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

ii

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: Emily Won

Department: Institute of Biomaterials and Biomedical Engineering

University: 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.

iii

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.

iv

Table of Contents

Acknowledgments ........................................................................................................................ iii

Table of Contents ......................................................................................................................... iv

List of Tables ............................................................................................................................... vii

List of Figures............................................................................................................................. viii

Chapter 1: Introduction ............................................................................................................... 1

1.1 Spinal Metastasis ...................................................................................................................... 1

1.2 Bone and Bone Remodeling ..................................................................................................... 2

1.3 Treatment of Spinal Metastasis................................................................................................. 3

1.3.1 Radiation Therapy....................................................................................................... 3

1.3.2 Chemotherapy /Hormone therapy ............................................................................... 3

1.3.3 Surgical Therapy ......................................................................................................... 4

1.3.4 Vertebral Augmentation .............................................................................................. 4

1.3.5 Systemic Bisphosphonates........................................................................................... 5

1.4 Photodynamic Therapy............................................................................................................. 7

1.4.1 Photosensitizers........................................................................................................... 8

1.4.2 Photodynamic Therapy for Spinal Metastasis............................................................. 9

1.5 Thesis Objectives.................................................................................................................... 11

1.6 Thesis Outline ......................................................................................................................... 12

1.7 References............................................................................................................................... 13

v

Chapter 2: Short and Intermediate Term Effects of Photodynamic Therapy in Healthy

Vertebrae........................................................................................................................... 15

2.1 Abstract ................................................................................................................................... 15

2.2 Introduction............................................................................................................................. 16

2.3 Materials and Methods............................................................................................................ 17

2.3.1 Photodynamic therapy............................................................................................... 17

2.3.2 µCT Image Analysis................................................................................................... 18

2.3.3 Mechanical Testing ................................................................................................... 19

2.3.4 Data Analysis ............................................................................................................ 20

2.4 Results..................................................................................................................................... 20

2.4.1 Photodynamic therapy............................................................................................... 20

2.4.2 Stereological analysis................................................................................................ 21

2.4.3 Mechanical testing .................................................................................................... 22

2.5 Discussion............................................................................................................................... 23

2.6 References............................................................................................................................... 25

vi

Chapter 3: Short Term Effects of Photodynamic Therapy and Bisphosphonates in Healthy

and Metastatic Vertebrae ................................................................................................ 28

3.1 Abstract ................................................................................................................................... 28

3.2 Introduction............................................................................................................................. 28

3.3 Methodology........................................................................................................................... 30

3.3.1 Study Design.............................................................................................................. 30

3.3.2 Animal Model ............................................................................................................ 30

3.3.3 Bisphosphonate Therapy ........................................................................................... 31

3.3.4 Photodynamic Therapy.............................................................................................. 31

3.3.5 Architectural Analysis ............................................................................................... 32

3.3.6 Histological Confirmation of Tumor Destruction ..................................................... 33

3.3.7 Mechanical Testing ................................................................................................... 33

3.3.8 Statistical Analysis .................................................................................................... 33

3.4 Results..................................................................................................................................... 33

3.5 Discussion............................................................................................................................... 39

3.6 References............................................................................................................................... 45

Chapter 4: Summary .................................................................................................................. 47

4.1 Effects of Photodynamic Therapy on Bone............................................................................ 47

4.2 Future Directions .................................................................................................................... 50

4.3 Conclusion .............................................................................................................................. 51

vii

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 ............................................................................................................ 21

Table 3.1. Summary of stereological parameters in healthy and tumor involved bone following

PDT and/or BP treatment...................................................................................................... 35

Table 3.2. Summary of mechanical parameters in healthy and tumor involved bone following

PDT and/or BP treatment...................................................................................................... 37

Table 3.3. Correlation coefficients between stereological and mechanical parameters ............... 37

viii

List of Figures

Figure 1.1. Mechanism of nitrogen-containing bisphosphonates ................................................... 6

Figure 1.2. Application of photodynamic therapy to the spine..................................................... 10

Figure 2.1. Administration of photodynamic therapy................................................................... 18

Figure 2.2. Volume shrinking threshold technique used to segment vertebral bone.................... 19

Figure 2.3. Triangulated surface of the vertebral body................................................................. 19

Figure 2.4. Force-displacement curve generated during axial compression testing ..................... 20

Figure 2.5. µCT slices of vertebrae............................................................................................... 22

Figure 2.6. Representative force-displacement curves ................................................................. 22

Figure 3.1. Monitoring tumor growth with bioluminescence imaging......................................... 32

Figure 3.2. Histology analysis of tumor burden ........................................................................... 36

Figure 3.3. Comparison of bone mass by µCT ............................................................................. 39

Figure 3.4. Inflammatory response post PDT............................................................................... 41

1

Chapter 1: Introduction

1.1 Spinal Metastasis

Bone metastasis occurs in up to 1/3 of all cancer patients, with the vertebral column being the

most common site of skeletal metastatic development1,2. 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 viability3. 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 marrow3.

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

mortality3,5,6.

Table 1.1. Rate of spinal metastases from common primary cancers in the United States4.

