Cancer cells decorate themselves with • bone sialoprotein (BSP) • Osteopontin and bone cells expressing integrins trap them → preferential adhesion

cancer cell

Osteopontin: →ανβ3 integrin: blocks apoptosis

blood vessel

osteoblasts

osteoclast

Fig. 3.4 The role of integrins and ostopontin in osteotropism

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reported that 62–71% of breast cancer patients had bone metastases at autopsy, suggesting that chemotherapy was not a preventive factor as far as bone metastases are considered [48, 49]. Breast, lung and prostate cancer have a definitive predilection to bone metastasis [50, 51]. Stromal interactions are important for cancer osteotropism. Through cell-surface adhesion molecules, such as the ανβ3 integrin, present in cancer cells, they bind to bone matrix proteins. Osteopontin (OPN) binding and signaling through ανβ3 integrins on breast cancer cells blocks apoptosis, providing a survival advantage to breast cancer cells in bone [52, 53]. Then, cancer cells decorate themselves with bone matrix proteins, such as bone sialoprotein (BSP) and OPN [52, 54, 55] enabling bone stromal cells expressing integrins to trap them. This adhesion of cancer cells to the bone marrow vasculature is preferential and depends on the phenotypic differentiation of endothelial cells across organs [56–59].

3.5 Angiogenic Molecules in Bone Metastasis 3.5.1 VEGF As already described, VEGF is a fundamental angiogenesis inducer secreted by cancer cells and by activated tumor- associated fibroblasts contributing to the growth of the primary and secondary tumors. VEGF expression is directly dependent on the transcriptional activity of hypoxia inducible factors 1 and 2 (HIF 1 and 2) [60]. Hypoxia decelerates the degradation for the HIF1a and HIF2a subunits of HIFs, leading to the accumulation of HIFs in the cytoplasm of cancer cells, entrance in the nuclei and binding to the hypoxia responsible element of VEGF-A and of genes involved in angiogenesis, anaerobic metabolism and apoptosis inhibition. VEGF binds to receptors on endothelial cells that are transmembrane tyrosine kinases and are coupled to the cellular regulatory network [9]. VEGF receptors are highly expressed on endothelial cells but also in cancer cells. VEGF acts by binding to three distinct VEGF receptors: VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1), and VEGFR3 (Flt-4) [60–62]. VEGFA isoforms are the basic angiogenic molecules since they induce most of angiogenesis steps (migration, protease production, and proliferation). Moreover, VEGF increases the permeability of blood vessels by stimulating the functional activity of vesicular-vacuolar organelles, clusters of cytoplasmic vesicles and vacuoles located in microvascular endothelial cells [62]. This is thought to facilitate tumor progression by generating an extravascular fibrin gel that acts as a substrate for endothelial and tumor cell growth [63]. VEGF also mediates endothelial cell survival by upregulating the phosphatidylinositol-3 kinase/Akt signal transduction pathway [64] and stimulating expression of the antiapoptotic proteins Bcl-2 and A1 [65]. VEGF has been proven to promote osteoblast activity and to induce initial differentiation of osteoblasts; however, it requires other factors to induce mineralization [66]. Furthermore, it has been shown to contribute to prostate cancer–induced osteoblastic activity in vivo [66].

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3.5.2 Heparenase Other growth-regulatory factors, involved in bone metastasis are heparan sulfates [67]. They are present within the tumor microenvironment as components of heparan sulfate proteoglycans or as free heparan sulfate chains. They act to fine-tune the activities of growth factors and chemokines and therefore regulate tumor cell growth, angiogenesis, and osteoclastogenesis [68]. One of them, syndecan-1, is a major heparan sulfate proteoglycan of breast cancer cells which is also found in the bone marrow, that correlates with poor prognosis [69]. Syndecan-1 is also induced in reactive stroma responding to breast cancer [70]. Interestingly, the heparan sulfate of tumors of the breast is substantially different from that found in normal breast tissues [67]. This may be due in part to the action of heparan sulfate–modifying enzymes, such as heparanase. Heparanase cleaves heparan sulfate chains with an endo-´ι-D-glucuronidase activity releasing activated fragments of heparan sulfate that mediate its growth and angiogenic effects by acting on tumor and endothelial cells [68]. These fragments interact with growth factors, while the cleavage of heparan sulfate contributes to erosion of basement membrane barriers, thereby facilitating invasion and metastasis [71, 72]. Indeed, heparanase has been directly implicated in promoting invasiveness, angiogenesis, and metastasis [73–76]. Heparanase is an important factor of breast carcinomas, correlating with greater metastatic potential [77]. It has been shown that the expression of heparanase in myeloma cells implanted in immunodeficient mice promotes tumor metastasis to bone [78]. Furthermore, that same effect has been shown in breast cancer, where a marked enhancement of osteoclastogenesis and bone turnover was discovered in animals bearing tumors that expressed heparanase compared with controls, suggesting a role for heparanase in promoting bone resorption even when tumors are not evident in the bone [67]. The underlying mechanism is the stimulation of osteoclastogenesis. Heparanase might hold a role in mediating the release of osteolytic agents into the circulation that then travel to act on bone [67]. This has also been shown in other studies emphasizing the role of heparan sulfate proteoglycans, such as syndecan-1, interleukin (IL)-8, hepatocyte growth factor, FGF, and osteoprotegerin [68, 79–84]. Interestingly, these findings are consistent with the fact that treatment with heparin, a highly sulfated form of heparan sulfate, causes bone resorption and decreased bone density [85]. It has further been hypothesized that tumors condition the bone marrow for metastases by first stimulating osteoclastogenesis and bone resorption and by releasing various factors stored in the bone that fuels further tumor growth, thereby leading to continued stimulation of osteoclasts [86, 87]. Functional interactions between metastatic cancer cells and bone cells are essential, that are being mediated by soluble stimulators of osteoclast activity [86, 87]. Anyhow, expression of heparanase by a tumor distal to the bone can have a dramatic impact on bone turnover by affecting skeletal integrity, preparing a growth-enriching bone microenvironment that will support metastatic tumor cells once they invade the bone.

