Structural Effects of Photodynamic Therapy and Bisphosphonates on Healthy and Metastatically Involved Vertebral Bone

Transcription

1 Structural Effects of Photodynamic Therapy and Bisphosphonates on Healthy and Metastatically Involved Vertebral Bone by Emily Won A thesis submitted in conformity with the requirements for the degree of Master of Applied Science Biomedical Engineering Graduate Department of the Institute of Biomaterials and Biomedical Engineering University of Toronto Copyright by Emily Won 2009

2 Thesis Title: Structural Effects of Photodynamic Therapy and Bisphosphonates on Healthy and Metastatically Involved Vertebral Bone Degree and Year: Master of Applied Science, 2009 Name: Department: University: Emily Won Institute of Biomaterials and Biomedical Engineering University of Toronto Abstract The vertebral column is the most common site of skeletal metastatic development secondary to breast cancer. Multiple clinical treatments are available for spinal metastasis, including systemic bisphosphonates and radiation therapy, however the success of current treatment approaches varies considerably. Alternative treatment strategies for spinal metastatic destruction must be aimed at both reducing tumor burden and restoring mechanical stability. Photodynamic therapy (PDT) has been shown to be successful at destroying osteolytic lesions in preclinical models of breast cancer spinal metastasis. However, the clinical feasibility of PDT for spinal metastasis is dependent on its potential effects on the structural integrity of vertebral bone. This thesis aims to determine the effects of PDT alone and in combination with bisphosphonate therapy on the structural architecture and mechanical properties of healthy and metastatically involved vertebrae. PDT was shown to have a positive effect on vertebral bone structure, alone and in combination with previous bisphosphonate therapy. ii

3 Acknowledgments Working towards this Master s degree has been an incredible experience filled with learning opportunities, challenges, and triumphs. I have encountered many individuals along the way who have enriched this experience and helped me succeed in my academic and personal endeavours. First and foremost, I would like to thank my supervisor, Dr. Cari Whyne, who has been an incredible mentor and continuously provided support on both academic and personal levels. Cari has always provided insight and ideas about the project and guided me through all the struggles encountered during my thesis. I am truly grateful for all her hard work and encouragement, which has been an integral aspect of the successful completion of this thesis. I would also like to thank Dr. Margarete Akens and Dr. Lisa Wise-Milestone for their assistance with the seemingly never-ending series of animal work. Margarete provided much needed veterinary experience and without her it would not have been possible to perform so many treatments. I am also extremely thankful for Lisa s help with all the animal work. Her assistance has made treatment days much more tolerable and enjoyable, particularly on weekends and holidays when we had to go into the lab to check on our sick patients. My committee members, Dr. Brian Wilson and Dr. Albert Yee, have provided invaluable expertise throughout the project. I am very thankful for their advice, which has undoubtedly improved the scientific and clinical merit of my research. I am thankful for having a vibrant group of lab members who were always willing to provide help with the infamous Amira program and brightened the work environment on a daily basis. I am particularly grateful to Asmaa and Meghan, with whom I have shared the joys and tribulations of life as we motivated each other every day with our box of inspirational quotes. Finally, I would like to thank my mom, my dad, Albert, and Jonathan who have provided much love and encouragement that gave me the strength to push through the toughest phases of my research. I never would have succeeded without their support, and for that I am forever grateful. iii

4 Table of Contents Acknowledgments...iii Table of Contents... iv List of Tables... vii List of Figures...viii Chapter 1: Introduction Spinal Metastasis Bone and Bone Remodeling Treatment of Spinal Metastasis Radiation Therapy Chemotherapy /Hormone therapy Surgical Therapy Vertebral Augmentation Systemic Bisphosphonates Photodynamic Therapy Photosensitizers Photodynamic Therapy for Spinal Metastasis Thesis Objectives Thesis Outline References iv

5 Chapter 2: Short and Intermediate Term Effects of Photodynamic Therapy in Healthy Vertebrae Abstract Introduction Materials and Methods Photodynamic therapy µct Image Analysis Mechanical Testing Data Analysis Results Photodynamic therapy Stereological analysis Mechanical testing Discussion References v

6 Chapter 3: Short Term Effects of Photodynamic Therapy and Bisphosphonates in Healthy and Metastatic Vertebrae Abstract Introduction Methodology Study Design Animal Model Bisphosphonate Therapy Photodynamic Therapy Architectural Analysis Histological Confirmation of Tumor Destruction Mechanical Testing Statistical Analysis Results Discussion References Chapter 4: Summary Effects of Photodynamic Therapy on Bone Future Directions Conclusion vi

7 List of Tables Table 1.1. Rate of spinal metastases from common primary cancers... 1 Table 2.1 Summary of stereological and mechanical parameters 1 week and 6 weeks following PDT on healthy bone Table 3.1. Summary of stereological parameters in healthy and tumor involved bone following PDT and/or BP treatment Table 3.2. Summary of mechanical parameters in healthy and tumor involved bone following PDT and/or BP treatment Table 3.3. Correlation coefficients between stereological and mechanical parameters vii

8 List of Figures Figure 1.1. Mechanism of nitrogen-containing bisphosphonates... 6 Figure 1.2. Application of photodynamic therapy to the spine Figure 2.1. Administration of photodynamic therapy Figure 2.2. Volume shrinking threshold technique used to segment vertebral bone Figure 2.3. Triangulated surface of the vertebral body Figure 2.4. Force-displacement curve generated during axial compression testing Figure 2.5. µct slices of vertebrae Figure 2.6. Representative force-displacement curves Figure 3.1. Monitoring tumor growth with bioluminescence imaging Figure 3.2. Histology analysis of tumor burden Figure 3.3. Comparison of bone mass by µct Figure 3.4. Inflammatory response post PDT viii

9 1.1 Spinal Metastasis Chapter 1: Introduction Bone metastasis occurs in up to 1/3 of all cancer patients, with the vertebral column being the most common site of skeletal metastatic development 1,2. The incidence of skeletal metastases is increasing due to improving cancer treatments that prolong patient life expectancy. The propensity for primary tumors to migrate, establish and thrive in bone is due to the fertile environment of bone marrow, which is rich in growth factors that support tumor cell adhesion, proliferation, and viability 3. Among the types of cancers that metastasize to bone, breast cancer is one of the most common primary tumors to metastasize to the spine (Table 1.1) 4. Spinal metastasis is prevalent in breast cancer pathology because breast cancer cells produce many factors that stimulate the production and activity of bone resorbing osteoclasts. Among these factors, parathyroid hormone-related peptide (PTHrP) is thought to be a key molecule in the metastatic spread of breast cancer cells to bone. PTHrP induces osteoclast formation, which leads to increased bone resorption and release of growth factors residing in the bone marrow 3. Transforming growth factor-β (TGF-β), fibroblast growth factor, and platelet-derived growth factor are among the many growth factors released, and they stimulate tumor cell growth and viability. TGF-β in particular, further upregulates PTHrP production, which perpetuates a cycle of bone destruction and tumor cell growth. Thereafter, the pathological condition becomes incurable, causing considerable consequences for patients in terms of morbidity and mortality 3,5,6. Table 1.1. Rate of spinal metastases from common primary cancers in the United States 4. Primary Cancer Site Number of Cases Number of Spinal Metastases (%) All sites 113,831 11,884 (100) Breast 13,977 2,592 (25.7) Lung 10,568 2,410 (22.8) Blood 12,907 1,213 (9.4) Prostate 6,975 1,137 (16.3) Urinary tract 5, (8.4) Skin 10, (3.4) 1

10 Mechanical stability is critical in the spine, and thus bony destruction resulting from spinal metastasis has considerable consequences in terms of patient quality of life. Metastasis establishes primarily in the vertebral bodies, which bear 80% of the mechanical loads sustained by the body 7. Thus tumorous lesions in metastatic vertebrae compromise the mechanical stability of the spine, and in 2/3 of patients, lead to skeletal related events (SREs) such as pathological fractures, skeletal or neurological pain, hypercalcaemia, and spinal cord compression, which significantly hinder the daily activities of patients 8,9,10. Current treatment strategies for spinal metastasis are not only aimed at reducing tumor burden, but also at restoring stability in the spinal column. It is therefore imperative to understand tumor behaviour in bone, its influence on bone tissue, and the impact of treatments on bone pathology. 1.2 Bone and Bone Remodeling The human skeleton is made up of 80% compact cortical bone and 20% trabecular bone, which is arranged in a complex spongy mesh structure 11. Despite differences in structure, the composition of both cortical and trabecular bone is very similar, consisting of organic and inorganic material. The majority of the inorganic component is made up of hydroxyapatite, which is a crystalline form of calcium phosphate. The organic component of bone consists of the extracellular matrix and osteogenic cells. The extracellular matrix is composed primarily of type I collagen, which imparts structure and strength to bone tissue. Osteogenic cells are responsible for bone remodeling activities, and include osteoclasts, osteoblasts, and osteocytes. Osteoclasts are large multinucleated cells that release degradative enzymes to resorb bone. Osteoblasts are responsible for laying down new bone matrix on the surface of existing bone. Once bone mineralizes around osteoblasts, completely encasing them in bone, the cells become osteocytes. Bone is constantly undergoing three stages of bone remodeling (resorption, reversal, and formation) to adapt to mechanical stimuli and maintain calcium homeostasis. During resorption, osteoclasts resorb bone at the site of remodeling. Following bone resorption, the reversal phase occurs in which pre-osteoblasts are recruited and prepare the bone surface for the subsequent bone formation phase. During bone formation, pre-osteoblasts mature into osteoblasts, which in turn deposit bone matrix and release alkaline phosphatase to mineralize the matrix to form mature bone. Throughout physiological bone remodeling, osteoclast mediated bone resorption and osteoblast mediated bone formation are in constant balance to maintain healthy bone mass. 2