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 5,692 478 (8.4)

Skin 10,844 369 (3.4)

2

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 body7. 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 patients8,9,10. 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 structure11. 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.

3

Bone resorption typically takes 3-4 weeks while bone deposition and mineralization is much

slower and takes approximately 3 months12. 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.

1.3.1 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 patients10. 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 procedures13,14. Moreover, there is also a high recurrence rate in spinal metastases,

which is difficult to treat with radiotherapy because surviving tumors become more radio-

resistant with repeated exposures while the spinal cord and surrounding tissues become more

susceptible to radio-toxicity.

1.3.2 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 cord15. 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

4

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 sites16. 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 fracture17.

1.3.3 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 cord18. 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 deficit18. 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.

1.3.4 Vertebral Augmentation

Vertebroplasty and kyphoplasty both involve injection of bone cement (polymethyl-

methacrylate, 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

5

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.

1.3.5 Systemic Bisphosphonates

Systemic bisphosphonate (BP) therapy is becoming a clinical standard for treating spinal

metastasis secondary to breast cancer19. Bisphosphonates alleviate symptoms of bone metastasis

primarily through inhibiting osteoclast function. Bisphosphonates are analogues of endogenous

proton pump inhibitors (PPi), with a P-C-P backbone and two covalently bonded side chains, R1

and R220. The backbone and the R1 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 R2 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.

6

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.

mevalonate

Mevalonate Pathway

isopentenyl pyrophosphate (IPP)

geranyl disphosphate (GPP)

farnesyl diphosphate (FPP)

geranylgeranyl diphosphate (GGPP)

geranylgeranylated proteins required for osteoclast function

e.g. Rho, Rac

FPP synthase

N-containing bisphosphonates

7

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 metastases19. Since zoledronic acid is administered intravenously and does not

undergo biotransformation prior to absorption into bone, only 4mg is required to achieve a

therapeutic effect21. 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

compliance21. 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 bone23. 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 metastasis24. 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 neoplasms25. PDT involves

8

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.

1.4.1 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 state25. 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 diameter25. 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 tissues26. 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) efficient generator of reactive singlet oxygen

iii) high specificity for targeted malignant tissue over surrounding normal healthy tissue

iv) 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

cancer26. The absorption in the red spectrum (required for deep tissue penetration) is low, thus

relatively high light doses of 100-200J/cm2 is required to induce a therapeutic effect. Clearance

9

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 keratosis26. 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.

1.4.2 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.

10

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 cord27. 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

11

tumor tissue should be eliminated to achieve long term tumor ablation, suggesting that vascular-

targeted 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.

12

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

13

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;15:1-4.

2. Jacobs WB, Perrin RG. Evaluation and treatment of spinal metastases: an overview. Neurosurg Focus 2001;11:e10.

3. Roodman GD. Mechanisms of bone metastasis. N Engl J Med 2004;350:1655-64.

4. 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;64:1611-8.

5. Houston SJ, Rubens RD. The systemic treatment of bone metastases. Clin Orthop Relat Res 1995:95-104.

6. Toma S, Venturino A, Sogno G, et al. Metastatic bone tumors. Nonsurgical treatment. Outcome and survival. Clin Orthop Relat Res 1993:246-51.

7. Ecker RD, Endo T, Wetjen NM, et al. Diagnosis and treatment of vertebral column metastases. Mayo Clin Proc 2005;80:1177-86.

8. Constans JP, de Divitiis E, Donzelli R, et al. Spinal metastases with neurological manifestations. Review of 600 cases. J Neurosurg 1983;59:111-8.

9. 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;88:1082-90.

10. Tombolini V, Zurlo A, Montagna A, et al. Radiation therapy of spinal metastases: results with different fractionations. Tumori 1994;80:353-6.

11. An YH. Orthopaedic Issues in Osteoporosised: Informa Health Care, 2002.

12. Rodan GA. Bone mass homeostasis and bisphosphonate action. Bone 1997;20:1-4.

13. Sundaresan N, Rothman A, Manhart K, et al. Surgery for solitary metastases of the spine: rationale and results of treatment. Spine 2002;27:1802-6.

14. Ghogawala Z, Mansfield FL, Borges LF. Spinal radiation before surgical decompression adversely affects outcomes of surgery for symptomatic metastatic spinal cord compression. Spine 2001;26:818-24.

15. Bartels RH, van der Linden YM, van der Graaf WT. Spinal extradural metastasis: review of current treatment options. CA Cancer J Clin 2008;58:245-59.

16. 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.

14

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;339:733-8.

18. Klimo P, Jr., Schmidt MH. Surgical management of spinal metastases. Oncologist 2004;9:188-96.

19. Senaratne SG, Colston KW. Direct effects of bisphosphonates on breast cancer cells. Breast Cancer Res 2002;4:18-23.

20. Clezardin P, Ebetino FH, Fournier PG. Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity. Cancer Res 2005;65:4971-4.

21. Coleman RE. Bisphosphonates in breast cancer. Ann Oncol 2005;16:687-95.

22. Ross JR, Saunders Y, Edmonds PM, et al. Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer. BMJ 2003;327:469.

23. 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;13:3490-7.