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Thus, the use of heparanase inhibitors as an early therapeutic approach to impede progression of breast cancer as well as other bone-homing tumors, might prove a useful approach [67].

3.5.3 TGF-β TGF is an important molecule deriving from the bone extracellular matrix that activates PTHrP-independent osteolytic pathways. It acts through a Smad-dependent signaling pathway and leads to the induction of increased synthesis and secretion of IL-11 by bone-homing breast cancer cells. IL-11 is a known cytokine with powerful osteolytic activity, which is thought of playing a critical role in the molecular signature of bone metastasizing breast carcinoma cells [41]. TGF-β is present in high concentrations in bone matrix [88] and is expressed by some breast cancers and cancer-associated stromal cells [89]. TGF-β is stored in bone and is released and activated during osteoclastic bone resorption [90]. Therefore, it is suggested that TGF-β is an important factor for bone metastasis of breast cancer through the TGF-β receptor- mediated signaling pathway and that its interaction to PTHrP accounts for the osteolytic matastases seen in breast cancer patients [91]. Fibroblasts are now being considered as critical players in tumor growth by regulating the phenotype of the tumor cells as well as the angiogenic response that supports them [92]. Fibroblasts and their activated counterpart, the myofibroblast, synchronize these events through the expression of extracellular matrix molecules, growth factors and morphogens, including FGF and TGF-β [92]. TGF-β also participates into smooth muscle cell accumulation during normal angiogenesis [93]. It has already been mentioned that TGF-β is an important factor considering vascular morphogenesis: while VEGF is essential for initiation of angiogenic sprouting, TGF-β regulates cell migration and proliferation and regulates extracellular matrix [24]. Overexpression of TGF-β is associated with metastasis and poor prognosis, and TGF-β antagonism has been shown to prevent metastasis in preclinical models with surprisingly little toxicity [94]. The efficacy of the anti-TGF-β antibody 1D11 in suppressing metastasis was shown to be dependent on a synergistic combination of effects on both the tumor parenchyma and microenvironment through enhancement of the CD8+ T-cell-mediated antitumor immune response, but also through the innate immune response and angiogenesis. This study suggested that elevated TGF-β expression in the tumor microenvironment modulates a complex network of intercellular interactions that promote metastasis, while TGF-β antibodies reverse this effect, and produce no major effect of TGF-β antagonism on any cell compartment, thus letting authors of the study to suggest there might exist a good therapeutic window and autoimmune complications could be avoided [94]. Moreover, TGF-β has been suggested to depend on cyclooxygenase-2, which displays a proangiogenic activity, mediated principally through its metabolite PGE2.

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A novel signalling route through which PGE2 activates the Alk5-Smad3 pathway in endothelial cells, involving the release of active TGF-β, through a process mediated by the metalloproteinase MT1-MMP has been recently identified [95].