11 Bone resorption typically takes 3-4 weeks while bone deposition and mineralization is much slower and takes approximately 3 months 12. Following injury or illness, such as metastasis, the balance is disrupted and may lead to excess bone resorption or formation. 1.3 Treatment of Spinal Metastasis Treatment for spinal metastasis is aimed at reducing tumor volume, growth and associated mechanical instability that may result in pathological fractures and damage the spinal cord. The current clinical strategy for patients with established breast cancer metastasis to the spine is a multimodality approach that includes systemic chemotherapy and bisphosphonates (BP) in conjunction with local therapies such as radiation therapy (Rx), vertebroplasty, kyphoplasty and surgical intervention. The following sections provide an overview of various clinical treatments, and outline their corresponding benefits and shortcomings Radiation Therapy Radiation therapy (Rx) is widely used as a palliative therapy to treat symptoms of pain in spinal metastasis patients. However, some tumors are resistant to radiation, leading to varying and unpredictable tumor responses across patients 10. Although up to 80% of patients experience some pain alleviation, the proportion of patients with complete pain relief is low (35%) 10. Furthermore, radiation therapy does not immediately improve the mechanical instability resulting from tumor burden in the bone; hence patients undergoing radiation therapy often require further treatments such as surgical intervention. Unfortunately, several studies have found that radiation therapy increases wound complication rates by 3- to 4-fold following surgical procedures 13,14. Moreover, there is also a high recurrence rate in spinal metastases, which is difficult to treat with radiotherapy because surviving tumors become more radioresistant with repeated exposures while the spinal cord and surrounding tissues become more susceptible to radio-toxicity Chemotherapy /Hormone therapy Chemotherapy can be administered as an anti-cancer treatment when spinal metastatic lesions cannot be completely treated by radiation therapy due to accumulated radiation exposure to the spinal cord 15. Although chemotherapy is rarely administered as a stand-alone treatment for spinal metastasis, it is sometimes given as an adjuvant therapy after surgery or radiation therapy. For 3

12 breast cancer patients, chemotherapy is typically initiated immediately after local treatment. Hormone therapy is usually administered following surgery to reduce the risk of recurrence at both local primary tumor and distant metastatic sites 16. With hormone therapy, symptomatic effects do not appear until weeks or months following commencement of treatment. Furthermore, although chemotherapy and hormone therapy are aimed at treating tumors, they rapidly cause estrogen depletion, which contributes to decline in bone quality and increases the risk of vertebral fracture Surgical Therapy Surgery is an invasive procedure, and is thus typically reserved for those patients who experience neurological compromise, spinal instability, or failed radiation therapy due to radio-resistant tumors or having reached the radiation tolerance in the spinal cord 18. Any combination of posterior, anterior, and posterolateral approaches provides circumferential access to the vertebral body to enable excision of the tumor, decompression of neural tissue, and reconstruction of the spine using fixation devices to stabilize the spinal column. Surgical intervention has palliative benefits for most epidural spinal metastasis patients, who can experience significant pain relief, improved neurological complications, and maintain mobility. Due to the invasiveness of surgery, there is a long recovery time and several potential complications including surgical wound infections, fixation device failure, and neurological deficit 18. Wound infection is the most prominent surgical complication, which motivates the development of minimally invasive surgical procedures that would significantly reduce post treatment risks and complications Vertebral Augmentation Vertebroplasty and kyphoplasty both involve injection of bone cement (polymethylmethacrylate, PMMA) into the vertebral body to stabilize the vertebral column, which serves to relieve pain and prevent pathological fracture. In vertebroplasty, the cement is injected into the vertebral body through a needle inserted transpedicularly under fluoroscopic guidance. Kyphoplasty is a modified version of vertebroplasty, in which a balloon is inserted and inflated in the vertebral body. This generates a cavity within the vertebral body where the cement is then injected into the balloon space. By creating an encapsulated space, kyphoplasty helps to prevent leakage of the cement into the epidural space. It also allows the cement to be injected under lower pressures, so that a more viscous form of PMMA can be injected compared to that of 4

13 vertebroplasty, which further reduces the risk of leakage. The advantage of injecting bone cement is that almost immediate stabilization is achieved with a low complication rate in metastatic disease (10%) 15. However, these procedures are not designed to ablate metastatic lesions, and local tumor growth following treatment can ultimately lead to failure in mechanical stabilization Systemic Bisphosphonates Systemic bisphosphonate (BP) therapy is becoming a clinical standard for treating spinal metastasis secondary to breast cancer 19. Bisphosphonates alleviate symptoms of bone metastasis primarily through inhibiting osteoclast function. Bisphosphonates are analogues of endogenous proton pump inhibitors (PP i ), with a P-C-P backbone and two covalently bonded side chains, R 1 and R The backbone and the R 1 side chain exhibit a strong binding affinity to the hydroxyapatite surface of bone, leading to the accumulation of BPs in regions of increased bone activity. The R 2 side chain determines the potency of a particular BP to inhibit osteoclast function. First generation nonnitrogen-containing bisphosphonates, such as clodronate, are metabolized into cytotoxic ATP analogues that inhibit ATP-dependent enzymes and ultimately lead to osteoclast death. Newer generations of nitrogen-containing bisphosphonates, such as alendronate and zoledronic acid, inhibit farnesyl diphosphate (FPP) synthase in the mevalonate pathway, which reduces the amount of geranylgeranyl disphosphate and the subsequent prenylation of small GTPases such as Rho, Rab, and Rac (Figure 1.1). These small GTPases are essential in many molecular pathways, and ultimately hinder osteoclast function and induce osteoclast apoptosis. Bisphosphonates may also impede the progression of bone metastasis through reduction of matrix metalloproteinase (MMP) activity, which acts to digest the basement membrane of bone for tumor cell invasion. By inhibiting bone resorption, BPs inhibit the release of bone-derived growth factors that stimulate tumor cell activity and ultimately reduce tumor cell survival in bone and further metastatic progression. 5

14 Mevalonate Pathway mevalonate N-containing bisphosphonates isopentenyl pyrophosphate (IPP) FPP synthase geranyl disphosphate (GPP) farnesyl diphosphate (FPP) geranylgeranyl diphosphate (GGPP) geranylgeranylated proteins required for osteoclast function e.g. Rho, Rac Figure 1.1. Mechanism of nitrogen-containing bisphosphonates: Zoledronic acid is a nitrogen-containing bisphosphonate, which inhibits farnesyl diphosphate synthase in the mevalonate pathway and decreases osteoclast function. There is gathering evidence that nitrogen-containing bisphosphonates may have direct anti-tumor effects in select breast cancer cell lines through suppressing cell growth, inducing cell apoptosis, and reducing cell adhesion and invasion into bone (e.g. MDA-MB-231, MCF-7) 19. The exact anti-tumor mechanism of BPs is unknown, however, it is proposed that the inhibition of prenylated small GTPases alters tumor cell activity and survival. The attenuation of prenylated RhoA leads to reduced tumor cell motility to drive cell invasion into bone. Zoledronic acid has been shown to inhibit integrin activation, which is required for cell adhesion to bone matrix. High doses of bisphosphonates have also been shown to inhibit mitogenic and anti-apoptotic pathways, which in turn lead to caspase activation to initiate apoptosis. However, anti-tumor effects are only evident in some cell lines, and there is no conclusive evidence that clinically relevant doses of bisphosphonates exhibit anti-tumor effects. Further investigation of varying bisphosphonate doses and dosing regimens in various types of tumor cells is required to fully elucidate the treatment protocol that would elicit direct anti-tumor effects. 6

15 Of the nitrogen-containing bisphosphonates studied, zoledronic acid is the most potent BP in inhibiting FPP synthase and influencing tumor cell viability, and will likely be the most effective BP to fight bone metastases 19. Since zoledronic acid is administered intravenously and does not undergo biotransformation prior to absorption into bone, only 4mg is required to achieve a therapeutic effect 21. This is much lower and less toxic than the 90mg of pamidronate, also administered intravenously. Furthermore, the BP dosage is administered over a 15 minute interval, which is more tolerable than the 2 hour infusion of pamidronate, and increases patient compliance 21. Patients are administered 4mg of zoledronic acid once every 3-4 weeks, which provides a convenient treatment schedule. A statistical analysis on clinical bisphosphonates trials showed that bisphosphonates significantly reduced the odds ratio for vertebral fractures compared to placebo (0.69, 95%CI: 0.57 to 0.84, p<0.0001) in spinal metastasis patients. However, in those patients whose spinal metastasis originated from breast cancer, there was no significant reduction in vertebral fractures compared to placebo (odds ratio 0.87, 95%CI: 0.71 to 1.06) 22. Studies in animal models of breast cancer metastasis have shown that BP administered in a preventative manner significantly reduced metastasis. However, BPs failed to impede growth of metastatic tumors that had already reached a threshold size in the bone 23. Therefore in cases where metastatic disease is detected in advanced stages of the disease, BP therapy may not be an effective treatment at impeding the progression of metastatic spread to the spine. Despite the arsenal of treatments available to patients, tumor response in the spine varies across patients, and no combination of therapies has consistently achieved a comprehensive effect on vertebral metastasis 24. Tumor recurrence also introduces treatment complications by limiting the number of repeated treatment sessions for therapies such as radiation and chemotherapy, which result in toxicity accumulation. As such, there is an increasing demand for novel approaches to treat metastatic lesions in the spine, particularly when existing therapies are ineffective or no longer viable options. 1.4 Photodynamic Therapy Photodynamic therapy (PDT) is a promising minimally invasive cancer treatment that has been utilized in treating various cancers where tumors are directly or endoscopically accessible by an external light source, such as skin, lung, bladder, and gastrointestinal neoplasms 25. PDT involves 7