24. Tokuhashi Y, Ogawa T. [Spinal metastases]. Clin Calcium 2007;17:1267-72.

25. Juarranz A, Jaen P, Sanz-Rodriguez F, et al. Photodynamic therapy of cancer. Basic principles and applications. Clin Transl Oncol 2008;10:148-54.

26. Triesscheijn M, Baas P, Schellens JH, et al. Photodynamic therapy in oncology. Oncologist 2006;11:1034-44.

27. 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;83:1034-9.

15

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.

16

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 involvement1. 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 compression2,3. 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 surgery4. Tumor responses are highly

variable across patients and there are risks and side effects associated with all current

modalities5. 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 applications6. 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 death6.

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 tract6. 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 model8,9. 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

17

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 PDT10. However, little is known about the

effects of PDT on the structural integrity of bone10, 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.

2.3.1 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).

18

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.

2.3.2 µCT Image Analysis

To examine changes in architectural and structural properties of the vertebrae, X-ray micro-

computed 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 model12.

19

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.

2.3.3 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/s13 (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).

20

Ultimate stress: ROIavg,

ultimate

ultimateA

F=σ (Eqn. 1)

Stiffness: ultimate

ultimate

d

F=K (Eqn. 2)

, where Fultimate=ultimate force, Aavg, ROI=average cross sectional area within region of interest

(including cortical shell) and dultimate=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.

2.3.4 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

2.4.1 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 cord7. 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

21

(tα=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.

2.4.2 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) 23 26 17

Bone Volume Fraction (%) 38.5 ± 6.4 43.2 ± 7.9 55.7 ± 11.1*

Trabecular Thickness (µm) 90 ± 20 100 ± 30 130 ± 40*

Trabecular Number (/mm) 4.28 ± 0.48 4.48 ± 0.50 4.51 ± 0.76

Trabecular Spacing (µm) 150 ± 10 130 ± 40* 100 ± 30*

Bone Surface/Bone Volume (/mm)

22.8 ± 4.5 21.6 ± 5.3 16.9 ± 5.0*

Ultimate Stress (MPa) 27.5 ± 9.5 34.1 ± 9.8 39.3 ± 11.3*

Ultimate Stiffness (N/mm) 306 ± 123 399 ± 150* 410 ± 113*

* indicates statistical significance of comparisons to the control group at α=0.05.

22

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.

2.4.3 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.

23

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 pre-

clinical 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 environment14. 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 discrepancies15.

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

24

shell or growth plate15. 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 bone16. 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 treatment17. PDT-induced ossification in growth plates following

BPD-MA PDT has been reported to occur15. 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 PDT18.

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 injury19,20. 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

bone21. VEGF may directly enhance bone quality by promoting chemotactic migration and

differentiation of osteoblasts to lay down new bone22. 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

25

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

effects25. 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;15:1-4.

2. Constans JP, de Divitiis E, Donzelli R, et al. Spinal metastases with neurological manifestations. Review of 600 cases. J Neurosurg 1983;59:111-8.

3. 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;88:1082-90.

4. Bartels RH, van der Linden YM, van der Graaf WT. Spinal extradural metastasis: review of current treatment options. CA Cancer J Clin 2008;58:245-59.

5. Tokuhashi Y, Ogawa T. [Spinal metastases]. Clin Calcium 2007;17:1267-72.

6. Juarranz A, Jaen P, Sanz-Rodriguez F, et al. Photodynamic therapy of cancer. Basic principles and applications. Clin Transl Oncol 2008;10:148-54.

26

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;23:995-1003.

8. 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;10:034011.

9. 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, 2008.

10. 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;83:1034-9.

11. Hardisty M, Skrinskis T, Gordon L, et al. A repeatable bone quality measurement technique using 3D stereology. Canadian Orthopaedic Association Meeting, 2006.

12. Feldkamp LA, Goldstein SA, Parfitt AM, et al. The direct examination of three-dimensional bone architecture in vitro by computed tomography. J Bone Miner Res 1989;4:3-11.

13. 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;1:405-11.

14. Rodan GA. Bone mass homeostasis and bisphosphonate action. Bone 1997;20:1-4.

15. Bisland SK, Johnson C, Diab M, et al. A new technique for physiodesis using photodynamic therapy. Clin Orthop Relat Res 2007;461:153-61.

16. Li XQ, Klein L. Decreasing rates of bone resorption in growing rats in vivo: comparison of different types of bones. Bone 1990;11:95-101.

17. 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;51:373-83.

18. Gomer CJ, Ferrario A, Luna M, et al. Photodynamic therapy: combined modality approaches targeting the tumor microenvironment. Lasers Surg Med 2006;38:516-21.

19. MacDonald IJ, Dougherty TJ. Basic principles of photodynamic therapy J Porphyr Phthalocya 2001;5:105-29.

20. 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;248:47-57.

21. 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;99:9656-61.

22. 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;64:994-9.

23. Probst A, Spiegel HU. Cellular mechanisms of bone repair. J Invest Surg 1997;10:77-86.

27

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;80:22-30.

25. 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;104:8989-94.