3.5.4 IGF Bone expresses large amounts of insulin-like growth factor IGF ligands, and the IGF system is required for normal bone physiology. In a study, high IGF-IR-expressing neuroblastoma cells have been shown to adhere tightly to bone stromal cells, flatten, and extend processes [96]. The IGF/IGF-IR system plays a major role in the pathogenesis and progression of Ewing’s sarcoma [97–99]. Moreover, a central role for IGF-1 in the pathophysiology of multiple myeloma has been established. IGF-I deriving from the tumor-microenvironment interaction may facilitate the migration, survival and expansion of the multiple myeloma cells in the bone marrow [100, 101]. Moreover, the inhibition of the IGF-1R-mediated signalling pathway has been suggested to be a possible new therapeutic principle in multiple myeloma. Targeting the IGF-1R using picropodophyllin has both antitumor activities and also influences the bone marrow microenvironment by inhibiting both angiogenesis and bone disease [100, 102]. Even more interestingly, evidence suggests that IGF family is a multi-component network of molecules involved in the regulation of both physiological and pathological growth processes in the prostate [103]. Their roles include participation in cellular metabolism, differentiation, proliferation, transformation and apoptosis, during normal development and malignant growth [103]. They also are essential in prostate cancer bone metastases, angiogenesis and androgen-independent progression. Therapeutic interventions in men with androgen independent progression targeting the IGF family are currently under intense research, such as reduction of IGF-I levels (growth hormone-releasing hormone antagonists, somatostatin analogs), reduction of functional IGF-I receptor levels (antisense oligonucleotides, small interfering RNA), inhibition of IGF-IR and its signalling (monoclonal antibodies, small-molecule tyrosine kinase inhibitors) and IGF Binding Proteins [103]. VEGF is produced by many cell types, including osteoblasts. IGF-I is a known osteogenic factor and it modulates VEGF expression in osteoblasts [104]. Therefore, IGF-I may enhance osteoblast synthesis of VEGF, which may then act locally on endothelium to stimulate angiogenesis [104]. Moreover, IGF-I has a local regulatory role in bone remodelling, regulates proliferation of bone-derived endothelial cells and has a role in skeletal angiogenesis. IGF-I induces growth and chemotactic responses in bone endothelium acting through the type-I IGF receptor, which might be part of a generalized response of bone cells to IGF-I that facilitates cell migration [105]. Nevertheless, the transcription factor runt-related gene 2 (RUNX2)/core binding factoralpha-1/acute myeloid leukemia 3/polyoma enhancer-binding protein 2alphaA/osteoblast-specific transcription factor 2 regulates osteoblast differentiation through cascades involving IGF [106]. RUNX2 might be important in IGF-I and extracellular matrix-regulated endothelial cell migration and differentiation [106].

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3.5.5 PDGF Another family of genes activated in response to HIF-1α signaling is this encoding the polypeptide chains of platelet-derived growth factor (PDGF) [107]. PDGF is a family of cationic homo-and heterodimers of disulfide-bonded A- and B-chains, which are synthesized as precursor molecules that assemble into dimers and undergo proteolytic processing [108,109]. To date, five PDGF isoforms have been identified: PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. These isoforms act by binding to two tyrosine kinase receptors, PDGF-Rα and PDGF-Rβ. What is interesting about PDGF is that its functional activity depends, to a large extent, on the anatomical location of the tumor in question. Tyrosine kinase receptors can play different roles in different vascular beds. Imatinib (STI571 or Glivec; Novartis Oncology), that selectively inhibits activation of PDGF-R signal transduction has changed the field in the treatment of chronic myelogenous leukemia [110]. In an orthotopic murine model of hormone-refractory human prostate cancer metastasis to the bone, enhanced tumor cell expression of PDGF-BB, and its receptor PDGF-Rβ has been noted [25]. The expression of these proteins was enhanced in lesions growing adjacent to bone. In contrast, these angiogenic proteins were poorly expressed in surrounding tissues, such as muscle [9]. They also noted that PDGF-Rβ was activated on both the prostate tumor cells and the tumor-associated endothelium, while phosphorylated PDGF-Rβ was not found in either of them, thus suggesting a paracrine as well as an autocrine action for PDGF-BB. This expression pattern of PDGF-Rβ suggested that it might be a good target for therapy because its inhibition could affect the malignant cell population as well as the blood vessels that support tumor growth. Indeed, treatment of mice with imatinib or the combination of imatinib plus paclitaxel led to induction of significant apoptosis of both tumor cells and tumor-associated endothelial cells, resulting in smaller tumors, fewer lymphatic metastases, and a significant reduction in bone lysis. These experiments demonstrated that tumor-associated endothelial cells express phosphorylated PDGF-R when confronted with tumor cells that secrete PDGF ligands and that inhibition of this activation, particularly in combination with chemotherapy, can produce a significant therapeutic effect.

3.5.6 Interleukin-8 (IL-8) IL-8 is a member of the alpha chemokine family of cytokines originally identified as a neutrophil chemoattractant. IL-8 has been shown to stimulate osteoclastogenesis and bone resorption and is characteristically expressed by tumors that tend to metastasize to bone [87]. Expression of IL-8 is significantly enhanced by lysophosphatidic acid, which is a product of activated platelets [111]. Breast cancer cells have been proven to induce platelet aggregation and stimulate the secretion of lysophosphatidic acid. It has been reported that elevated levels of IL-8 correlated with increased bone metastasis in breast cancer [87].

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IL-8 is thought of playing a major role in VEGF-independent tumor angiogenesis [112]. Induction of IL-8 preserved the angiogenic response in HIF1-α-deficient colon cancer cells, suggesting that IL-8 mediates angiogenesis, independently of VEGF [113]. IL-8, a member of the CXC chemokine family exerts its angiogenic properties on endothelial cells through interaction with its receptors CXCR1 and CXCR2 [114, 115]. Overexpression of IL-8 is associated with advanced disease, poor prognosis and tumor recurrence in several cancer types [116, 117]. The synthesis and secretion of IL-8 depends, among others, on EGFR signalling [118]. In addition, activation of endothelial growth factor receptor EGFR is involved in the pathogenesis of bone metastases, through stimulation of bone marrow stromal cells to produce osteoclastogenic factors and to sustain osteoclast activation [118].

