Mediated Photochemical Internalization of Bleomycin and Concurrent Hyperthermia of Multicellular Tumor Spheroids

UNLV Theses, Dissertations, Professional Papers, and Capstones

5-1-2013

AlPcS(2a)-Mediated Photochemical Internalization of Bleomycin and Concurrent Hyperthermia of Multicellular Tumor Spheroids

Aaron Andersen University of Nevada, Las Vegas

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Repository Citation Andersen, Aaron, "AlPcS(2a)-Mediated Photochemical Internalization of Bleomycin and Concurrent Hyperthermia of Multicellular Tumor Spheroids" (2013). UNLV Theses, Dissertations, Professional Papers, and Capstones. 1794. http://dx.doi.org/10.34917/4478188

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This Thesis has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. ALPCS(2A)–MEDIATED PHOTOCHEMICAL INTERNALIZATION OF

BLEOMYCIN AND CONCURRENT HYPERTHERMIA OF MULTICELLULAR

TUMOR SPHEROIDS

Aaron David Andersen

Bachelor of Science in Physics Utah State University 2010

A thesis submitted in partial fulfillment of the requirements for the

Master of Science in Health Physics

Department of Health Physics and Diagnostic Sciences College of Allied Health Sciences The Graduate College

University of Nevada, Las Vegas May 2013

THE GRADUATE COLLEGE

We recommend the thesis prepared under our supervision by

Aaron David Andersen

entitled

ALPCS(2A)-Mediated Photochemical Internationalization of Bleomycin and Concurrent Hyperthermia of Multicellular Tumor Spheroids

be accepted in partial fulfillment of the requirements for the degree of

Master of Science in Health Physics Department of Health Physics and Diagnostic Sciences

Steen Madsen, Ph.D., Committee Chair

Ralf Sudowe, Ph.D., Committee Member

Gary Cerefice, Ph.D., Committee Member

Daniel Young, D.P.T., Graduate College Representative

Thomas Piechota, Ph.D., Interim Vice President for Research & Dean of the Graduate College

May 2013

ABSTRACT

Treatments for glioblastoma multiforme and invasive ductal carcinoma involve surgical resection and adjuvant or concurrent targeted therapies. The goal of the secondary treatment is to reduce the cancer cells in the resection margin and other areas of suspected involvement. Photochemical internalization is a targeted therapy using light to activate photosensitizers embedded in cellular structures to improve the delivery of macromolecules within the cell.

In a previous study, optimized treatment parameters including radiant exposure, and irradiance, concentration of AlPcS(2a), temperature, and the concentration of bleomycin were determined for human ACBT glioma multicell spheroids. The current study investigates the effectiveness of the ACBT–optimized concurrent PCI/hyperthermia treatment for F98 rat glioma and MCF-7 human invasive ductal carcinoma spheroids.

The combination of photochemical internalization and hyperthermia was shown to be more effective than either treatment modality alone and, in all cases, the combined treatments interacted in a synergistic manner.

iii

ACKNOWLEDGEMENTS

Special thanks go to my thesis advisory committee chair, Dr. Steen Madsen, for his knowledge concerning the subject matter and his support in helping me complete my research.

I would also like to thank my thesis advisory committee members, Dr. Steen

Madsen, Dr. Ralf Sudowe, Dr. Gary Cerefice and Dr. Daniel Young for their time and service.

Additionally I would like to thank Christina Schlazer for teaching me laboratory protocols and Stephanie Molina for assisting with sub-culturing and measurements.

TABLE OF CONTENTS

ABSTRACT ...... ii ACKNOWLEDGEMENTS ...... iv LIST OF FIGURES ...... vii

CHAPTER 1 INTRODUCTION ...... 1 1.1 Brain Cancer ...... 1 1.2 F98 Cell Line ...... 3 1.3 Breast Cancer ...... 5 1.4 MCF-7 Cell Line ...... 7 1.5 Monolayer vs. Multicellular Tumor Spheroids ...... 8 1.6 Targeted Therapies ...... 12 1.7 Photodynamic Therapy (PDT) ...... 13 1.8 Aluminum Phthalocyanine Disulfonate ...... 18 1.9 Photochemical Internalization (PCI) ...... 20 1.10 Bleomycin ...... 24 1.11 Hyperthermia ...... 25 1.12 Scope of work...... 26