16 administration of a photosensitizer, which circulates through the vasculature and is preferentially taken up by malignant tissue. Once activated by light at a specific wavelength, the photosensitizer produces highly reactive singlet oxygen that causes cell toxicity and death Photosensitizers Photosensitizers are non-toxic dyes that become activated by light energy at a specific wavelength. When a photosensitizer in the ground state absorbs a photon of light, it becomes excited to a singlet state 25. At this point, it can either return to ground state and emit a fluorescent photon or undergo intersystem crossing to a triplet state. From the triplet state, it can either return to the ground state and emit a phosphorescent photon, or it can transfer its energy to another molecule by radiationless transition. In the presence of oxygen, the photosensitizer easily transfers its energy to ground state molecular oxygen, creating highly reactive singlet oxygen that subsequently causes cytotoxic effects. Due to the extremely high reactivity of singlet oxygen, it is depleted within 0.04µs and its toxic effects only reach a volumetric space of 0.04µm in diameter 25. Thus PDT-induced oxidative damage is highly localized and damage to surrounding tissue is minimized. There are various types of photosensitizers, and the selection of a photosensitizer for a specific cancer treatment depends on the excitation wavelength (dictating the depth of tissue penetration) and the biodistribution of the photosensitizer in various tissues 26. The ideal photosensitizer for oncological purposes possesses the following properties: i) an excitation energy at a wavelength that corresponds to the depth of tissue penetration ii) iii) iv) efficient generator of reactive singlet oxygen high specificity for targeted malignant tissue over surrounding normal healthy tissue rapid clearance from the system to reduce skin photosensitivity following treatment Photosensitizers have been modified to achieve these ideal characteristics as they have continued to develop for various applications. Porfimer sodium (Photofrin) is a first generation photosensitizer, and was the first to be approved for PDT to treat superficial papillary bladder cancer 26. The absorption in the red spectrum (required for deep tissue penetration) is low, thus relatively high light doses of J/cm 2 is required to induce a therapeutic effect. Clearance 8

17 of Photofrin is low, leading to a long skin sensitivity period of 4-12 weeks following treatment. 5-aminolevulinic acid (5-ALA, Levulan) is a second generation photosensitizer approved for the treatment of actinic keratosis 26. Although ALA itself is not photosensitive, it is a precursor in the heme pathway in which ferrochelatase converts protoporphyrin IX (PpIX) to heme. Since many tumors have low ferrochelatase activity compared to normal tissue, administration of ALA leads to an accumulation of PpIX in malignant tissue. PpIX has photosensitizing properties, and thus elicits photodynamic effects upon light activation. Although PpIX has low absorption of red light, it is advantageous over older generation porfimer sodium because it enables higher tumor selectivity, and clears rapidly from the system, resulting in only a 1-2 day skin photosensitivity period. New generation photosensitizers such as benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) are designed to absorb light at higher wavelengths, distribute more selectively in tumor tissue, and clear rapidly from the system to reduce the period of skin photosensitivity following treatment. BPD-MA (commercially available as Visudyne) is an FDA-approved photosensitizer for treating age related macular degeneration, and it has been used safely to treat patients worldwide with minimal toxic side effects. Visudyne is delivered in the body via lipoproteins and is excited by light at 690nm, which allows deeper penetration to reach the target tissue. Since BPD-MA is cleared from the system 24 hours following injection, skin sensitivity following treatment is reduced. PDT is an attractive alternative for cancer therapy because the photosensitizer accumulates preferentially in tumorous tissue and is activated only in the presence of light. Hence therapeutic effects can be targeted locally and selectively ablate malignant cells with minimal damage to surrounding tissues Photodynamic Therapy for Spinal Metastasis PDT can be applied to the spine by adapting a minimally invasive technique developed for vertebroplasty to deliver light to the vertebral body (Figure 1.2). Previous studies in preclinical porcine and rat models have shown that a single PDT treatment using this technique can be successful in ablating vertebral metastases secondary to breast cancer. The extent of therapeutic effect was found to be proportional to the dosage of light energy. However, high light dosages applied at 3 hours (when concentration of photosensitizer in the tumor was high) were associated with increased incidence of paralysis. 9

18 Figure 1.2. Application of photodynamic therapy to the spine: Light is administered adjacent to the vertebral body (outlined in blue) using optical fibers guided through an 18 gauge needle. A preclinical human MT-1 breast cancer cell rnu/rnu rat model of breast cancer metastasis has been established that is suitable for studying the effects of photodynamic therapy in the metastatic spine. In this model, human MT-1 breast cancer cells are inoculated via intracardiac injection. Since rnu/rnu rats are immunodeficient, the cells are able to survive and circulate through the vasculature to establish in the spine. Although the tumor cells are injected directly into the bloodstream and bypasses the requirement for primary cells to detach from the primary site, this model uses a breast cancer cell line that originates from humans, which is better able to mimic the clinical response of the tumor population to PDT treatment. Following tumor cell inoculation, the rats can survive for 21 days before ethical endpoints are reached, providing a total period of 3 weeks for treatment and assessment. In a study comparing benzoporphyrin derivative monoacid ring A (BPD-MA) to 5- aminolevulinic acid (5-ALA), only BPD-MA was taken up by malignant tissue preferentially over healthy tissue in the vertebrae and caused minimal damage to the spinal cord 27. With a short drug-light interval (15 minutes between BPD-MA administration and light activation), the majority of the BPD-MA remains in the vasculature, and cytotoxic effects act on the endothelium of blood vessels to cut off the nutrient supply to the tumor (termed vascular-targeted PDT). In contrast, following longer drug-light intervals (3 hours between drug administration and activation), the photosensitizer absorbs into the tumor tissue and causes cytotoxicity directly to the tumor cells upon light activation. Studies have shown that the vasculature feeding the 10

19 tumor tissue should be eliminated to achieve long term tumor ablation, suggesting that vasculartargeted PDT may be more successful at achieving long term treatment effect. With previously demonstrated success of PDT with BPD-MA in destroying tumors, PDT has great potential as a treatment for spinal metastasis. Since the success of current treatment options varies across patients with spinal metastases, the increasing demand for novel treatment alternatives signifies that PDT can potentially have a significant impact on the clinical care of breast cancer patients. Hence it would be of great value to develop PDT as a minimally invasive intervention that would serve as either a stand-alone or additive treatment to existing therapies, aimed at reducing the risk of fracture and improving quality of life for patients with spinal metastases. However, the effects of PDT on the structural integrity of bone remain to be determined in order to evaluate its structural safety and clinical feasibility. If PDT proves to be effective at ablating tumor and improving the mechanical stability of the spine, then it will be an attractive minimally invasive therapy for spinal metastasis. 1.5 Thesis Objectives The purpose of this thesis is to evaluate the clinical potential of photodynamic therapy as a minimally invasive treatment for spinal metastasis secondary to breast cancer by understanding the impact of photodynamic therapy alone and in combination with bisphosphonate treatment on vertebral bone. The validity of PDT for spinal metastasis is evaluated based on an initial feasibility study that investigates the short term (1 week) and intermediate term (6 weeks) effects of PDT on the structural integrity of healthy vertebral bone. The potential of PDT is further analyzed by characterizing its effects both alone and in combination with bisphosphonates on the structural integrity and mechanical strength of non-pathologic and metastatically involved vertebrae. The purpose of studying PDT in combination with BPs is to determine the compatibility of PDT with existing therapies to emulate the clinical setting in which PDT will be administered. Understanding the potential impact of this therapy on spinal tumor burden and stability, particularly when traditional approaches such as BP have not been successful, is essential to ensuring the clinical success of this novel treatment. 11

20 1.6 Thesis Outline This thesis determines the clinical translational capacity of photodynamic therapy as a treatment for spinal metastasis. In evaluating the efficacy of photodynamic therapy, it is important to not only determine its effect on tumor, but to ensure that it can be safely applied to the spine without compromising the structural integrity of the vertebral bone. While PDT has been shown to be effective at treating metastatic tumors, a negative impact on the structural integrity of bone would be a disadvantage, whereas a positive impact on bone would aid in mechanical stabilization. In determining the clinical feasibility of PDT, the short and intermediate term impact of PDT on healthy vertebral bone will be investigated. Then the short term effects of PDT alone and in combination with traditional systemic bisphosphonates treatment will be determined in both healthy and tumor involved vertebrae. The work presented in this thesis is a compilation of two studies. The first has been accepted for publication (Spine, May 2009), and the second is a modified version of a manuscript that has been prepared for submission for review (Breast Cancer Research and Treatment). Chapter 2 investigates the effects of a single photodynamic therapy treatment on the bone architecture and mechanical integrity of healthy vertebral bone. Post treatment effects are examined 1 week and 6 weeks following treatment to determine the short term and intermediate term impact of PDT on vertebral bone quality. This study was of critical importance in evaluating the feasibility of PDT as a clinical therapy for spinal metastasis and provides guidance for future research endeavors. Chapter 3 focuses on the effects of PDT in metastatically involved vertebral bone. Since PDT will likely be administered to patients with previous exposure to currently available clinical treatments such as bisphosphonates and radiation therapy, it is also important to understand any interactions between PDT and existing therapies. This study will elucidate the effects of PDT in combination with prior bisphosphonate therapy on the structural and mechanical integrity of healthy and metastatically involved bone, and will provide insight into its translational capacity to the clinic where it may be administered alone or as a component of multi-modal treatment strategies. The final chapter will summarize the major findings from each study and discuss the implications on the clinical care of patients with spinal metastasis. It will also provide future 12