3.5.7 Interleukin-6 (IL-6) Mesenchymal stem cells (MSC) are a predominant fibroblast cell population within the bone marrow and are among the first cell types to encounter metastatic breast cancer cells. Hormone-responsive (i.e., estrogen receptor- alpha (ERα)-positive) tumors have a much stronger metastatic predilection for bone than their ERα-negative counterparts [119–121]. Although hormone-responsive breast cancer patients tend to have a more favorable clinical prognosis than hormone unresponsive patients, those presenting with bone metastasis and increased serum IL-6 levels face high mortality rates [122, 123]. Elevated IL-6 serum levels directly correlate with disease staging and unfavorable clinical outcomes in women with metastatic breast cancer [124]. Activated fibroblasts produce elevated levels of IL-6 [125]. The pleiotropic cytokine IL-6 has many homeostatic functions and serves as a growth factor for several cancers including multiple myeloma and prostate cancer [124, 126]. Among other cytokines exerting an important role in multiple myeloma, interleukin 6 (IL-6), IGF-1, VEGF and others are the most critical. They are secreted from stromal, endothelial cells and/or osteoclasts, and promote myeloma cell growth as well as paracrine cytokine secretion and angiogenesis in the bone marrow [101]. IL-6 is considered among the major osteoclastogenic cytokines, along with IL-1β and TGF-β. In multiple myeloma, B-cell plasmacytomas stimulate bone resorption and angiogenesis, resulting in osteolytic lesions. An interaction between multiple myeloma cells and mesenchymal stem cells from bone marrow stroma results in the formation of osteolytic metastases. It has been shown that an IL-6-neutralizing antibody blocks this effect [127]. There is a mutual stimulation between VEGF and IL-6, suggesting paracrine interactions between myeloma and marrow stromal cells [128]. Breast cancer cells interact with bone marrow cells and result in increased production of IL-6 that enhances bone destruction and angiogenesis within the bone [129, 130]. IL-6 is produced by macrophages, T cells, B cells, endothelial cells and

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tumour cells. It promotes tumour growth by upregulating anti-apoptotic and angiogenic proteins in tumour cells [131]. In a recent study it was shown that serum IL-6 levels have independent prognostic value in patients with metastatic breast cancer and that they are correlated with the extent of disease [131].

3.6 General Principles of Antiangiogenic Therapy Early in the mid ‘90s, when antiangiogenic therapy first became a realistic option in cancer treatment, some first guidelines where proposed by Folkmann: “(i) since antiangiogenic therapy is directed at specific targets of angiogenesis (migrating and proliferating endothelial cells), it is not likely to cause bone marrow suppression, gastrointestinal symptoms, or hair loss, as observed with chemotherapy. (ii) antiangiogenic therapy needs to be administered for several months to a year or more, since it down-regulates neovascularization by inhibiting the proliferation and migration of endothelial cells rather than by killing them and this is a much slower process than the lysis of tumor cells. (iii) antiangiogenic agents are not likely to cause resistance phenomena similar to those observed with classic chemotherapeutic agents. (iv) combining antiangiogenic and cytotoxic therapy or radiotherapy may be more effective than either type alone. (v) antiangiogenetic therapy does not have to cross the blood–brain barrier” [1]. Understanding the regulatory mechanisms of the angiogenic process allows for the development of compounds and monoclonal antibodies suitable for antiangiogenic therapy. Monoclonal antibodies that bind and block the actions of VEGF have been developed, as well as compounds interfering with signal transduction of VEGF receptors. Moreover, the knowledge of morphogenesis of new vessels provides important targets for antiangiogenic treatment. For example, sprouting capillaries express the integrins alpha-v-beta-3 αv β3 or alpha-v-beta-5 αv β5 . Abrogation of these integrins evokes programmed cell death (apoptosis) of the new endothelial cells and dramatically impairs neovascularization of tumors [132,133]. Anti-integrin antibodies and interfering RDG peptides are in development as candidates for antiangiogenic therapy [9]. Another biological principle is the interrelationship between tumor angiogenesis and modulation of cell death. The vasculature has been suggested to be a paracrine regulator of apoptosis [9]. Therefore, not only does inadequate vasculature promote necrotic cell death, but it also can elicit tumor cell apoptosis. Therefore, angiogenesis obtains a pivotal role in anticancer treatment. Angiogenesis inhibitors are already an important component of therapeutic strategies targeting metastatic tumors. Moreover, antiangiogenic treatments already emerge as efective adjuvant treatments as they may prolong the dormant state of micrometastasis improving the progression- free survival. Finally, as methods for early cancer detection improve, interference to cancer growth through downregulation of the angiogenic switch can become another option of both cancer treatment and prevention [9].