CHAPTER 2 MATERIALS AND METHODS ...... 28 2.1 Cell Lines ...... 28 2.2 Multicellular Tumor Spheroid Formation ...... 29 2.3 Photodynamic Therapy ...... 29 2.4 Photochemical Internalization ...... 30 2.5 Hyperthermia ...... 30 2.6 Measurements...... 30 2.7 Statistical analysis ...... 31

CHAPTER 3 RESULTS ...... 32 3.1 F98 Spheroid Viability ...... 32 3.2 F98 Spheroid Growth Kinetics ...... 33

3.3 MCF-7 Spheroid Viability ...... 36 3.4 MCF-7 Spheroid Growth Kinetics ...... 38

CHAPTER 4 DISCUSSION ...... 42

CHAPTER 5 CONCLUSION...... 46

REFERENCES ...... 48

VITA ...... 57

LIST OF FIGURES

Figure 1. GBM pre- and post- tumor resection ...... 2 Figure 2. F98 tumor cells ...... 5 Figure 3. MCF-7 spheroid and intracellular junctions ...... 9 Figure 4. Regions and gradients in MCTS...... 10 Figure 5. Tumor growth curve...... 11 Figure 6. Photosensitizer excitation process ...... 15 Figure 7. Type I and Type II reactions...... 16 Figure 8. Aluminum Phthalocyanine Disulfonate structure ...... 18 Figure 9. AlPcS(2a) absorption spectrum...... 19 Figure 10. Photochemical internalization process ...... 21 Figure 11. “Light first” PCI process...... 22 Figure 12. Structure of bleomycins...... 25 Figure 13. F98 spheroid viability as a function of days post-treatment...... 33 Figure 14. Growth kinetics of F98 spheroids subjected to HT...... 34 Figure 15. Growth kinetics of F98 spheroids at low radiant exposures...... 35 Figure 16. Growth kinetics of F98 spheroids – PCI effect...... 35 Figure 17. Growth kinetics of F98 spheroids subjected to combination treatments ...... 36 Figure 18. MCF-7 spheroid formation...... 37 Figure 19. MCF-7 spheroid elongation...... 37 Figure 20. MCF-7 spheroid viability as a function of days post-treatments...... 38 Figure 21. Growth kinetics of MCF-7 spheroids subjected to HT...... 39 Figure 22. Growth kinetics of MCF-7 spheroids at low radiant exposures ...... 40 Figure 23. Growth kinetics of MCF-7 spheroids – PCI effect...... 40 Figure 24. Growth kinetics of MCF-7 spheroids subjected to combination treatments.. . 41

vii

CHAPTER 1

INTRODUCTION

1.1 Brain Cancer

The central nervous system (CNS) is associated with several types of benign and malignant tumors. Each year, approximately 35,000 Americans are diagnosed with primary brain tumors. Of those 35,000 new diagnoses just over 18,000 are malignant brain tumors which account for approximately 2% of all cancers in the United States and nearly 13,000 deaths per year (CBTRUS).

CNS tumors in pediatric patients are 50% more likely to be malignant when compared to adults (Khoshnevisan 2012). The central brain tumor registry of the United

States (CBTRUS) estimates that 1 in every 1300 children will develop a primary brain tumor by age 20 (CBTRUS 1998). The incidence rate has increased from 5.85 per

100,000 person-years in 1975 to 6.4 per 100,000 person-years in 2003 (Chandana et al.

2008).

Unfortunately, it is much more difficult to treat primary and secondary malignant

CNS tumors as compared to benign CNS tumors due to the lack of effective treatments.

In the past decade, however, advances in several fields including stem cell biology, genomic medicine and molecular biology have revolutionized the understanding of brain tumorigenesis. These advancements have led to many new therapies that target molecular processes in primary malignant CNS tumors (Khoshnevisan 2012).

The most frequent primary brain tumor is a glioma. Gliomas account for 49% of primary brain tumors (Wrensch et al. 2002).

Of the various central nervous system malignancies glioblastoma multiforme

(GBM; Figure 1) has a very poor prognosis second only to brain stem gliomas.

Improvements in the standard of care over the past 7 years have led to only a minor increase in the median survival time for patients after diagnosis from 10 to 14 months.

Current treatments include surgical resection of the tumor followed by various concurrent or subsequent treatments such as radiosurgery, chemo-radiotherapy and anti-angiogenic therapy (Van Meir et al. 2010). The goal of subsequent treatments is to eliminate the malignant cells residing in the margin surrounding the resection cavity thereby reducing recurrence of the malignant glioma (Angell-Petersen et al. 2006) (Hirschberg et al. 2004).