21 directions for this research to increase our understanding of PDT and how PDT may be optimized to ensure its successful translation into clinical application for the treatment of cancer in the spine. 1.7 References 1. Wong DA, Fornasier VL, MacNab I. Spinal metastases: the obvious, the occult, and the impostors. Spine 1990;15: Jacobs WB, Perrin RG. Evaluation and treatment of spinal metastases: an overview. Neurosurg Focus 2001;11:e Roodman GD. Mechanisms of bone metastasis. N Engl J Med 2004;350: Walsh GL, Gokaslan ZL, McCutcheon IE, et al. Anterior approaches to the thoracic spine in patients with cancer: indications and results. Ann Thorac Surg 1997;64: Houston SJ, Rubens RD. The systemic treatment of bone metastases. Clin Orthop Relat Res 1995: Toma S, Venturino A, Sogno G, et al. Metastatic bone tumors. Nonsurgical treatment. Outcome and survival. Clin Orthop Relat Res 1993: Ecker RD, Endo T, Wetjen NM, et al. Diagnosis and treatment of vertebral column metastases. Mayo Clin Proc 2005;80: Constans JP, de Divitiis E, Donzelli R, et al. Spinal metastases with neurological manifestations. Review of 600 cases. J Neurosurg 1983;59: Lipton A, Theriault RL, Hortobagyi GN, et al. Pamidronate prevents skeletal complications and is effective palliative treatment in women with breast carcinoma and osteolytic bone metastases: long term follow-up of two randomized, placebo-controlled trials. Cancer 2000;88: Tombolini V, Zurlo A, Montagna A, et al. Radiation therapy of spinal metastases: results with different fractionations. Tumori 1994;80: An YH. Orthopaedic Issues in Osteoporosised: Informa Health Care, Rodan GA. Bone mass homeostasis and bisphosphonate action. Bone 1997;20: Sundaresan N, Rothman A, Manhart K, et al. Surgery for solitary metastases of the spine: rationale and results of treatment. Spine 2002;27: Ghogawala Z, Mansfield FL, Borges LF. Spinal radiation before surgical decompression adversely affects outcomes of surgery for symptomatic metastatic spinal cord compression. Spine 2001;26: Bartels RH, van der Linden YM, van der Graaf WT. Spinal extradural metastasis: review of current treatment options. CA Cancer J Clin 2008;58: Houghton J. Initial adjuvant therapy with anastrozole (A) reduces rates of early breast cancer recurrence and adverse events compared with tamoxifen (T) - data reported on behalf of the ATAC Trialists' Group [Abstract]. Ann Oncol. 2006;17:243PD. 13

22 17. Cummings SR, Browner WS, Bauer D. Endogenous hormones and the risk of hip and vertebral fractures among older women. N Engl J Med 1998;339: Klimo P, Jr., Schmidt MH. Surgical management of spinal metastases. Oncologist 2004;9: Senaratne SG, Colston KW. Direct effects of bisphosphonates on breast cancer cells. Breast Cancer Res 2002;4: Clezardin P, Ebetino FH, Fournier PG. Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity. Cancer Res 2005;65: Coleman RE. Bisphosphonates in breast cancer. Ann Oncol 2005;16: Ross JR, Saunders Y, Edmonds PM, et al. Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer. BMJ 2003;327: Kaijzel EL, van der Pluijm G, Lowik CW. Whole-body optical imaging in animal models to assess cancer development and progression. Clin Cancer Res 2007;13: Tokuhashi Y, Ogawa T. [Spinal metastases]. Clin Calcium 2007;17: Juarranz A, Jaen P, Sanz-Rodriguez F, et al. Photodynamic therapy of cancer. Basic principles and applications. Clin Transl Oncol 2008;10: Triesscheijn M, Baas P, Schellens JH, et al. Photodynamic therapy in oncology. Oncologist 2006;11: Akens MK, Yee AJ, Wilson BC, et al. Photodynamic therapy of vertebral metastases: evaluating tumor-to-neural tissue uptake of BPD-MA and ALA-PpIX in a murine model of metastatic human breast carcinoma. Photochem Photobiol 2007;83:

23 Chapter 2: Short and Intermediate Term Effects of Photodynamic Therapy in Healthy Vertebrae This work has been accepted for publication in Spine, entitled "Effects of Photodynamic Therapy on the Structural Integrity of Vertebral Bone". 2.1 Abstract Spinal metastasis develops in one-third of all cancer patients, compromising the mechanical integrity of the spine and thereby increasing the risk of pathological fractures and spinal cord damage. There is a need for more effective local therapies to treat pre-critical, high-risk vertebral lesions. Photodynamic therapy (PDT) is a minimally invasive treatment that involves the administration of a photosensitizer that is activated by light at a specific wavelength and causes tumor cell and /or tumor microvascular destruction. PDT has recently been adapted to ablate metastatic tumors in the spine in preclinical animal models. The present study investigates its short-term (1 week) and intermediate-term (6 weeks) effects on the mechanical and structural properties of bone following a single treatment using the photosensitizer benzoporphyrin derivative in a normal rat model, at photosensitizer and light doses known to be effective in rats bearing human breast cancer metastases. Changes in trabecular architecture and global stiffness and strength of vertebrae post PDT were quantified using µct stereological analysis and axial compression testing. At 6 weeks, there was a significant increase in bone volume fraction (to 0.557±0.111 versus 0.385±0.064, p<0.001) and decrease in bone surface area-to-volume ratio (16.9±5.0/mm versus 22.8±4.5/mm, p=0.001), attributed to trabecular thickening (0.13±0.04 mm versus 0.09±0.02mm, p<0.001). Although not statistically significant, there was a similar stereological trend at 1 week following PDT. There was a significant increase in stiffness from control (306±123 N/mm) to 1 week (399±150 N/mm, p=0.04) and 6 weeks (410±113 N/mm, p=0.05) post PDT. There was a positive trend towards increased yield stress at 1 week, which became statistically significant at 6 weeks compared to control (39.3±11.3 MPa versus 27.5±9.5MPa control, p=0.002). These positive effects of PDT on bone have important implications for spinal metastasis patients. Not only may PDT be successful in ablating metastatic tumor tissue in the spine but, through its effects on bone remodeling, it may also improve the mechanical stability of vertebrae weakened by metastatic involvement. 15

24 2.2 Introduction Bone metastasis occurs in approximately one-third of all cancer patients (from breast, prostate, colon and other primary tumors), with the vertebral column being the most common site of skeletal involvement 1. Metastatic lesions compromise the mechanical stability of the spine and, in two-thirds of patients, lead to skeletal related events (SREs), such as pathological fractures and neurological complications arising from metastatic spinal cord compression 2,3. Treatment for spinal metastasis is aimed at reducing tumor volume, growth and associated mechanical instability that may damage the spinal cord. The current clinical strategy is a multimodality approach that includes systemic chemotherapy and bisphosphonates in conjunction with local therapies such as radiation therapy, vertebroplasty and surgery 4. Tumor responses are highly variable across patients and there are risks and side effects associated with all current modalities 5. Hence, there is an unmet need for more effective minimally invasive and low-risk procedures, either as stand-alone therapies or as adjuvants to existing treatments. Photodynamic therapy (PDT) with various photosensitizers is approved or under active investigation for a range of solid tumors, as well as non-oncological applications 6. PDT involves administration of a photosensitizer, either systemically or topically. A degree of tumor selectivity is usually achieved by photosensitizer uptake and/or clearance relative to normal host tissues, while increased target specificity is achieved by accurate delivery of the light to the tumor mass. Once excited by light at a wavelength that is specific to the photosensitizer, highly reactive singlet oxygen is generated that causes cell toxicity and tissue death 6. The use of optical fibers for light delivery has increased the range of primary tumor sites that are amenable to PDT, including lung, bladder, prostate and the gastrointestinal tract 6. In the case of spinal metastases, transpedicular placement of optical fibers to deliver light to the vertebral body has the potential to locally debulk lesions. This procedure can also facilitate other surgical interventions aimed at mechanical stabilization, such as vertebroplasty or kyphoplasty. A previous study has demonstrated the efficacy of PDT in a nude rat model of spinal metastases of human breast cancer, using the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA) 7. The safety profile of PDT treatment using transpedicular fiber-optic light delivery has been characterized in a normal porcine model 8,9. Rodent studies have also shown that BPD- MA induced PDT with light administered at a total energy dose of 75J and 15 minutes post drug 16

25 administration (1.0 mg/kg body weight) to the lumbar spine is more ideal regarding optimal therapeutic window (in safety and efficacy) when compared to ALA-PpIX (aminolevulinic acid-induced protoporphyrin IX) induced vertebral PDT 10. However, little is known about the effects of PDT on the structural integrity of bone 10, which is critical for safety of the treatment as maintenance of spinal stability is desirable. Hence, this study was designed to determine the effects of BPD-PDT on bone tissue by quantifying the structural and mechanical properties of PDT treated versus untreated vertebral bone. 2.3 Materials and Methods Twenty-five healthy female Wistar rats (three months; Harlan Sprague-Dawley, Indianapolis, IN) were randomly assigned to control (N=5 no drug/light only, N=3 drug only/no light), 1 week (N=9) or 6 weeks (N=8) treatment groups. All procedures were carried out with institutional approval from University Health Network, Toronto Photodynamic therapy Rats were placed under general anaesthesia with 2% isoflurane/oxygen. Animals in the PDT treatment groups received an intravenous (i.v.) injection of 1.0 mg/kg body weight BPD-MA photosensitizer (verteporfin, Visudyne; Novartis, Dorval, Canada) dissolved in 200 µl of 5% dextrose. Control animals received i.v. injection of 200 µl saline solution. After 15 min, the drug was activated by light from a 690 nm diode laser delivered through a 400 µm outer diameter, flat-cut optical fiber inserted percutaneously via an 18 G needle adjacent to the third lumbar vertebra, L3. Light was delivered at 150 mw for 16.7 min for a total energy dose of 150 J. X-ray fluoroscopic guidance was used to place the fiber accurately (Figure 2.1). Following treatment, 2.0 mg/kg meloxicam analgesic was administered. The animals were sacrificed 1 week (control, 1 week group) or 6 weeks (6 weeks group) later by an overdose of pentobarbital (120 mg/kg Euthanol; Bimedia-MTC, Cambridge, Canada). Immediately following euthanasia, the intact spine including L2-L4 was excised and frozen following soft tissue removal. The vertebrae adjacent to L3 were included in the sample, since there is significant scattering of the 690 nm light to these, due to the small size of the vertebrae (~ 5 mm axial thickness). 17