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3.6.1 Treatment of Bone Metastases with Anti-Angiogenetic Agents 3.6.1.1 Biphosphonates as Anti-Angiogenic Agents Skeletal complications of bone metastases increase the risk of death and undermine patients’ quality of life [134]. The role of biphosphonates, which are essential in the symptomatic palliation of bone metastasis is currently being investigated in the prevention of bone metastases. Recent evidence indicates that benefits of bisphosphonates may extend beyond the treatment of metastatic bone lesions and, through their potential anti-tumour activity, they may prevent bone metastasis. This antitumor activity is thought to be due to induction of apoptosis, inhibition of tumour cell invasion and tumour growth reduction [134]. Therefore, it is currently being suggested that patients with early-stage disease may benefit from early bisphosphonate therapy, before bone metastasis develops [134]. Part of the antitumor activity of bisphosphonates may be attributed to an antiangiogenic effect [135]. Treatment of endothelial cells with biphosphonates reduced proliferation, induced apoptosis, and decreased capillary-like tube formation in vitro, while reducing the quantity of blood vessels in bone biopsy specimens from patients with Paget’s disease [136]. It is possible that biphosphonates, particularly zoledronic acid and pamidronate, could represent a powerful tool for angiogenesis inhibition. However, these findings are still preliminary and cannot be affirmed unless properly tested in prospective clinical trials. Nitrogen-containing bisphosphonates lead to caspase-dependent apoptosis, inhibit matrix metalloproteinases and downregulate αv β3 and αv β5 integrins suppressing angiogenesis [137]. It has been shown that zolendronic acid at therapeutic dose levels markedly inhibits in vitro proliferation, chemotaxis and capillarogenesis of bone marrow endothelial cells of patients with multiple myeloma [138]. Zoledronic acid also reduces angiogenesis in the in vivo chorioallantoic membrane assay [138]. These effects are partly sustained by gene and protein inhibition of VEGF and VEGFR2 in an autocrine loop. At the clinical level, it has been shown that this antiangiogenic activity of zolendronic acid could be attributed, at least patially, to a transient reduction of VEGF, bFGF and MMP-2 circulating levels after infusion [139]. In another study the changes in VEGF and markers of bone resorption were assessed in a cohort of patients with metastatic bone disease following a single infusion of zoledronic acid [140]. The majority of patients developed a significant reduction in circulating levels of beta-CTX at just 1 day after the single zoledronic acid infusion and a statistically significant correlation between median VEGF and beta-CTX was noted. In another study, after a single dose infusion of zolendronic acid, the MMP-2, VEGF and bFGF basal values showed a statistically significant decrease in their circulating levels [139]. Even more interestingly, the VEGF- related zolendronic acid modifications were shown to correlate with time to- first skeletal-related event, time to- bone progression disease, and time to- worsening of performance status, thus suggesting that the VEGF modifications may represent a surrogate marker [141]. Alendronate also possesses an antiangiogenic effect, possibly deriving from its direct antiangiogenic effects on intra-tumor endothelial cells [142]. Moreover,

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neridronate was shown to have antiangiogenic properties in vitro [143]. In a recent study, treatment with clodronate encapsulated in liposomes (clodrolip) efficiently depleted tumor-associated macrophages in the murine F9 teratocarcinoma and human A673 rhabdomyosarcoma mouse tumour models resulting in significant inhibition of tumour growth [144]. Tumour inhibition was accompanied by a substantial reduction in blood vessel density in the tumour tissue and the strongest effects were observed with the combination therapy of clodrolip and a VEGF-neutralising antibody, whereas free clodronate was not significantly active. Minodronate inhibited melanoma growth and improved survival in nude mice by suppressing the tumorassociated angiogenesis and macrophage infiltration [145]. After treating patients with disodium pamidronate infusion, basal VEGF levels decreased significantly, while interferon-gamma (IFN-γ) and IL-6 levels increased [146]. 3.6.1.2 Targeted Anti-Angiogenic Therapies VEGF-A and its receptors play a role in both osteoclastogenesis and tumor growth. In a study, systemic (i.v.) treatment of nude mice bearing intrafemoral prostate (PC-3) tumors with the vascular ablative agent VEGF121 /recombinant gelonin (rGel) strongly inhibited tumor growth. Thus, VEGF121 /rGel inhibits osteoclast maturation in vivo and is probably important in the suppression of osteolytic lesions. This is a novel mechanism of action for this class of agents, suggesting a potentially new approach for treatment or prevention of tumor growth in bone [147]. Megavoltage irradiation and anti-VEGF monoclonal antibodies have been shown to have a beneficial effect on bone destruction [148]. Renal cancer is another disease with a known predilection to bone metastasis and interferon- α (IFN-α) -based therapies are among the standard agents used in the treatment of metastatic renal cell cancer. IFN-α has, among others, antiangiogenic properties; it has been implied there could be an effect of IFN-α on tumorinduced osteoclast differentiation and bone angiogenesis. In a recent study, it was shown that IFN-α has a wide spectrum of activities on renal cancer-induced bone disease, in addition to its recognized role as a cytotoxic and immunomodulatory agent, namely its ability to reduce bone resorption and to impair tumor-associated angiogenesis [149]. Vitronectin receptor, an ανβ3 integrin, is required for osteoclasts to adhere to the bone surface. Interactions between vitronectin receptor and the Arg-Gly-Asp tripeptide sequence found in several bone matrix proteins lead to osteoclast attachment, activation, and the release of cathepsins into the resorption lacuna [150]. Several small-molecule inhibitors of the vitronectin receptor specifically reduce the angiogenic activity and also inhibit bone resorption in vitro and in vivo, which makes them candidates for clinical testing [150–152]. Endothelin-1 binds to the G-protein–coupled endothelin-A receptor and initiates signaling pathways that lead to vasoconstriction, cell proliferation, and angiogenesis. Endothelin-1 is highly secreted from prostate cancer cells and stimulates osteoblast proliferation, leading to osteoblastic bone metastases. Inhibiting the endothelin-A receptor may prevent the formation of osteoblastic metastasis in patients with