28

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 skeleton1. Vertebral metastases commonly occur

in patients with primary breast cancer and compromise the mechanical stability of the spine. In

29

2/3 of patients, spinal metastases lead to skeletal related events such as bone pain,

hypercalcaemia, pathological fracture and spinal cord compression2,3. Eighty percent of these

lesions are found in the vertebral bodies with the remaining metastases established in the

posterior elements4. 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

therapy4. 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 intervention4.

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 cancer5. In vivo studies

in preclinical animal models have also shown that BPs do not inhibit metastatic progression once

vertebral tumors have reached a sufficient size6. 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.

30

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 metastasis7. It has also been shown that PDT

administered to healthy 3 month old rats led to increased vertebral bone formation and

mechanical strength8. 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

3.3.1 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).

3.3.2 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

2 × 106 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

31

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.

3.3.3 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.

3.3.4 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

32

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.

3.3.5 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.

33

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.

3.3.7 Mechanical Testing

For mechanical testing, the L2 vertebral body from each sample was potted in polymethyl-

methacrylate (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.

3.3.8 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).

34

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.

35

Table 3.1. Summary of stereological parameters in healthy and tumor involved bone following PDT and/or BP treatment.

* indicates statistical significance of comparisons to the corresponding control group at α=0.05.

Output Parameter

Tumor Healthy

Control PDT BP BP+PDT Control PDT BP BP+PDT

N 7 9 6 9 9 11 11 9

Bone Volume Fraction (%)

25.2 ± 9.4 36.9 ± 5.7* 41.6 ± 5.5* 44.4 ± 4.4* 44.0 ± 3.4 46.1 ± 8.0 48.5 ± 5.5 52.2 ± 5.5*

Trabecular Thickness (µm)

61.4 ± 10.2 66.4 ± 6.7 75.8 ± 12.2* 77.5 ±7.6* 78.8 ± 6.2 76.9 ± 11.4 90.8 ± 13.9 97.4 ± 19.7*

Trabecular Number (/mm)

4.0 ± 0.9 5.5 ± 0.5* 5.5 ± 0.2* 5.7 ± 0.2* 5.6 ± 0.2 6.0 ± 0.3 5.4 ± 0.3 5.4 ± 0.6

Trabecular Spacing (µm)

201 ± 72 115 ± 20* 106 ± 8* 97 ± 9* 100 ± 7 91 ± 17 96 ± 9 88 ± 10

Bone Surface/Bone Volume (/mm)

33.3 ± 5.5 30.4 ± 3.1 26.9 ± 4.1* 26.0 ± 2.5* 25.5 ± 2.0 26.5 ± 3.8 22.4 ± 2.7 21.2 ± 3.8*

Trabecular BMD (mg/cc)

711 ± 18 734 ± 24 752 ± 36 747 ± 51 713 ± 41 740 ± 32 767 ± 30 752 ± 51

Trabecular volumetric BMD (mg/cc)

179 ± 66 271 ± 46* 313 ± 47* 332 ± 42* 316 ± 27 340 ± 61 352 ± 23 391 ± 34*

Cortical BMD (mg/cc)

593 ± 61 666 ± 25* 719 ± 48* 697 ± 54* 703 ± 27 653 ± 53 736 ± 35 704 ± 62

Cortical shell mass fraction (%)

1.4 ± 0.4 1.0 ± 0.2* 0.8 ± 0.1* 0.8 ± 0.1* 1.0 ± 0.4 1.0 ± 0.3 1.0 ± 0.3 0.8 ± 0.3

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.

37

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 4 5 3 5 5 6 6 5

Ultimate Force (N)

87 ± 30 147 ± 40† 158 ± 32* 139 ± 14† 180 ± 36 194 ± 52 181 ± 31 231 ± 43

Ultimate Stress (MPa)

14.8 ± 6.0 26.5 ± 8.2† 30.9 ± 4.4* 26.5 ± 4.2† 34.1 ± 7.8 35.8 ± 9.0 34.6 ± 6.2 43.9 ± 8.0

Stiffness (N/mm)

108 ± 69 227 ± 98 359 ± 59* 197 ± 73 327 ± 105 298 ± 91 339 ± 58 387 ± 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 0.69 0.66 0.57

Trabecular vBMD 0.70 0.68 0.56

Cortical BMD 0.50 0.57 0.48

38

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% - 46.6%). 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.

39

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 tumors7,11,12, 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

40

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 treatment8. 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

collapse13. 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 homeostasis13-

15. Hypoxia in the local region of damage induces hypoxia-inducible factor-1α subunit (HIF-1α),

which in turn promotes vascular endothelial growth factor (VEGF) expression16,17. Both HIF-1α

and VEGF play important roles in angiogenesis, an essential process for recruiting osteogenic

cells and cytokines during bone remodeling and repair18.

41

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 repair16,17. Several studies have

shown VEGF stimulates bone repair and remodeling through enhancing both osteoclast and

osteoblast differentiation and activity16,18,19. 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 mineralization18. 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

a b c 1mm

100 µm

42

may also be due to primary bone formation in response to tissue injury, which would account for

the resulting trabecular formation. Further studies on the effects of PDT on osteogenic cells may

elucidate the molecular mechanism through which PDT induces bone formation.