26 Figure 2.1. Administration of photodynamic therapy: PDT treatment showing A. light delivery and B. placement of the optical fiber guided by X-ray fluoroscopy. The white-light appearance surrounding the fiber tip in A is due to saturation of the CCD camera used to take the image µct Image Analysis To examine changes in architectural and structural properties of the vertebrae, X-ray microcomputed tomographs (µct) were taken of intact L2-L4 vertebrae suspended in agar gel at a resolution of 34.7 µm x 34.7 µm x 34.7 µm /voxel (GE Explore Locus; GE, Fairfield, CT), at 80 kvp and 90 µa, with 907 projections per 360 view. Stereological parameters were measured using a volume-shrinking threshold algorithm (Amira 4.1.1; TGS, Berlin, Germany) to define the volume of interest, namely the trabecular bone centrum, excluding the cortical shell and growth plates (Figure 2.2) 11. A triangulated surface of the volume of interest was generated using the SurfaceGen function in Amira (Figure 2.3), and the bone volume and surface area were determined using the TissueStatistics function. Using these parameters, the bone surface area over bone volume ratio (BS/BV), bone volume over total volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular spacing (Tb.Sp) were estimated, based on a parallel plate model

27 Figure 2.2. Volume shrinking threshold technique used to segment vertebral bone: A. the vertebral body seen on a µct slice is distinguished from the dorsal elements by a cylindrical region, B. the vertebral bone is defined applying a threshold intensity, C. the cortical shell is excluded using a shrinking technique and the trabecular bone within the vertebral body is segmented by applying a threshold intensity. Figure 2.3. Triangulated surface of the vertebral body: The surface outlining the trabecular bone (black) and bone marrow (grey) space within the trabecular bone centrum of the vertebral body is shown (500 µm scale). Using the triangulated surface representation of the vertebral body, the bone surface area can be calculated and other trabecular architecture parameters can be determined Mechanical Testing Following CT scanning, L2-L4 vertebral levels were separated and the dorsal elements of each vertebrae were resected. Each vertebral body was individually potted in polymethyl-methacrylate (PMMA) and loaded to failure under axial compression at a strain rate of 0.5 mm/s 13 (MTS Bionix 858; Eden Prairie, MN). Ultimate stress and stiffness were calculated for each vertebral body from the load displacement curves (Figure 2.4, Equations 1, 2). 19

28 Ultimate stress: F ultimate σ ultimate= (Eqn. 1) Aavg,ROI Stiffness: F d ultimate K = (Eqn. 2) ultimate, where F ultimate =ultimate force, A avg, ROI =average cross sectional area within region of interest (including cortical shell) and d ultimate =ultimate displacement Figure 2.4. Force-displacement curve generated during axial compression testing: The ultimate force and ultimate displacement are extracted from the curve and used to calculate ultimate stress and stiffness Data Analysis To test for differences in the individual stereological and mechanical parameters among the control, 1-week and 6-week treatment groups, multivariate analysis of variance followed by Tukey post-hoc multiple comparisons was used (α=0.05). 2.4 Results Photodynamic therapy Two rats were prematurely euthanized due to paralysis which likely occurred as a side-effect from the para-vertebral placement of the optical fiber (mechanical trauma) and the resulting proximity of the light to the spinal cord 7. Skin lesions were observed on two treated rats, associated with the PDT-induced inflammatory response on the surrounding soft tissue. There was no significant difference between no drug/light only and drug only/no light control groups 20

29 (t α=0.05 =2.046, p>0.05, β<0.2), so the two groups were combined into a single no-treatment group. The diameter of effect of the laser focal spot was about 2 cm and encompassed the adjacent L2 and L4 vertebrae, in addition to the targeted L3 vertebra. Since there was no significant difference between levels L2 to L4 for any of the measured parameters (p>0.05 in all cases), the L2-L4 vertebrae in each animal were grouped to increase the statistical power of the study Stereological analysis Architectural parameters were calculated from µct images for L2-L4 in the control and two treatment groups (Table 2.1). At 6 weeks post treatment, differences in bone architecture were clearly seen on the µct images compared to control (Figure 2.5). BS/BV decreased significantly from 22.8±4.5 /mm to 16.9±5.0 /mm (p=0.001), corresponding to a significant increase in BV/TV from 38.5±6.4% to 55.7±11.1% (p<0.001). The increase in bone growth was attributed to an increase in trabecular thickness from 90±20 µm to 130±40 µm (p=0.004). Although not statistically significant, similar trends were observed 1 week following treatment, with BV/TV increasing to 43.2±7.9% (p=0.15). While trabecular number remained constant in both groups (4.48±0.50 /mm(1wk), 4.51±0.76 /mm(6wks)) compared to control (4.28±0.48 /mm) (p=0.4(1wk), p=0.4(6wks)), the trabecular thickening led to a significant decrease in trabecular spacing from 150±10 µm in the control group to 130±40 µm at 1 week (p=0.013) and to 100±30 µm at 6 weeks (p<0.001). Table 2.1 Summary of stereological and mechanical parameters 1 week and 6 weeks following PDT on healthy bone. Output Parameter Control 1 week post-pdt 6 weeks post-pdt N (vertebral levels) Bone Volume Fraction (%) 38.5 ± ± ± 11.1* Trabecular Thickness (µm) 90 ± ± ± 40* Trabecular Number (/mm) 4.28 ± ± ± 0.76 Trabecular Spacing (µm) 150 ± ± 40* 100 ± 30* Bone Surface/Bone Volume (/mm) 22.8 ± ± ± 5.0* Ultimate Stress (MPa) 27.5 ± ± ± 11.3* Ultimate Stiffness (N/mm) 306 ± ± 150* 410 ± 113* * indicates statistical significance of comparisons to the control group at α=

30 Figure 2.5. µct slices of vertebrae: Slices from control (left), 1 week (center), and 6 weeks (right) groups. Trabecular thickening can be seen in the 6 weeks post PDT group compared to control Mechanical testing Following PDT treatment, the vertebrae became stiffer and stronger (Table 2.1, Figure 2.6). Compared to the control group, there was a trend towards increased ultimate stress from 27.5±9.5 MPa to 34.1±9.8 MPa at 1 week (p=0.07), which became statistically significant (p=0.002) at 39.3±11.3 MPa by 6 weeks post PDT. A significant increase in stiffness was observed at both time points: from 306±123 N/mm to 399±150 N/mm (p=0.04) to 410±113 N/mm (p=0.05). Figure 2.6. Representative force-displacement curves: Each curve is generated from one representative sample for the control and 1 and 6 weeks post PDT groups. 22

31 2.5 Discussion A novel finding of our study is that PDT therapy that is directed towards vertebral metastatic tumor ablation can also enhance vertebral spinal mechanical stability as demonstrated in our preclinical stereologic and mechanical loading experiments. There is little literature on the effects of PDT on bone structure including the spine, which is important to consider in potentially extending the clinical indications for PDT in cancer therapy to bone and spinal lesions. Negative effects on the biomechanical properties of bone would be a distinct disadvantage of this approach, while PDT-induced strengthening of bone could further aid in stabilization. This study demonstrates that PDT, applied with the specific photosensitizer dose, light dose, drug-light time interval and light delivery system used, enhanced the mechanical and structural integrity of bone within the 6 weeks post treatment assessment interval. Hence, in contrast to existing treatment alternatives, PDT appears to be unique in offering both targeted ablation of tumor mass and improved structural integrity of surrounding bone. As far as is known at this time, using PDT would also not preclude the use of additional (neo)adjuvant treatment for improved local disease control. Stereologically, PDT caused an increase in bone density, which seems to be due mainly to trabecular thickening rather than to the formation of new trabeculae. This process is typical of physiological remodeling to maintain bone homeostasis, where bone resorption and deposition occur by trabecular thinning or thickening, respectively, to maintain calcium balance and adapt to the mechanical environment 14. The present results suggest that PDT promotes bone deposition and/or slows down bone resorption, while not over-stimulating bone turnover. It is interesting to consider other orthopaedic applications that might exploit this phenomenon, such as using PDT to induce ossification in the growth plate to correct leg length discrepancies 15. Mechanically, the PDT-treated vertebrae were stiffer and stronger than the control group. The stereological and mechanical findings at 6 weeks post PDT were associated with significant increases in stiffness and ultimate stress, corresponding to a significant increase in bone volume fraction and trabecular thickening. There was no significant increase in ultimate strength at 1 week post treatment, similar to the stereological findings. However, there was a statistically significant increase in stiffness at 1 week. This may indicate that additional changes to the bone occurred, which were not captured by the stereological analysis, such as changes to the cortical 23

32 shell or growth plate 15. It is unlikely that significant changes would have taken place in the cortical bone after only 1 week following treatment, because of its low turnover rate compared to trabecular tissue in vertebral bone 16. Since the growth plates in rat vertebrae do not fuse in adulthood and remain a site of regular bone growth, it is possible that a response to PDT is occurring at this site following treatment 17. PDT-induced ossification in growth plates following BPD-MA PDT has been reported to occur 15. Although we did not observe changes in the growth plates by µct imaging, planned studies using histology may reveal osseous changes after PDT. Nevertheless, the growth plates are unlikely to greatly influence the ultimate strength, since they are not the sites of fracture and are small relative to the length of the whole bone. However, changes in the growth plates are more likely to affect apparent stiffening of the whole bone and may possibly explain the significant increase in stiffness at 1 week in the absence of significant growth in the µct images. In terms of the time scale for the observed changes, in general the effects of PDT begin after only a short time interval and may persist for a long period of time following treatment. As shown in the stereological analysis, there was a trend in bone deposition at 1 week following treatment that became significant at 6 weeks. Indications of bone growth and strengthening also appeared in the mechanical testing at 1 week, with even more significant growth found at 6 weeks. The bone formation found in the stereological analysis, coupled with the increase in mechanical strength, reveal that the newly-formed bone was strong and healthy rather than sclerotic or brittle and so offered structural support and strength. The immediate and sustained effect of PDT on bone remodeling revealed here may potentially be explained by the upregulation of vascular endothelial growth factor (VEGF) following PDT 18. Several studies have found that PDT with light applied while the photosensitizer is still in the circulation damages vascular endothelial cells, which triggers an inflammatory response similar to that found after tissue injury 19,20. It is likely that VEGF has a regulatory role in bone remodeling, as it has been shown to promote ossification and new bone maturation in fractured bone 21. VEGF may directly enhance bone quality by promoting chemotactic migration and differentiation of osteoblasts to lay down new bone 22. It may also indirectly improve bone quality by promoting angiogenesis, leading to greater delivery of nutrients to bone cells to enhance bone remodeling 23. The abundance of VEGF following PDT and its role in accelerating bone turnover is consistent with the bone growth observed following PDT in this study. Future 24