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prostate cancer. Atrasentan (ABT-627) is an inhibitor of the endothelin-A receptor that promotes bone formation in vitro and inhibits osteoblastic metastases in mice [153]. Atrasentan is in phase III trials in patients with prostate cancer and bone metastases [154,155], as well as in patients with increasing prostate-specific antigen levels who are expected to develop bone metastases. c (Pfizer, The receptor tyrosine kinase (RTK) inhibitor SU11248 (sutent malate ), Inc.) is a multitargeted kinase inhibitor that inhibits VEGF receptor (R)-1, 2 and 3, PDGFR-α and β, Flt3, RET, and Kit [156]. Its inhibitory effects against osteoclast formation have been reported, suggesting that it may be an effective and tolerated therapy to inhibit growth of breast cancer bone metastases, with the additional advantage of inhibiting tumor-associated osteolysis [157]. In another experimental model of bone metastasis, an angiogenesis inhibitor, angiostatin, was showed to produce a marked inhibition in the extent of skeletal lesions through a direct inhibition of osteoclast activity and generation, suggesting that, besides its anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy [158]. Neovastat (AE-941), a naturally occurring multi-functional inhibitor of angiogenesis was also tested with respect to its antimetastatic bone cancer properties [159]. Neovastat prevented the degradation of osteoid-like radiolabeled extracellular matrices, while it inhibited the gelatinolytic activity of matrix metalloproteinase, producing, however a small decrease in the number of osteolytic lesions. c an inThe systemic administration of STI571 (imatinib mesylate, Gleevec ), hibitor of phosphorylation of PDGFR, in combination with paclitaxel, produced apoptosis of tumor cells and bone- and tumor-associated endothelial cells. Thus, by inhibiting angiogenesis, a decrease in tumor incidence and weight, and a decrease in incidence of lymph node metastasis were shown [160]. This therapeutic activity was correlated with inhibition of osteoclast function, inhibition of tumor cell proliferation, and induction of apoptosis in tumor-associated endothelial cells and tumor cells. The molecular players (angiogenic molecules) in bone metastasis and the possible emerging agents with anti-metastatic properties to bone are described in Fig. 3.5.

•

• • • • • • •

Interplaying molecules of angiogenesis in bone metastasis: VEGF Heparanase TGF-β IGF PDGF IL-8 IL-6

Agents with possible anti-metastatic properties to bone • • • • • • • • •

Anti-integrin antibodies (interfering RGD peptides) Biphosphonates (zolendronic acid, alendronate, nerindronate, clondronate, minodronate, disodium pamidronate) Vascular ablative agent VEGF 121 recombinant gelonin (rGel) IFN-α Vitronectin receptor (ανβ3 integrin) Endothelin-1 (atresentan ABT-627) Receptor tyrosine kinase (RTK) inhibitor, SU 11248 (sutent malate) Neovastat (AE-941) STI 571 (imatinib mesylate (Gleevec©), inhibitor of PDGFR

Fig. 3.5 Angiogenic molecules and emerging agents with antimetastatic properties to bone

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Chapter 4

NATURAL HISTORY, PROGNOSIS, CLINICAL FEATURES AND COMPLICATIONS OF METASTATIC BONE DISEASE Vassilios Vassiliou, Edward Chow and Dimitrios Kardamakis Department of Radiation Oncology, University of Patras Medical School, 26504 Patras, Greece, e-mail: [email protected]

Abstract:

The survival and prognosis of patients with metastatic bone disease varies widely and depends on many factors including the histologic type and grade of the primary tumor, performance status and age of patients, presence of extraosseus metastases, level of tumor markers and extend of skeletal disease. Bone metastases are inevitably associated with considerable morbidity and suffering, and severe complications such as pain, pathological fractures, spinal cord or nerve root compression, impaired mobility, bone marrow infiltration and hypercalcemia of malignancy. All aforementioned complications are thoroughly discussed, giving emphasis to associated symptomatology, clinical features and patient evaluation. The last part of the chapter deals with symptom clusters that occur in patients with bone metastases before and after treatment. Such symptoms are pain, depression, fatigue, drowsiness, anxiety, shortness of breath, nausea, poor sense of well being and poor appetite.