BPs significantly reduced tumor induced osteolysis, which was not surprising since it is well

established that bisphosphonates inhibit osteoclast activity and subsequent bone resorption. The

resulting bone deposition in the absence of equally balanced bone resorption led to an increase in

bone mineral density, which is similar to the findings of other studies20-22. The positive effect of

BP on bone was not as pronounced in the healthy (non-metastatically involved) vertebrae. Since

the bone was initially within normal homeostatic range, 2 weeks may have been insufficient time

to observe the effects of BP, since the healthy bone may have counteracted the action of BPs by

also reducing osteoblastic bone formation to maintain homeostatic bone mass23. Despite some

evidence that bisphosphonates have direct anti-tumor effects on some breast cancer cell lines,

there is no evidence that clinically relevant dosages of BPs directly inhibit tumor growth21.

Histological examination of the vertebrae showed no evidence that zoledronic acid had direct

anti-tumor effects in this particular MT-1 human breast cancer metastasis model.

The application of PDT is not precluded by the use of existing clinical BP treatments, since

administering PDT following BP treatment led to net bone formation. In fact, previous work

showed that pre-treatment of the MT-1 tumor cells with BP in vitro rendered these cells more

susceptible to PDT-induced cytotoxicity24. In both healthy and tumor involved vertebrae,

combination BP+PDT treatment led to increased bone volume fraction resulting from trabecular

thickening. In the presence of both BP and PDT, it is expected that PDT-induced bone

remodeling with completely functional osteoblasts and BP inhibited osteoclasts would result in

greater net bone deposition compared to either treatment alone. Although there was no direct

statistical evidence that combination BP+PDT was more effective than BP alone, the inclusion of

multiple treatment groups in the study design resulted in a lack of power to detect significant

differences specifically between these two treatments. Nonetheless, only combination BP+PDT

was significantly better than untreated controls in the healthy group, which suggests that an

enhanced combinatory effect indeed exists when BP and PDT are administered together.

43

There are likely numerous interactions that occur between BP and PDT treatments, since

inhibition of GTPase proteins by BPs affects many other signalling pathways involved in cell

activity and viability. For example, BPs have been shown to be anti-angiogenic by reducing the

levels of circulating VEGF, which in turn inhibits tumor development and delays bone

metastasis25. Although the inhibition of VEGF theoretically may interfere with the upregulation

of bone remodeling induced by PDT, studies have found that anti-angiogenic therapy in

combination with PDT enhances its ablative effects on tumor17,26,27. Examination of the µCT

images of BP+PDT treated bone also revealed callus formation in response to inflammation,

indicating that the anti-angiogenic effects of BP did not interfere with the bone enhancing effects

of PDT. Thus PDT direct tumor cell kill in combination with BP’s anti-angiogenic impedance of

tumor recurrence may lead to enhanced, long-term tumor ablation and ultimately decrease

secondary metastatic bone destruction.

While stereological findings showed bone formation following treatment, mechanical testing

corroborated that the newly formed bone provided mechanical support. In the tumor bearing

vertebrae, trends of increasing bone strength and stiffness were observed in all treatment groups.

Increases in ultimate force, stress, and stiffness were statistically significant in the BP treated

group, which was likely a result of increased mineralization20. Trends in increased ultimate stress

and ultimate force were also observed in the PDT and combined BP+PDT treated groups,

although no statistically significant differences in mechanical properties were detected in the

healthy treated versus untreated groups.

Due to the study design, only half of the samples from each treatment group were mechanically

tested under load to failure. The small sample size in combination with the large variation

inherent to axial compressive testing resulted in a lack of statistical power to detect significant

differences between treated and untreated controls. Nonetheless, several studies have found that

stereological parameters of bone are able to explain the variation in its corresponding mechanical

properties28,29. vBMD is commonly used as a predictor of bone mechanics since it is an index

that reflects both structural and material properties of bone. The cortical shell also contributes to

mechanical strength, and bears increasingly more load as trabeculae fail. Thus the stereological

parameters of the cortical shell are expected to correlate to the mechanical properties of the

whole bone, particularly in vertebrae with large osteolytic lesions. In previous studies, cortical

BMD and vBMD have been shown to be strongly correlated to the overall strength and stiffness

44

of the pathologic vertebrae28,30. In this study, Pearson analyses between stereological and

mechanical parameters of those specimens mechanically tested showed that there was a

significant correlation between the stereological architecture and corresponding strength and

stiffness of the whole vertebrae. Thus, the trends seen in improved mechanical integrity are

supported by the significant differences seen in the stereologic parameters.

The cortical shell mass fraction was significantly lower in tumor bearing untreated controls,

which reflects the presence of large osteolytic lesions in the trabecular centrum. This left the

cortical shell constituting most of the bone mass and bearing most of the load. However, the

cortical shell BMD was also significantly lower in the untreated control versus the treated

groups, which strongly indicates that the treated vertebrae have a greater capacity to bear load

prior to fracture. This is further supported by the increase in vBMD following any of the

treatments. In the healthy groups, a significant increase in vBMD in the combined BP+PDT

group relative to control indicates that the observed increase in ultimate force (28%) is consistent

with the increased bone formation reflected by the stereology measurements.