33 work examining the histology of bone and bone marrow cells while tracking VEGF may elucidate the specific mechanism(s) through which PDT strengthens vertebral bone. The fact that PDT makes vertebrae stiffer and increases their strength should result in greater weight-bearing capacity, which would be particularly beneficial for spinal metastasis patients, whose spines have weakened from metastatic lesions. An important caveat is, however, that to date we have only examined these effects in normal bone that is not tumor-involved. Further studies in vertebrae that have metastatic lesions will be required to determine if the beneficial effects seen in normal bone translate to tumor-associated bone. Longer term studies would be of value to determine if this enhanced bony response to PDT is sustained. In addition, several other parameters should be examined, including: the cellular and molecular effects of PDT; the effects over a longer time period; the use of repeated, fractionated or metronomic (low dose rate) 24 PDT treatment regimens; and the effect of different photosensitizers targeting either vasculature and/or tumor cells on tumor ablation and bone stability. Ongoing evaluation in both metastatic vertebrae and tumor cell-targeted PDT (using molecular beacons, i.e. photosensitizers that can be switched on by tumor-specific enzymes or mrna) will enhance our understanding of observed effects 25. If PDT is able to both destroy tumor cells (directly and/or indirectly) and alter the balance between bone destruction and bone deposition favouring bone strengthening, it will provide a very attractive minimally-invasive treatment option for patients with spinal metastases. 2.6 References 1. Wong DA, Fornasier VL, MacNab I. Spinal metastases: the obvious, the occult, and the impostors. Spine 1990;15: Constans JP, de Divitiis E, Donzelli R, et al. Spinal metastases with neurological manifestations. Review of 600 cases. J Neurosurg 1983;59: Lipton A, Theriault RL, Hortobagyi GN, et al. Pamidronate prevents skeletal complications and is effective palliative treatment in women with breast carcinoma and osteolytic bone metastases: long term follow-up of two randomized, placebo-controlled trials. Cancer 2000;88: Bartels RH, van der Linden YM, van der Graaf WT. Spinal extradural metastasis: review of current treatment options. CA Cancer J Clin 2008;58: Tokuhashi Y, Ogawa T. [Spinal metastases]. Clin Calcium 2007;17: Juarranz A, Jaen P, Sanz-Rodriguez F, et al. Photodynamic therapy of cancer. Basic principles and applications. Clin Transl Oncol 2008;10:

34 7. Burch S, Bisland SK, Bogaards A, et al. Photodynamic therapy for the treatment of vertebral metastases in a rat model of human breast carcinoma. J Orthop Res 2005;23: Burch S, Bogaards A, Siewerdsen J, et al. Photodynamic therapy for the treatment of metastatic lesions in bone: studies in rat and porcine models. J Biomed Opt 2005;10: Akens MK, Hardisty, MR, Wilson, BC, et al. Evaluation of the Therapeutic Window of Photodynamic Therapy Treatment (PDT) of Breast Cancer Metastases in the Spine. Orthopaedic Research Society Meeting, Akens MK, Yee AJ, Wilson BC, et al. Photodynamic therapy of vertebral metastases: evaluating tumor-to-neural tissue uptake of BPD-MA and ALA-PpIX in a murine model of metastatic human breast carcinoma. Photochem Photobiol 2007;83: Hardisty M, Skrinskis T, Gordon L, et al. A repeatable bone quality measurement technique using 3D stereology. Canadian Orthopaedic Association Meeting, Feldkamp LA, Goldstein SA, Parfitt AM, et al. The direct examination of threedimensional bone architecture in vitro by computed tomography. J Bone Miner Res 1989;4: Pelker RR, Friedlaender GE, Markham TC, et al. Effects of Freezing and Freeze-Drying on the Biomechanical Properties of Rat Bone. J Orthop Res 1984;1: Rodan GA. Bone mass homeostasis and bisphosphonate action. Bone 1997;20: Bisland SK, Johnson C, Diab M, et al. A new technique for physiodesis using photodynamic therapy. Clin Orthop Relat Res 2007;461: Li XQ, Klein L. Decreasing rates of bone resorption in growing rats in vivo: comparison of different types of bones. Bone 1990;11: Roach HI, Mehta G, Oreffo RO, et al. Temporal analysis of rat growth plates: cessation of growth with age despite presence of a physis. J Histochem Cytochem 2003;51: Gomer CJ, Ferrario A, Luna M, et al. Photodynamic therapy: combined modality approaches targeting the tumor microenvironment. Lasers Surg Med 2006;38: MacDonald IJ, Dougherty TJ. Basic principles of photodynamic therapy J Porphyr Phthalocya 2001;5: Osaki T, Takagi S, Hoshino Y, et al. Antitumor effects and blood flow dynamics after photodynamic therapy using benzoporphyrin derivative monoacid ring A in KLN205 and LM8 mouse tumor models. Cancer Lett 2007;248: Street J, Bao M, deguzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci USA 2002;99: Jinlu D, Kitagawa Y, Zhang J, et al. Vascular endothelial growth factor contributes to the prostate cancer-induced osteoblast differentiation mediated by bone morphogenetic protein. Cancer Res 2004;64: Probst A, Spiegel HU. Cellular mechanisms of bone repair. J Invest Surg 1997;10:

35 24. Bisland SK, Lilge L, Lin A, et al. Metronomic photodynamic therapy as a new paradigm for photodynamic therapy: rationale and preclinical evaluation of technical feasibility for treating malignant brain tumors. Photochem Photobiol 2004;80: Zheng G, Chen J, Stefflova K, et al. Photodynamic molecular beacon as an activatable photosensitizer based on protease-controlled singlet oxygen quenching and activation. Proc Natl Acad Sci U S A 2004;104:

36 Chapter 3: Short Term Effects of Photodynamic Therapy and Bisphosphonates in Healthy and Metastatic Vertebrae This chapter is a modified version of a paper that has been compiled for submission to Breast Cancer Research and Treatment. The manuscript that will be submitted for review is entitled "Beyond bisphosphonates: Photodynamic Therapy Structurally Augments Metastatically Involved Vertebrae and Destroys Tumor Tissue". 3.1 Abstract Breast cancer patients commonly develop metastases in the spine, which compromises its mechanical stability and can lead to skeletal related events. The current clinical standard of treatment includes the administration of systemic bisphosphonates (BP) to reduce metastatically induced bone destruction. However, response to BPs can vary both within and between patients, which motivates the need for additional treatment options for spinal metastasis. Photodynamic therapy (PDT) has been shown to be effective at treating metastatic lesions secondary to breast cancer in an athymic rat model, and is proposed as a treatment for spinal metastasis. The objective of this study was to determine the effect of PDT, alone or in combination with previously administered systemic BPs, on the structural and mechanical integrity of both healthy and metastatically involved vertebrae. Human breast carcinoma cells (MT-1) were inoculated into athymic rats (day 0). At 14 days, a single PDT treatment was administered, with and without previous BP treatment at day 7. In addition to causing tumor necrosis in metastatically involved vertebrae, PDT significantly reduced bone loss, resulting in strengthening of the vertebrae compared to untreated controls. Combined treatment with BP+PDT further enhanced bone architecture and strength in both metastatically involved and healthy bone. Overall, the ability of PDT to both ablate malignant tissue and improve the structural integrity of vertebral bone motivates its consideration as a local minimally invasive treatment for spinal metastasis secondary to breast cancer. 3.2 Introduction Up to 1/3 of all cancer patients develop bone metastases, and the vertebral column is the most common site of metastatic development in the skeleton 1. Vertebral metastases commonly occur in patients with primary breast cancer and compromise the mechanical stability of the spine. In 28

37 2/3 of patients, spinal metastases lead to skeletal related events such as bone pain, hypercalcaemia, pathological fracture and spinal cord compression 2,3. Eighty percent of these lesions are found in the vertebral bodies with the remaining metastases established in the posterior elements 4. The vertebral column bears large mechanical loads, as such, clinical treatments are aimed at achieving mechanical stability in addition to reducing tumor burden. Current treatment for spinal metastasis involves a multi-modal approach that includes systemic chemotherapy and bisphosphonates as adjuvant therapies to local treatments such as radiation therapy 4. Tumor response varies considerably across patients, and there are side effects associated with each treatment. There remains a need for alternative treatments for those patients who continue to respond poorly to existing therapies, prior to open or minimally invasive (vertebroplasty/kyphoplasty) surgical intervention 4. Systemic bisphosphonate (BP) treatment is considered a clinical standard of care for inhibiting osteoclast-mediated bone resorption and further metastatic progression in breast cancer patients with skeletal disease. However, clinical studies have demonstrated that while BPs significantly reduce the odds of vertebral fracture in all spinal metastasis patients, BPs are unable to reduce the odds of fracture in spinal metastases arising specifically from breast cancer 5. In vivo studies in preclinical animal models have also shown that BPs do not inhibit metastatic progression once vertebral tumors have reached a sufficient size 6. This suggests that BPs may be less effective at treating patients with more advanced cancers, further motivating the need for the development of alternative therapies to impede metastatic growth and subsequent fracture. Photodynamic therapy (PDT) has been used to treat a variety of cancers where the tumor is accessible by an external light source. PDT involves topical or systemic administration of a photosensitizer that accumulates preferentially in tumor tissue through differential uptake/clearance relative to normal tissue, and becomes activated by light at a wavelength specific to the drug. Activation of the photosensitizer in the presence of molecular oxygen leads to generation of highly reactive singlet oxygen, which in turn causes tumor cell toxicity and tissue necrosis. Since both photosensitizer and light are simultaneously required for a photodynamic effect, therapeutic effects can be achieved locally without causing substantial damage to the surrounding tissues. 29