A B

Figure 1. T1-weighted post-contrast magnetic resonance images of GBM (a) pre- and (b) post-tumor resection. (Lacroix et al. 2001)

Animal tumor models have been used extensively to determine the efficacy of emerging therapies. In the 1970’s it was discovered that intravenous injections of N- methylnitrosourea (MNU) or a single dose of N-ethyl-N-nitrosourea (ENU) could induce

2 reproducible CNS tumors in adult rats. Since the mid 1970’s rat brain tumor models have been used extensively for this purpose. Although these animal models are useful in evaluating treatment methods, existing models cannot exactly simulate human glioblastoma multiforme or anaplastic astrocytomas (Barth and Kaur 2009).

There is a general consensus outlining the criteria that must be met in order for a brain tumor model to be considered valid. The criteria are as follows “(1) They should be derived from glial cells; (2) it should be possible to grow and clone them in vitro as continuous cell lines and propagate them in vivo by serial transplantation; (3) tumor growth rates should be predictable and reproducible; (4) the tumors should have glioma- like growth characteristics within the brain including neovascularization, alteration of the blood-brain barrier (BBB), an invasive pattern of growth, and lack of encapsulation; (5) host survival time following intracranial tumor implantation should be of sufficient duration to permit therapy and determination of efficacy; (6) for therapy studies, the tumors should be either non or weakly immunogenic in syngeneic hosts; (7) they should not grow into the epidural space or extend beyond the brain and finally, (8) their response or lack thereof to conventional treatment should be predictive of the response in human brain tumors,” (Barth and Kaur 2009).

1.2 F98 Cell Line

This study investigates various treatments on the F98 rat glioma as GBM model.

The F98 glioma (ATCC # CRL-2397) was originally cultured from a subcutaneous tumor on a male Fischer rat. F98 gliomas behave similarly to human glioblastomas exhibiting low immunogenicity, invasive growth patterns and overexpression of similar genes. F98

3 cells have also been used to produce brainstem tumors in Fischer rats with similar histopathological and radiobiological characteristics of primary human brainstem tumors.

Because of the characteristics of the F98 glioma it has been used extensively as a brain tumor model in experimental neuro-oncology for pre-clinical testing of therapies (Barth and Kaur 2009).

Table 1. Rat brain tumor models (Barth and Kaur 2009)

The F98 glioma is composed of two different shaped cells. Spindle-shaped cells with fusiform nuclei make up the majority while polygonal cells with oval nuclei make up the minority of the composition. The tumor mass is surrounded by pools of tumor cells that have extended various distances into the normal brain tissue (Barth and Kaur

2009).

The F98 glioma shares several traits with the human GBM including the overexpression of PDGFβ and Ras, and increased expression of EGFR, cyclin D1, cyclin

D2 and Rb when compared to rat astrocytes (Sibenaller et al. 2005). Other characteristics of the F98 glioma include decreased expression of BRCA1 and the lack of BRCA1 foci induced by radiation and cisplatin (Bencokova et al. 2008). F98 gliomas exhibit necrotic

4 cores, non-glomeruloid neovascular proliferation and, like human GBM, are characterized by extensive infiltration into normal brain (Figure 2) (Mathieu et al. 2007).

Studies have shown that the F98 glioma is radio-resistant, probably due to its functionally impaired BRCA1 status (Bencokova et al. 2008), making it a model for evaluating the efficacy of combination therapies. The F98 model can be used to determine which therapies should go on to further clinical trials and which therapies would likely have little to no effect and should be discarded.

Figure 2. Hematoxyllin and Eosin-stained histological section of Fischer rat brain illustrating the diffuse and infiltrative nature of F98 tumor cells. (Barth and Kaur 2009).

1.3 Breast Cancer

The second type of cancer used for evaluating treatments in this study is breast cancer. According to the Center for Disease Control (CDC), breast cancer is the second

5 most common cancer in women and one of the leading causes of cancer related death among women. The most recent statistics show that in 2009 over 200,000 women in the

United States were diagnosed with breast cancer and over 40,000 women in the United

States died from breast cancer. One in every eight women will be diagnosed with breast cancer in her lifetime (U.S. Cancer Statistics Working Group 2013).