Notwithstanding the encouraging results of this preclinical work, the metastatic model utilized in

this investigation may be limited in its clinical applicability, as immunodeficiency and rapid

bone remodeling in the young rats may affect the response of bone to PDT. Moreover, further

investigation is required to elucidate how a positive pro-angiogenic influence on bone

remodeling due to PDT could affect tumor recurrence. It is also important to investigate the long-

term outcome of PDT to determine whether these positive effects on bone are sustained.

The short-term benefits of photodynamic therapy on bone stereology and mechanical strength

signify that it is a promising therapy for spinal metastasis. Clinically, treatment with PDT can be

particularly beneficial for patients who have recurring tumors untreatable by conventional

treatments, such as radiation and chemotherapy, since PDT induces the production of anti-tumor

antigens to fight tumor recurrence and does not generate cumulative toxic side effects31. PDT can

be targeted to specific spinal levels, or other skeletal sites, as it acts locally and can be easily

administered (through a minimally invasive outpatient vertebroplasty-like cannulated approach)

in combination with other treatments. Overall, this study has further motivated PDT as a viable

and attractive treatment for spinal metastasis secondary to breast cancer, either as an alternative

or an adjuvant therapy compatible with existing treatments.

45

3.6 References

1. Klimo P, Jr., Schmidt MH. Surgical management of spinal metastases. Oncologist 2004;9:188-96.

2. Coleman RE. Metastatic bone disease: clinical features, pathophysiology and treatment strategies. Cancer Treat Rev 2001;27:165-76.

3. 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;88:1082-90.

4. Ecker RD, Endo T, Wetjen NM, et al. Diagnosis and treatment of vertebral column metastases. Mayo Clin Proc 2005;80:1177-86.

5. Ross JR, Saunders Y, Edmonds PM, et al. Systematic review of role of bisphosphonates on skeletal morbidity in metastatic cancer. BMJ 2003;327:469.

6. Kaijzel EL, van der Pluijm G, Lowik C. Whole-body optical imaging in animal models to assess cancer development and progression. Clin Cancer Res 2007;13:3490-7.

7. Akens MK, Hardisty MR, Wilson BC, et al. Defining the therapeutic window of vertebral photodynamic therapy in a murine pre-clinical model of breast cancer metastasis using the photosensitizer BPD-MA (Verteporfin). Breast Cancer Res Treat 2009.

8. Won E, Akens MK, Hardisty MR, et al. Effects of Photodynamic Therapy on the Structural Integrity of Vertebral Bone. Spine 2009.

9. Hardisty M, Gordon L, Agarwal P, et al. Quantitative characterization of metastatic disease in the spine. Part I. Semiautomated segmentation using atlas-based deformable registration and the level set method. Med Phys 2007;34:3127-34.

10. Feldkamp LA, Goldstein SA, Parfitt AM, et al. The direct examination of three-dimensional bone architecture in vitro by computed tomography. J Bone Miner Res 1989;4:3-11.

11. 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;23:995-1003.

12. 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;83:1034-9.

13. Korbelik M. PDT-associated host response and its role in the therapy outcome. Lasers Surg Med 2006;38:500-8.

14. van Duijnhoven FH, Aalbers RI, Rovers JP, et al. The immunological consequences of photodynamic treatment of cancer, a literature review. Immunobiology 2003;207:105-13.

15. 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;248:47-57.

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16. Dai J, Rabie AB. VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res 2007;86:937-50.

17. Gomer CJ, Ferrario A, Luna M, et al. Photodynamic therapy: combined modality approaches targeting the tumor microenvironment. Lasers Surg Med 2006;38:516-21.

18. 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 U S A 2002;99:9656-61.

19. Zelzer E, Olsen BR. Multiple roles of vascular endothelial growth factor (VEGF) in skeletal development, growth, and repair. Curr Top Dev Biol 2005;65:169-87.

20. Turner CH. Biomechanics of bone: determinants of skeletal fragility and bone quality. Osteoporos Int 2002;13:97-104.

21. Daubine F, Le Gall C, Gasser J, et al. Antitumor effects of clinical dosing regimens of bisphosphonates in experimental breast cancer bone metastasis. J Natl Cancer Inst 2007;99:322-30.

22. Berenson JR, Rosen LS, Howell A, et al. Zoledronic acid reduces skeletal-related events in patients with osteolytic metastases. Cancer 2001;91:1191-200.

23. Rodan GA. Bone mass homeostasis and bisphosphonate action. Bone 1997;20:1-4.

24. Akens MK, Karotki A, Wilson BC, et al. Effect of photodynamic therapy (PDT) on bisphosphonate pre-treated breast cancer in-vitro. Orthopaedic Research Society Meeting, 2007:0972.

25. Lipton A. Emerging role of bisphosphonates in the clinic--antitumor activity and prevention of metastasis to bone. Cancer Treat Rev 2008;34:S25-30.

26. Kosharskyy B, Solban N, Chang SK, et al. A mechanism-based combination therapy reduces local tumor growth and metastasis in an orthotopic model of prostate cancer. Cancer Res 2006;66:10953-8.

27. Dimitroff CJ, Klohs W, Sharma A, et al. Anti-angiogenic activity of selected receptor tyrosine kinase inhibitors, PD166285 and PD173074: implications for combination treatment with photodynamic therapy. Invest New Drugs 1999;17:121-35.