38 Using a transpedicular approach adapted from vertebroplasty to deliver light to the vertebral body, PDT can be utilized to treat spinal metastatic lesions. Previous work has shown that a single PDT treatment using 1.0mg/kg of benzoporphyrin derivative monoacid ring A (BPD-MA) photosensitizer, a 15 minute drug-light interval, and a total light dose of 75J at a power output of 100mW is the optimized setting for safe and effective ablation of vertebral tumors in a preclinical model of breast cancer spinal metastasis 7. It has also been shown that PDT administered to healthy 3 month old rats led to increased vertebral bone formation and mechanical strength 8. This suggests that PDT may be able to ablate malignant tumor tissue and simultaneously improve the mechanical integrity of metastatically involved vertebrae. However, it is unknown whether these bone enhancing effects would similarly occur in metastatically involved bone and in bone previously treated with BPs. Hence this study was designed to examine the effects of PDT on the structural and mechanical properties of both healthy and tumor involved bone, alone and in combination with systemic BPs. 3.3 Methodology Study Design The effects of PDT alone and in combination with BP treatment were evaluated in 31 rnu/rnu rats with metastatically involved vertebrae, randomly assigned to 4 distinct treatment groups: (i) untreated control (N=7), (ii) PDT only (N=9), (iii) BP only (N=6), and (iv) PDT following BP treatment (N=9). Treatments were also administered to 40 healthy control rnu/rnu rats to examine the independent and combined effects of PDT on bone tissue: untreated control (N=9), PDT only (N=11), BP only (N=11), PDT following BP treatment (N=9). Institutional animal care committee approval was obtained for all procedures (University Health Network, Toronto, Canada) Animal Model A 5-6 week old athymic rat model was used in all groups (rnu/rnu, Harlan Sprague Dawley, Indianapolis, IN). Vertebral metastasis was induced in the tumor group at day 0 by intracardiac injection of luciferase transfected MT-1 human breast cancer carcinoma cells into the left ventricle. Under general anaesthesia (2% isoflurane/oxygen), animals were inoculated with cells in 200µl of RPMI 1640 media, which metastasize to the spine. These cells are stably transfected with luciferase to make the cells confer bioluminescence and allow in vivo 30

39 detection of their spatial distribution. Following injection of the tumor cells, animals were returned to their cages and given free access to food and water Bisphosphonate Therapy BP treatment was administered on day 7. Animals were subcutaneously injected with 60 µg/kg of zoledronic acid (Zometa; Novartis, Dorval, Canada) dissolved in 0.9% sodium chloride solution at 80 µg/ml. This represents an equivalent dose to the clinical treatment given to humans undergoing systemic BP therapy for skeletal metastasis Photodynamic Therapy PDT was administered on day 14. Rats were placed under general anaesthesia throughout the duration of the PDT treatment. Verification of spinal metastasis was first conducted in animals injected with the bioluminescent tumor cells (Figure 3.1a). Animals received an intraperitoneal (i.p.) injection of 80 mg/kg luciferin substrate (Caliper LifeScience; Hopkinton, MA) dissolved in 0.9% sodium chloride solution at a concentration of 40 mg/ml, followed after a 5 minute interval by image acquisition in the left lateral and ventral positions using an IVIS Bioluminescent imaging system (Caliper LifeScience) All animals were then administered an intravenous injection (i.v.) of 1.0 mg/kg BPD-MA photosensitizer (verteporfin, Visudyne; Novartis, Dorval, Canada) dissolved in 200 µl of 5% dextrose. Under fluoroscopic guidance, a 400 µm outer diameter, flat-cut optical fiber was inserted percutaneously (through an 18G needle) adjacent to the target second lumbar vertebra, L2 (found previously to be the most common level of metastatic involvement in this rat model). Following a 15 minute drug-light interval, when the photosensitizer remains largely in the vasculature, 75J of light energy was delivered from a 690nm diode laser inserted through the optical fiber, at a power output of 100mW for a total treatment time of 12.5 minutes. One week following treatment (day 21, or earlier pending signs of neurological deficit), final bioluminescent images were acquired in animals injected with the tumor cells, immediately prior to euthanasia, to identify final tumor burden following treatments (Figure 3.1b). All animals were euthanized with an overdose of pentobarbital (120 mg/kg Euthanol; Bimedia-MTC, Cambridge, Canada). Immediately following sacrifice, the spines were excised and half the 31

40 samples were frozen for subsequent mechanical testing while the other half were fixed in 4% paraformaldehyde for histological evaluation. Figure 3.1. Monitoring tumor growth with bioluminescence imaging: tumor burden was visualized (a) before and (b) after treatment. The metastatic lesion identified prior to PDT treatment was reduced by endpoint imaging, which was in contrast to surrounding rapidly growing untreated metastases Architectural Analysis All spines were suspended in agar gel and imaged with a specimen micro-computed tomography (µct) scanner (GE Explore Locus; GE, Fairfield, CT) to determine structural and architectural properties of the vertebrae. Images were acquired at an isotropic 13.7 µm/voxel resolution, at 80 kvp and 90 µa. Calibration phantoms with hydroxyapatite densities of 250 mg/cc and 750 mg/cc were used to standardize all images. Trabecular bone within the L2 vertebral body was segmented using a semi-automated thresholding technique developed and validated in our group (Amira 4.1.1; TGS, Berlin, Germany) 9. From the vertebral segmentation, bone surface (BS), bone volume (BV), and bone mineral density (BMD) were determined. Stereological parameters were quantified using the geometric and material measurements extracted from the µct images: trabecular bone volume fraction (BV/TV), trabecular architecture (thickness, spacing, number) 10, trabecular bone surface to bone volume fraction (BS/BV), trabecular BMD, trabecular volumetric bone mineral density (vbmd), cortical BMD, and cortical shell mass fraction. 32

41 3.3.6 Histological Confirmation of Tumor Destruction Samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) and stained with haematoxcyclin and eosin (H&E) to evaluate cell and tissue morphology. Samples bearing tumors were stained by immunohistochemistry using a mouse-anti-human epidermal growth factor receptor (hegfr) antibody (Zymed Laboratories Inc., San Francisco, CA) to permit simple visualization of human derived tumor cells in the vertebrae Mechanical Testing For mechanical testing, the L2 vertebral body from each sample was potted in polymethylmethacrylate (PMMA) and loaded to failure in axial compression at a strain rate of 1mm/min (MTS Bionix 858; Eden Prairie, MN). Ultimate force, stress, and stiffness of each vertebral body were determined from the force-displacement curves generated from each test Statistical Analysis Multivariate analysis of variance followed by Tukey post-hoc multiple comparisons (α=0.05) was carried out separately for the healthy and tumor bearing rats to test for differences in stereological and mechanical properties between the treatment groups. Relationships between stereological and mechanical properties were determined using Pearson s product moment at a significance level of α=0.05. Normality of the measures was verified prior to correlation analyses. All data are represented as mean ± standard deviation. 3.4 Results In the tumor bearing vertebrae, the application of PDT alone significantly increased bone volume fraction by 46% (p=0.01; Table 3.1). PDT was found to significantly decrease trabecular spacing by 43% (p<0.001), increase trabecular number by 38% (p<0.001), and increase trabecular vbmd by 15% (p=0.006) compared to untreated controls. The ratio of the cortical shell to the trabecular centrum was significantly reduced in the PDT group compared to that of the control group (29% decrease in cortical mass fraction, p=0.02), while the cortical BMD was greater by 12% (p=0.03). Histological examination demonstrated destruction of the tumor, with necrotic MT-1 cells in the PDT treated vertebrae (Figure 3.2b, f). 33

42 As expected, BP treatment alone also significantly increased bone mass compared to untreated controls in the tumor bearing vertebrae. Bone volume fraction increased by 65% (p<0.001), leading to a corresponding 19% decrease in bone surface area to bone volume ratio (p=0.03). The bone growth was attributed to trabecular thickening by 23% (p=0.04), which consequently resulted in a 47% decrease in the spacing between trabecular struts (p<0.001). As a result of increased BMD and bone volume fraction, trabecular vbmd increased significantly by 75% (p<0.001). BP treatment demonstrated a significantly higher number of trabeculae (an increase of 17%, p<0.001) and a significantly lower cortical shell mass fraction (a decrease of 12%, p=0.002). There was a significant BMD increase in the cortical shell (21%, p<0.001). Histological stains revealed abundant viable tumor cells remaining in the vertebrae following BP treatment alone (Figure 3.2c, g). Together, the combined BP+PDT treatment resulted in the largest increases in bone volume fraction (76%, p<0.001), trabecular thickness (26%, p<0.001), trabecular number (43%, p<0.001), trabecular vbmd (85%, p<0.001), and decrease in trabecular spacing (52%, p<0.001) when compared to untreated controls. While the combined treatment did not yield statistically significant improvements in bone architecture over the BP or PDT treatment groups alone, the data clearly demonstrate that previous BP treatment does not inhibit the positive effect of PDT on either bone stereology or tumor ablation (Table 3.1, Figure 3.2d, h). Mechanically, metastatically involved vertebrae generally became stronger following treatment when compared to untreated controls (Table 3.2). A significant increase in ultimate force was found in the BP treated group (81% increase, p=0.04), with similar trends in the PDT (69% increase, p=0.05) and combination BP+PDT (60% increase, p=0.1) treated groups compared to control. A similar result was found when comparing ultimate stress: 109% increase in BP (p=0.02), 79% increase in PDT (p=0.06), and 79% increase in BP+PDT (p=0.06). A significant increase in stiffness was also found in the BP treated group compared to control (232% increase, p=0.005). No significant differences were detected between any of the 3 treatment groups. 34