Accounting for about 80% of all breast cancers, invasive ductal carcinoma is the most common type of breast cancer. This infiltrating cancer begins in the milk ducts which act as a connection between the milk producing lobules and the nipple. From there it invades the other areas of the breast and possibly the lymph nodes and other areas of the body. This type of cancer is more common among older women with two thirds of reported cases being in women age 55 and older. On average in the United States there are over 180,000 cases of invasive breast cancer each year. The signs and symptoms associated with ductal carcinoma include: swelling or pain in the breast region, a lump in the underarm area or thickening of the breast skin. Diagnosis is performed by physical examination and the use of imaging modalities such as mammography, ultrasound and

MRI. Biopsies are performed for any suspicious findings from the image results. Staging for ductal carcinoma is based on tumor size, extent of the spread to the lymph nodes and the extent of spread to other parts of the body.

Treatment options for breast cancer are based on the stage and type of the cancer.

Stage 0 is classified as in situ and diagnosis at stage 0 has a 100% five year survival rate.

Patients with stage 0 lobular carcinoma are advised to undergo rigorous breast cancer surveillance (National Comprehensive Cancer Network 2009). Ductal carcinoma in situ

(DCIS) is more serious and unlike lobular carcinoma in situ can progress to invasive

6 breast cancer. Ductal Carcinoma has a 100% survival rate if diagnosed in situ. Treatment of DCIS involves breast conserving surgery or mastectomy depending on the extent of the disease and radiation therapy (Maughan et al. 2010).

As the stage of the breast cancer advances from stage I to stage IV the five year survival rate decreases from 98 to 23.4% respectively. Treatment for later stage breast cancer involves evaluation of lymph nodes by dissection, breast conserving surgery or mastectomy, radiation therapy and adjuvant therapies depending on the hormone receptors and the expression of ERBB2 (Maughan et al. 2010). Radiation therapy following breast conserving surgery has shown a significant reduction of the five year local recurrence rate and an apparent decrease in the 15 year mortality risk (Clarke et al.

2005). Adjuvant systemic therapies such as chemotherapy, endocrine therapy, and tissue targeted therapies are most beneficial to patients with node-positive disease. These therapies are able to substantially reduce cancer recurrence by enhancing local therapies such as surgery, radiotherapy or both (National Comprehensive Cancer Network 2009).

Induction systemic therapies are performed on patients with locally advanced breast cancer. Induction chemotherapy is able to facilitate the preoperative reduction of the tumor by more than 50% for 75% of patients thus allowing for breast conserving surgery

(Mauri et al. 2005). New and developing treatment options involve targeted therapies which are specifically directed toward the tumor characteristics.

1.4 MCF-7 Cell Line

The MCF-7 cell line was used in this study as an invasive ductal carcinoma model. The MCF-7 breast cancer cell line was originally acquired from a 69 year old

Caucasian woman in 1970. This cell line is particularly relevant since it was the first mammary cell line that was capable of surviving more than a few months.

MCF-7 is one of three breast cancer cell lines (BCCL) that account for 80% of the

35,000 publications involving BCCL. In the last decade MCF-7 has been the most used appearing in 53% of all journal articles that mention BCCL. It was acquired from pleural effusion of an invasive ductal carcinoma (Soule et al. 1973). This method facilitates the recovery and growth of the cell line easier than extracting cells directly from tumors or metastasis due to the large number of isolated tumor cells and low yield of contaminant cells. The estrogen receptor (ER) and progesterone receptor (PgR) status of the MCF-7 cell line is ER+ and PgR+ (Bana and Bagrel 2011).

Extensive studies with the MCF-7 line over the past 40 years has led to the establishment of complete genetic and proteinic profiles allowing this cell line to become a reference model in the field of breast cancer research (Bana and Bagrel 2011).

1.5 Monolayer vs. Multicellular Tumor Spheroids

Two-dimensional cultures have been used as in vitro models for cancer research for many years. There are several advantages to monolayers including ease of set up and good viability of cells. However being one of the simplest models, they lack the complexity that better represents solid tumors. Transport is limited in 2D models due to the lack of structure. Cells often exhibit characteristics when cultured in a monolayer that differ from their in vivo counterparts (Bissel 1981). Organ cultures are also a useful tool for modeling cancer due to the fact that they have an already established tumor

8 microenvironment, including cell-cell interactions and cell-matrix interactions. They are however difficult to obtain and exhibit poor viability in culture (Kim 2005).

Multicellular tumor spheroids (MCTS) were developed to bridge the gap between monolayer cultures and animal models. They include cell-cell interactions and extracellular matrix interactions that are vital to the growth and propagation of tumors. The surface and intracellular junctions of an MCF-7 spheroid are shown in Figure 3.