Key words: Bone metastases · Natural history · Prognosis · Morbidity · Complications · Clinical features · Symptom clusters · Hypercalcemia

4.1 Introduction Bone metastases are not only common in the event of malignancy, but their development is of particular clinical importance, since they can bring about severe complications such as pain, pathological fractures, spinal cord compression and hypercalcemia [1]. These events can be detrimental not only for the quality of life and performance status of cancer patients, but may also be life threatening [2]. In D. Kardamakis et al. (eds.), Bone Metastases, Cancer Metastasis – Biology and Treatment 12, DOI 10.1007/978-1-4020-9819-2 4, C Springer Science+Business Media B.V. 2009

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the current chapter we discuss the natural history, prognosis, clinical picture and complications of metastatic bone disease. Symptom clusters occurring in cancer patients with metastatic bone lesions are also presented.

4.2 Natural History and Prognosis Due to the high prevalence, marked osteotropism and the relatively long clinical course of breast and prostate cancer, bone metastases are most often seen in patients with such malignancies. Bone metastases are also frequent in other tumors such as lung, kidney and thyroid. The survival from the time of development of bone metastases varies considerably among the different types of tumors. In the case of prostate and breast cancer, the median survival from the time that bone metastases are diagnosed is measured in years [3, 4], whereas the corresponding survival in patients with advanced lung cancer is measured in months [5]. Through several studies it has been shown that certain tumor characteristics were associated with an increased risk of developing either bone or extaosseus metastases. In breast cancer patients the incidence of metastases to bone was found to be significantly higher in tumors which produce parathyroid hormone related peptide (PTHrP) [6] and are either estrogen receptor positive [7] or well differentiated [3,8]. A significant association between histological high grade tumors and a development of intrapulmonary, liver and para-aortic lymph node metastases has also been reported [8]. In a different study by James et al., a significant correlation between the development of bone metastases and the degree of lymph node involvement by the primary tumor was also found [9]. In a trial involving 2,240 consecutive patients with localized breast carcinoma, 30% relapsed after a median follow up period of 5 years, with 8% developing metastasis to bone. The median survival after the recurrence in bone was 20 months, whereas the survival in women who developed metastasis to liver was only 3 months [3]. The survival of patients with bone metastases from breast cancer was also influenced by the subsequent formation of extraosseus metastases. The median survival of such patients was shown to be 1.6 years as compared to 2.1 years for patients with metastases confined to the skeleton [10]. In the same study it was found that older, post menopausal women with lobular carcinoma or ductal grade III tumors were more likely to have disease that remains confined to skeleton [10]. The same was true for women with minimal axillary lymph node involvement [10]. Survival in women with bone metastases is also dependent on other clinical and histopathological factors such as the metastasis free survival interval, additional sites of metastatic disease other than bone, estrogen receptor status and serological tumor marker levels. Multivariate analysis has shown that all of these factors independently contributed to survival from the time of bone metastases formation [9]. In a different study by Coleman et al., multivariate analysis showed that age, menopausal status, bone disease at initial presentation and histological grade and type, were also important prognostic factors after the diagnosis of metastatic bone disease [10]. Important factors of good prognostic significance were lobular or ductal grade I or

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Table 4.1 Prognostic factors in patients with metastatic breast or prostate cancer Primary cancer

Breast

Prostate

Extraosseus metastases Estrogen receptor status Metastasis free survival Performance status Age Serological tumor marker levels Histologic type (lobular vs ductal) Histologic grade (ductal) Bone metastases at presentation

Performance status Histologic grade Baseline prostatic specific antigen Hemoglobin level Alkaline phosphatise Lactate dehydrogenase AST Extent of bone disease Age

Data from James et al. [9], Coleman et al. [10], Robson and Dawson [11], Sabbatini et al. [12], Eisenberger et al. [13], Matzkin et al. [14], Armstrong et al. [15]

II carcinomas, age < 70 years, disease free interval ≥ 3 years, bone disease at presentation and positive estrogen receptor status [10]. Established prognostic factors in women with bone metastases from breast cancer are presented in Table 4.1. Patients with prostatic carcinoma also have a relatively long clinical course. In men with metastases confined to the axial skeleton, good performance status and under androgen blockade, the duration of disease control was found to be 4 years [11]. Survival in patients with metastatic prostatic cancer is dependent on several prognostic factors such as tumor grade, baseline prostate specific antigen (PSA), PSA doubling time, hemoglobin, alkaline phosphatase (ALP), aspartate aminotranferase (AST), lactate dehydrogenase (LDH), performance status, number of metastatic sites and extent of metastatic bone disease (Table 4.1) [11–15]. It may be worth to note that the extent of metastatic bone disease in prostatic cancer may be quantified by using the bone scan index (BSI). In this system each bone is evaluated individually and assigned a numeric score. The score represents the product of the percentage of the involved bone with tumor times the known weight of the bone that is derived from the reference man [16]. It has been shown that in patients with BSI values 5.1%, median survivals were 18.3, 15.5 and 8.1 months respectively [12]. Survival in patients with multiple myeloma ranges from a few months to more than a decade [17]. With modern, intensive therapy involving autologous hematopoietic stem cell transplantation, the median survival is approximately 5 years [18]. Many prognostic factors have been reported in the scientific literature, the most important ones being albumin, beta2-microglobulin, chromosomal karyotype, renal function, hemoglobin, performance status, calcium, interleukin 6 (IL6), C-reactive protein (CRP), low plasma cell percentage in bone marrow and a positive response to treatment [17–19]. Renal cell cancer also shows remarkable osteotropism. Metastases from renal carcinoma are usually lytic in type, highly vascular and are associated with severe morbidity [20]. In a series with 209 patients with renal cell carcinoma, bone metastases developed in 22% of patients and bone was the second commonest site of metastases after lung (37%) [21]. In a recent study by Toyoda Y et al. it was reported