43 Table 3.1. Summary of stereological parameters in healthy and tumor involved bone following PDT and/or BP treatment. Output Parameter Tumor Healthy Control PDT BP BP+PDT Control PDT BP BP+PDT N Bone Volume Fraction (%) Trabecular Thickness (µm) Trabecular Number (/mm) 25.2 ± ± 5.7* 41.6 ± 5.5* 44.4 ± 4.4* 44.0 ± ± ± ± 5.5* 61.4 ± ± ± 12.2* 77.5 ±7.6* 78.8 ± ± ± ± 19.7* 4.0 ± ± 0.5* 5.5 ± 0.2* 5.7 ± 0.2* 5.6 ± ± ± ± Trabecular Spacing (µm) 201 ± ± 20* 106 ± 8* 97 ± 9* 100 ± 7 91 ± ± 9 88 ± 10 Bone Surface/Bone Volume (/mm) Trabecular BMD (mg/cc) Trabecular volumetric BMD (mg/cc) Cortical BMD (mg/cc) Cortical shell mass fraction (%) 33.3 ± ± ± 4.1* 26.0 ± 2.5* 25.5 ± ± ± ± 3.8* 711 ± ± ± ± ± ± ± ± ± ± 46* 313 ± 47* 332 ± 42* 316 ± ± ± ± 34* 593 ± ± 25* 719 ± 48* 697 ± 54* 703 ± ± ± ± ± ± 0.2* 0.8 ± 0.1* 0.8 ± 0.1* 1.0 ± ± ± ± 0.3 * indicates statistical significance of comparisons to the corresponding control group at α=0.05.

44 36 Figure 3.2. Histology analysis of tumor burden: Histologic sections with hegfr staining of tumor cells and corresponding H&E staining demonstrate viable tumor in (a, e) untreated controls and (c, g) following BP treatment alone. PDT treatment (b, f) alone and (d, h) combined BP+PDT treatment induced tumor cell necrosis.

45 Table 3.2. Summary of mechanical parameters in healthy and tumor involved bone following PDT and/or BP treatment. Output Parameter Tumor Healthy Control PDT BP BP+PDT Control PDT BP BP+PDT N Ultimate Force (N) Ultimate Stress (MPa) 87 ± ± ± 32 * 139 ± ± ± ± ± ± ± ± 4.4 * 26.5 ± ± ± ± ± Stiffness (N/mm) 108 ± ± ± 59 * 197 ± ± ± ± ± 57 * indicates statistical significance of comparisons to the corresponding control group at α=0.05. indicates trend towards statistical significance of comparisons to the corresponding control group at α=0.1. Table 3.3. Correlation coefficients between stereological and mechanical parameters (p<0.01 for all). Ultimate Force Ultimate Stress Stiffness Bone volume fraction Trabecular vbmd Cortical BMD

46 In the non-tumor bearing healthy groups, combination BP+PDT was the only treatment that induced significant bone formation (Table 3.1). Compared to controls, BP+PDT significantly increased bone volume fraction by 19% (p=0.03) and increased trabecular thickness by 24% (p=0.009), subsequently reducing the separation between trabeculae by 12% (p=0.1). In healthy bone, there were no significant changes in bone stereology with PDT treatment alone, with the exception of a trend indicating that PDT increased trabecular number by 7% (p=0.07). The effect of BP alone on bone formation was also less apparent in the healthy group as there were no statistically significant differences in bone architectural parameters. There was however a strong trend showing an 8% increase in trabecular BMD compared to untreated controls (p=0.07). In the healthy group, no significant differences were found in the vertebral mechanical properties following any of the treatments. In pooling all data from this study (tumor, healthy, controls and treated groups), statistically significant relationships were found between the stereological and mechanical properties of the vertebrae (Table 3.3). Ultimate force was significantly correlated with bone volume fraction, volumetric bone mineral density, and cortical bone mineral density. Similarly, these stereologic variables also had significant correlations with ultimate stress and stiffness (p<0.01 for all). Finally, our data suggest that combination BP+PDT treatment is able to restore bone volume fraction in metastatically involved vertebrae to healthy (non-tumor bearing control) levels (Figure 3.3). The 95% confidence interval of the mean bone volume fraction of BP+PDT treated tumor animals directly overlapped with that of untreated healthy animals (BP+PDT tumor mean BV/TV: 44.4%, 95% CI: 41.0% 47.8% versus control healthy mean BV/TV: 44.0%, 95% CI: 41.4% %). Further studies with larger sample sizes or fewer treatment groups, however, would be required in order to increase the power to make a statistical conclusion regarding this observation. 38

47 Figure 3.3. Comparison of bone mass by µct: Sagittal µct images showing (a) healthy untreated, (b) tumor untreated, and (c) tumor BP+PDT treated bone. Comparing the images, BP+PDT treated bone is comparable to normal healthy controls. 3.5 Discussion Mechanical stability is critical in the spinal column, and thus it is important to consider the impact of new and existing therapies aimed at metastatic disease on the structural and mechanical integrity of vertebral bone. Previous studies have proven the efficacy of photodynamic therapy in treating spinal metastatic tumors 7,11,12, and the findings of this study further support its application in this pathology. Histological examination revealed that PDT was able to destroy tumor tissue, and stereologic analysis and mechanical testing showed that PDT simultaneously improved the structural integrity of the surrounding bony tissue. This study has also shown that PDT is effective at treating spinal metastatic tumors and impeding bone destruction following previous exposure to bisphosphonate therapy with zoledronic acid, a standard clinical treatment for spinal metastasis. While BP and PDT are both able to significantly impede metastatic bone loss, only PDT offers the additional benefit of eradicating tumor tissue, which will further impede tumor invasion into bone. Although not significant, there was a general trend that bone stereological parameters (bone volume fraction and trabecular thickness) in the BP alone treated vertebrae were strengthened in comparison to PDT alone treated vertebrae. This may be explained by the fact that while BP was administered at day 7, PDT was not administered until day 14, allowing tumor growth and metastatic bone destruction (erosion of trabecular surfaces) to take effect for an extra week before any intervention in the PDT treated animals. Thus the PDT treatment alone cohort 39

48 was likely at a structural disadvantage on day 14, requiring more recovery in a shorter time span (1 week) as compared to the BP alone and combined BP+PDT groups. The increase in bone volume fraction following PDT in healthy bone was not statistically significant at 1 week following treatment, which is consistent with the previous study of PDT in Wistar rats that showed stereological parameters of vertebral bone did not significantly improve until 6 weeks post treatment 8. Although stiffness was found to increase significantly 1 week following PDT in the previous study, the limited sample size for mechanical testing in this study resulted in a lack of statistical power to detect this treatment effect. Significant findings at 1 week in the combined BP+PDT treatment healthy group are exciting, suggesting potential combinatory effects of these two therapies on bone formation. This may have future potential outside the area of metastatic disease, in the treatment of localized areas requiring bone augmentation, such as fracture healing. Observation of dendritic reticulation and proliferation of fibroblasts in H&E stained vertebrae, as well as presence of a lower density callus in the µct images, suggest that PDT induced an inflammatory reaction (Figure 3.4). As such, the mechanism for PDT induced bone growth may be due to an inflammatory response following tissue injury at the local treatment site. Activation of the photosensitizer while it remains largely in the vasculature leads to generation of reactive oxygen species that induces endothelial cell death, vasoconstriction, and complete vessel collapse 13. The depletion of oxygen upon photosensitizer activation in combination with the mass of damaged tumor tissue elicits a strong inflammatory response in an attempt to contain/remove necrotic tissue and promote local healing to restore normal tissue homeostasis Hypoxia in the local region of damage induces hypoxia-inducible factor-1α subunit (HIF-1α), which in turn promotes vascular endothelial growth factor (VEGF) expression 16,17. Both HIF-1α and VEGF play important roles in angiogenesis, an essential process for recruiting osteogenic cells and cytokines during bone remodeling and repair

49 1mm a b c 100 µm Figure 3.4. Inflammatory response post PDT: Inflammation induced bone remodeling resulting in callus formation following PDT is shown as seen on a (a) µct image and (b) distinct bone morphology in the corresponding histological section. Closer examination of histological section (c) reveals migration of fibroblasts that play a role in bone remodeling following tissue damage. VEGF is known to be upregulated following vascular-pdt, and subsequently promote angiogenesis to enhance bone remodeling, ossification, and repair 16,17. Several studies have shown VEGF stimulates bone repair and remodeling through enhancing both osteoclast and osteoblast differentiation and activity 16,18,19. More specifically, VEGF induces chemotactic migration and differentiation of osteoclasts to promote bone resorption in preparation for bone formation, and activates osteoblasts to increase nodule formation and alkaline phosphatase activity to induce ossification and callus mineralization 18. The upregulation of bone remodeling and net bone formation during inflammation was evident in the µct images where bone calluses were formed within the vertebral body and on the periosteum surface. As bone remodeling continues, it is expected that the soft woven bone will be replaced by mature mineralized laminar bone. In contrast to BP treated vertebrae, PDT seemed to increase bone volume fraction by inducing the formation of new trabeculae rather than solely through trabecular thickening. The increase in trabecular number suggests that PDT induces bone formation through a mechanism distinct from upregulating bone remodeling on existing trabecular bone surfaces. Since both osteoblasts and osteoclasts are functional in PDT treated vertebrae, trabecular thickening by osteoblasts would be balanced with trabecular thinning by osteoclasts to a greater degree during bone remodeling in comparison to BP treated vertebrae. Thus it is possible that the bone formation following PDT 41