Figure 3. MCF-7 spheroid, left (scale bar: 100 µm); intracellular junctions indicated by the arrows, right (scale bar: 5 µm). (Ho et al. 2012)

Spheroids were developed for cancer research to better understand the effects of radiation and chemotherapeutic agents on 3D cultures (Wheldon 1994) (Yuhas et al.

1978). There are several advantages to 3D cultures compared to monolayers. There is a higher degree of cellular heterogeneity in 3D cultures compared to the homogeneity of monolayers. The 3D structure presents obstacles in nutrient transport which creates cellular proliferation gradients. Regions that are amply supplied with O2 have proliferating cells adjacent to microvessels. In regions where nutrient diffusion and waste removal is limited, the cell cycle times are increased. This environment produces

9 quiescent cells that are at rest in G0 phase but remain clonogenic. Towards the center of the spheroid, non-proliferating cells can be found (Mueller-Kleiser 1987). This proliferation gradient sets spheroids apart from monolayers and resembles the volume growth kinetics of solid tumors. The various regions of the multicellular spheroid are shown in Figure 4.

Figure 4. Regions and gradients in MCTS. (Lin and Chang 2008)

Mueller-Kleiser observed three general phases of multicellular spheroid growth as shown in Figure 5. In the initial phase there is exponential growth as the cells proliferate until a diameter of 50-200 µm is achieved. After this initial growth phase the cell cycle distribution changes with an increase in non-proliferating cells (Durand and Sutherland

1975). This, along with the peripheral cell detachment greatly reduces the number of proliferating cells. The second phase exhibits a linear increase in the spheroid diameter versus time. Volume increase is significantly impaired in the third growth phase as the

10 spheroids reach a maximum diameter ranging from 1 – 4 mm (Sutherland and Durand

1976). Spheroids will maintain this diameter indefinitely without vascularization which leads to exponential growth. After vascularization, growth saturation is possible assuming the disease doesn’t terminate the host first (Steele 1977). These growth phases resemble the avascular growth of tumors and show that cellular aggregates can remain dormant until vascularization induces exponential growth.

Figure 5. Tumor growth curve. (Mueller-Kleiser 1987)

3D cultures exhibit increased resistance to treatments and show similar drug sensitivity patterns to in vivo tumors (Ohmori et al. 1998). As cells begin to form into 3D spheroids their cell shape and environment change mimicking that of glandular tumors as they transition from early to intermediate adenoma stages (Rak et al. 1995).

The extra cellular matrix (ECM) is much more extensive in spheroids as compared to monolayers. Monolayers have a reduced ability or even inability to synthesize the materials required for the extracellular matrix. Conversely, spheroids retain much of the structural and functional differentiation of solid tumors (Mueller-

Kleiser 1987). The ECM along with cell to cell interactions can have an impact on the distribution and function of drugs and physiological factors which can result in cellular growth, death and differentiation (Kim 2005).

In order to use MCTS for various applications they must be easily and reliably reproduced. There are several methods for MCTS generation with associated advantages and disadvantages. Factors that should be considered when deciding upon a generation technique should include: MCTS size uniformity, effect on cellular physiology, convenience and efficiency (Lin and Chang 2008).

1.6 Targeted Therapies

MCTS can be used to evaluate targeted therapies. Both cancers included in this study have surgical resection as the primary mode of treatment with secondary concurrent or adjuvant targeted therapies.

There has been increased interest in recent years in targeted therapies involving the clinical use of macromolecular drugs, such as cytotoxic agents. This is partly due to the advances in production technology and to a better understanding of the utilization of these types of drugs. Macromolecular drugs have the advantage of being much more specific for therapeutic targets than traditional drugs (Hogset et al. 2004). Many of the intended targets for specific drug therapies are intracellular and therefore difficult for the

12 macromolecule to reach. This lends to the need for the development of technologies that allow for the delivery of such drugs into the target cells. For cancer therapy, specificity is very important in determining the therapeutic outcome.

The structure of macromolecular drugs prevents them from passing directly through the plasma membrane making it especially difficult for them to reach specific intracellular targets. These drugs usually enter the cell through the process of endocytosis. As the drug is engulfed by the cell it resides inside endocytic vesicles such as endosomes. Eventually the drug is degraded and loses its ability to cause a biological effect on the intended target. In order to obtain an effect from the drug there must be a means of escape from the endosomal membrane before degradation in lysosomes.