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that median survival in patients with bone metastases from renal carcinoma was 12 months and overall survival at 2 years was 37%. In the same study it was found that clinical features correlating with longer survival were a long interval between the time of diagnosis and development of bone metastases (greater than 24 months) and the absence of extraosseus metastatic disease [22]. The median survival of patients with none of the above favorable factors was 5 months and for those with both factors 30 months [22].

4.3 Morbidity and Complications of Metastatic Bone Disease Bone metastases are accompanied by considerable morbidity and suffering. About two thirds of patients with breast cancer and metastases to bone will subsequently develop complications such as pain, pathological fractures, spinal cord or nerve root compression, impaired mobility, bone marrow infiltration and hypercalcemia of malignancy [23–25]. Table 4.2 summarizes the potential complications associated with bone metastases. From the presented complications (Table 4.2), pathological fractures, hypercalcemia of malignancy, spinal cord compression, surgery to bone and radiation to bone are known as skeletal related events (SREs). These events are composite end points used in the majority of trials involving treatment with bisphosphonates. Pain and impaired mobility are evident in 65–75% of patients with bone metastases [26] and metastatic bone lesions have been reported to be the commonest cause of cancer-related pain [27]. Bone pain may be nociceptive [28, 29], or neuropathic [28–30]. In the former case pain is produced via simulation of nociceptors in the endostium by chemical mediators such as prostaglandins, leukotrienes, substance P, bradykinine, interleukins 1 and 6, endothelins and tumor necrosis factor-a (TNF-a). Nociceptive pain may also result due to stretching of periostium resulting from tumor infiltration or increase in size, or fracture. Neuropathic pain may result from direct infiltration and destruction of nerves by tumors. In two recent trials pain was found to be the major factor affecting the quality of life and performance status of cancer patients with bone metastases [31, 32]. The level of morbidity differed between patients with different types of metastatic bone lesions (lytic, mixed, sclerotic) [31]. Figures 4.1–4.3 present typical examples Table 4.2 Complications that may accompany metastatic bone disease Pathological fracture Bone pain Hypercalcemia of malignancy Nerve root compression Impaired mobility Surgery to bone Radiation to bone Spinal cord compression Infiltration of bone marrow

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Fig. 4.1 Lytic bone metastases in the right (a) and left (b) iliac bones in two patients with renal carcinoma. This figure is reprinted from Clin. Exp. Metastasis 24:49–56, Fig. 3, copyright 2007, with kind permission of Springer Science + Business Media B

of lytic, mixed and sclerotic bone metastases. Patients with osteolytic lesions had the highest mean pain scores with 8.1 points (visual analogue scale, 0–10) and the least mean scores for quality of life (QOL-EORTC C30, physical functioning scale, 0–100) and Karnofsky performance status (KPS, 0–100) with 31.4 and 58.6 points respectively. This group of patients was also found to have the highest percentage and mean opioid consumption (measured in daily oral morphine equivalents, mg) and the least mean bone density with 116.3 Hounsfield Units (HU, measured by Computer Tomography). On the contrary the group with osteosclerotic bone lesions had the least mean pain score with 4.6 points, the highest mean scores for QOL and

Fig. 4.2 Typical mixed bone lesions in the second (a) and fifth (b) lumbar vertebrae, due to metastatic breast carcinoma in two separate patients. The above figure is reprinted from Clin. Exp. Metastasis 24:49–56, Fig. 4, copyright 2007, with kind permission of Springer Science + Business Media B

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Fig. 4.3 Osteosclerotic bone metastases in two different breast cancer patients, in the eighth thoracic (a) and fourth cervical (b) vertebrae. This figure is reprinted from Clin. Exp. Metastasis 24:49–56, Fig. 5, copyright 2007, with kind permission of Springer Science + Business Media B

KPS with 61.1 and 66.6 points respectively, the least percentage and mean opioid requirement and the highest mean bone density with 444 HU. Table 4.3 presents the mean values of the clinical and radiological evaluations of the 3 groups of patients taking part in the study [27]. Interestingly, this study also showed that bone density had a strong, negative, statistically significant correlation with pain and a strong, positive, statistically significant correlation with QOL (partial correlation coefficients −0.57 and 0.64 respectively) (Table 4.4). These results showed that Table 4.3 Summary of results of clinical and radiological evaluations Pts with lytic bone Pts with mixed bone Pts with sclerotic p value : lesions (n = 32) lesions (n = 30) bone lesions (n = 18) Pain score (0–10): Quality of life (0–100): Performance status (0–100): Bone density: (Hounsfield units) Opioid consumption: (%)

8.1 ± 2.2 31.4 ± 14.6

6.6 ± 1.7 45 ± 10.9

4.6 ± 1.3