28. Nazarian A, von Stechow D, Zurakowski D, et al. Bone volume fraction explains the variation in strength and stiffness of cancellous bone affected by metastatic cancer and osteoporosis. Calcif Tissue Int 2008;83:368-79.

29. Eswaran SK, Gupta A, Adams MF, et al. Cortical and trabecular load sharing in the human vertebral body. J Bone Miner Res 2006;21:307-14.

30. Forwood MR, Vashishth D. Translational aspects of bone quality--vertebral fractures, cortical shell, microdamage and glycation: a tribute to Pierre D. Delmas. Osteoporos Int 2009;20 Suppl 3:S247-53.

31. Castano AP, Demidova TN, Hamblin MR. Mechanisms in photodynamic therapy: part two -- cellular signalling, cell metabolism and modes of cell death. Photodiagnosis Photodyn Ther 2005;2:1-23.

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Chapter 4: Summary

4.1 Effects of Photodynamic Therapy on Bone

Advancements in breast cancer treatments have prolonged patient survival times and

subsequently increased the incidence of spinal metastasis. There is no clinical standard of care

for spinal metastasis because treatment response varies across patients. Therefore there is great

motivation to develop photodynamic therapy as a treatment for spinal metastasis that can treat

secondary metastatic lesions and simultaneously preserve the structural integrity of the spine.

Determining the effects of photodynamic therapy on the structural and mechanical properties of

healthy vertebral bone was an important first step in evaluating the feasibility of PDT as a

treatment for spinal metastasis. The first main study component of the thesis was simply a

feasibility study to ensure PDT did not catastrophically damage healthy bony tissue, and thus 3

month old wistar rats were utilized. The impact of photodynamic therapy on healthy vertebral

bone was examined both 1 week and 6 weeks following a single PDT treatment to examine

effects in the bone shortly after treatment and after a more prolonged period of time. Structural

properties were determined using three dimensional micro-computed tomography image analysis

to quantify bone volume and architecture, and mechanical integrity was tested with axial

compression load to failure studies.

Stereological analyses revealed that PDT did not compromise the structural integrity of bone, but

in fact, PDT induced trabecular thickening and an increase in bone volume fraction 6 weeks

following PDT. Mechanical loading of the vertebrae demonstrated that the ultimate strength and

stiffness increased significantly compared to controls, which indicates that the newly formed

bone was able to provide structural support. Although not statistically significant, similar trends

of improving stereology and mechanical properties were found 1 week following treatment. It is

postulated that the positive effects of PDT on bone may have been due to upregulation of VEGF

following treatment, which in turn upregulated bone remodeling and resulted in net bone

deposition.

The implications of this study were important in proceeding with further investigations of PDT

on bone. It provided the first indication that PDT has a positive effect on bone quality, which

supports the clinical application of PDT in the spine and motivates further research pursuits to

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gain a better understanding of the impact of PDT in metastatically involved bone. This study also

showed that 1 week may be insufficient to detect significant changes in bone, which is the

limited time interval over which PDT effects can be observed in a spinal metastatic model.

However, this study has shown significant effects at 6 weeks, which suggests that trends in bone

formation at 1 week would become more dramatic and statistically significant beyond the 1 week

period. This therapeutic effect would be particularly beneficial for spinal metastasis patients

whose spines are weakened by metastatic tumor induced bone destruction. Overall, this study has

demonstrated the structural safety of PDT, and has further supported its clinical translation as a

treatment for spinal metastasis.

Following the promising findings of the effects of PDT on healthy bone, the second main study

component of this thesis focused on the effects of PDT in metastatic vertebrae. This study was

built upon previous work that proved the efficacy of PDT in eradicating spinal tumors and

demonstrated that PDT induces bone formation in healthy vertebral bone. The effects of PDT on

metastatically involved bone is largely unknown, and thus the aim of this study was to quantify

the effects of PDT on the stereological architecture and bulk mechanical strength of

metastatically involved vertebrae. Furthermore, as many patients will have undergone systemic

bisphosphonate treatment (a standard clinical therapy for spinal metastasis), a second objective

of this study was to investigate the effects of PDT following previous bisphosphonates treatment

to determine whether active bisphosphonates will affect the action of PDT on healthy and

metastatically involved bone. Similar to the previous study in healthy vertebral bone,

stereological and mechanical properties were determined using µCT image analysis and

compression testing. However, treatments were carried out in rnu/rnu rats, which are athymic

and therefore reliably establish metastasis in the spine. To fully elucidate the direct impact of

PDT alone and in combination with BP on bone, this investigation includes their evaluation in

healthy (non-pathologic) rnu/rnu rats.

Stereology analysis showed that treatment with PDT alone significantly impeded metastatic bone

destruction, with increased bone volume fraction and trabecular number. Not surprisingly, BP

treatment was also effective at reducing bone resorption by increasing bone volume fraction,

trabecular number, and trabecular thickness. Previous treatment with bisphosphonates did not

affect the inhibitory action of PDT on metastatic bone destruction, but rather, combination

BP+PDT treatment seemed to have an enhanced effect compared to that with BP or PDT alone

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by increasing bone volume fraction, trabecular number, and trabecular thickness. Although direct

statistical comparison between BP and combination BP+PDT treatment groups did not show a

significant advantage of BP+PDT over BP alone, combination BP+PDT was the only treatment

group that induced a significant increase in bone mass in healthy bone, which suggests that there

is an enhanced combinatory effect when BP and PDT are administered together.

Mechanically, BP induced a statistically significant increase in vertebral stiffness and strength in

tumor involved bone, with similar trends of increasing strength following PDT or BP+PDT

treatment. No statistically significant improvement in mechanical properties were found in the

healthy bone following any treatment, but this was likely due to the small number of samples

that were mechanically loaded to failure (since half of the samples were decalcified for

histological analysis). Nonetheless, Pearson correlation analysis showed that bone volume

fraction, volumetric bone mineral density, and cortical bone mineral density were significantly

correlated to stiffness and strength of the whole bone. Thus the significant improvement in

stereological parameters provides strong indication that the newly formed bone provided

structural support and strengthened the mechanical integrity of the whole bone.

Histology analysis revealed that only PDT had direct anti-tumor effects in the treated vertebrae.

Thus, although BP and PDT were comparable in their effects on impeding metastatic bone

destruction, PDT is able to simultaneously ablate tumor tissue to further reduce tumor osteolysis.

This study has shown that patients who have responded poorly to bisphosphonates can attempt

PDT treatment, with similar bone preserving effects as BP. More importantly, we have shown

that previous exposure to bisphosphonates does not hinder the therapeutic effects of PDT. In fact,

since combination BP+PDT treatment seems to have an enhanced combinatory effect, it may be

worthwhile for patients who are responding well to bisphosphonates to also undergo PDT for

additional benefits of tumor eradication and further restoration of bone mass. PDT is a minimally

invasive procedure and therapeutic effects are local, which minimizes damage to tissue that

surrounds the treatment area. Thus it would be easily applied in combination with other

therapies, such as injection of bone cement using the same cannula to first de-bulk lesions and

then provide immediate mechanical stabilization. The relatively few side effects of PDT imply

that it can be safely administered with other treatments (as seen with conjunctive administration

with BPs in this study) for additional therapeutic efficacy.

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4.2 Future Directions

This thesis has shown that PDT is a structurally safe and effective treatment for spinal

metastasis. Although PDT is proving to be a promising treatment that can ablate tumor and

simultaneously strengthen the structural integrity of bone, it is also necessary to further

investigate the actions of PDT to understand the mechanisms through which it induces bone

formation. Particularly, in vitro studies of PDT on osteogenic cells would be of great value in

understanding the direct effects of PDT on bone on a cellular level. The therapeutic effect of

PDT can be further optimized by adjusting light dosages and photosensitizers pertaining to the

specific application, such as exploring metronomic (continuous low dose) PDT, and molecular

beacons that bind to biomarkers on the tissue of interest to increase the specificity of PDT to

selectively treat target tissue areas and minimize effects in surrounding tissues. Greater

understanding of the molecular mechanisms of PDT would allow researchers and clinicians to

develop strategies to optimize its therapeutic outcomes.

The finding that PDT did not interact antagonistically with BP treatment shows some indication

that PDT can be administered in combination with existing therapies, or used as an alternative

therapy following poor response to other treatments. Since radiation therapy is considered a

mainstay treatment for spinal metastasis, it would be clinically relevant to study the effects of

PDT in combination with prior radiation therapy to determine how PDT can be incorporated into

current treatment strategies.

PDT alone induced significant effects on bone stereology and strength in healthy bone at 6

weeks rather than 1 week following treatment. Thus the actions of PDT on bone take effect for a

longer period of time, and warrants further study of prolonged effects of BP and PDT on both

healthy and metastatically involved bone. The animal model used in this study has a limited

survival time of only 1 week following treatment, but longer term studies are feasible in healthy

rats, which will elucidate the direct impact of PDT and BP on bone independent of tumor

influences. The development of new preclinical tumor models utilizing a slower proliferating cell

line would extend animal survival times and permit long term studies in metastatically involved

vertebral bone. It would also be of clinical interest to study the effect of PDT on vertebral bone

with mixed osteolytic and osteoblastic lesions, since the widespread use of BP treatment has

been accompanied by the onset of mixed lesions.

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4.3 Conclusion

The findings from this body of work have further substantiated the application of photodynamic

therapy as a minimally invasive treatment for spinal metastasis. A single PDT treatment

administered alone or with previous bisphosphonates treatment directly induced tumor cell

necrosis. In addition to eradicating vertebral tumors, PDT alone and in combination with BP also

significantly reduced tumor induced osteolysis and bone destruction. The increased bone mass

translated to mechanical strengthening of the whole vertebrae. PDT was also found to have direct

effects on bone, as combination BP+PDT treatment induced significant bone formation in

healthy vertebrae after 1 week and PDT alone had positive effects on healthy bone 6 weeks after

treatment. As PDT is able to treat secondary metastatic lesions and simultaneously improve the

mechanical stability of the spinal column, it provides an attractive novel treatment for spinal

metastasis secondary to breast cancer.