1.7 Photodynamic Therapy (PDT)

The idea of using light and chemicals to cause cytotoxic effects is not new. In

1900 Oscar Raab showed that the combination of acridine and specific wavelengths of light could prove lethal for a species of Paramecium. In modern PDT, light activates a photosensitizer, both non-toxic components individually, which in turn transfers energy to oxygen creating a reactive oxygen species (ROS). This process induces cytotoxic effects (Figure 6). Direct cytotoxicity, inflammatory and immunological responses and vascular damage contribute to the therapeutic effect of PDT. These mechanisms will be discussed at a later point (Dolmans et al. 2003).

The photosensitizer is the first component in PDT. The clinically relevant properties that make a suitable photosensitizer will be discussed. Photosensitizers for the clinical setting and the related metabolites should be non-toxic, and non-carcinogenic.

The photosensitizer should exhibit reliable activation by the appropriate light wavelength and minimal activation by other wavelengths. It is crucial that the photosensitizer accumulates in the proper tissues and localize in the desired targets. Depending on the intracellular localization, the damage caused by the photoreaction could induce various responses and mechanisms of cell death. The solubility of the photosensitizer will determine how easily it travels through the body. The absorption spectrum of the photosensitizer determines the wavelength of the incident light which affects the depth in tissue that can be treated. Lastly, a photosensitizer should not prevent or restrict other treatments that are used in conjunction with PDT such as radiotherapy, chemotherapy or surgery (Allison et al. 2004).

Photodynamic therapy has been approved by the FDA for the treatment of esophageal cancer and non-small cell lung cancer using the photosensitizing agent porfimer sodium (Photofrin®). Clinical trials are currently underway to evaluate PDT for cancers involving the brain, skin, prostate, cervix and peritoneal cavity (NCI 2011).

The photosensitizing agent can be administered intravenously or topically depending on the need. It is important to remember that the method used to administer the photosensitizer greatly affects the bio-distribution of the agent. The effects of PDT can then be regulated based on the timing of the light exposure and the bio-distribution of the molecule over time (Dolmans et al. 2003). The process of activation involves the absorption of light to raise the photosensitizer from the ground state to a very short, excited singlet state and then to a longer excited triplet state (Henderson and Dougherty

1992).

Figure 6. Excitation of the photosensitizer resulting in the production of free radicals in the presence of oxygen. (Dolmans et al. 2003)

The excited triplet state then undergoes a type I or II reaction (Figure 7). In type I reactions a hydrogen atom is transferred from the excited photosensitizer to a substrate forming radicals which then interact with oxygen to produce oxygenated products. A type II reaction involves the formation of singlet oxygen from the direct energy transfer between the excited photosensitizer and oxygen. This oxygen dependence prevents photosensitization in oxygen depleted regions. The rate of occurrence of type I and II reactions is dependent on several factors including the photosensitizer being used, the binding affinity of the photosensitizer to the substrate and the relative concentrations of the substrate and oxygen.

Figure 7. Type I and Type II reactions. (Dolmans et al. 2003)

The damage caused by the ROS is local due to its high reactivity and relatively short half- life of <0.04 µs. Studies have shown the radius of action of singlet oxygen is <0.02 µm.

The light exposure dose and dose rate in combination with the type of photosensitizer, its localization and the tissue oxygen concentration affect the extent of photodamage and resultant cytotoxicity (Moan and Berg 1991).

PDT is able to induce tumor damage through three primary mechanisms: direct cytotoxicity, vascular damage and activation of an immunological response (Dougherty

1998).

Direct cytotoxicity is a result of the damage caused by the generation of ROS.

The difficulty with this mechanism is that it is directly related to the localization of the photosensitizer. It has been shown that, in cases of intravenously administered

16 photosensitizer, cytotoxicity decreases with increasing distance between tumor cells and the vasculature (assuming a homogenous distribution of the sensitizer and readily available oxygen within the tumor). In order to minimize the rapid depletion of oxygen in the tumor, and thus the diminution of the direct cytotoxic effect, fractionation methods and/or low light fluence rates can be used to facilitate re-oxygenation (Messmann 1995).

Alternatively, PDT can cause a therapeutic effect by damaging the tumor vasculature. This in effect cuts off the nutrient supply to the tumor. Several studies have been published that indicate microvascular collapse, vasoconstriction and thrombus formation leading to hypoxia and ultimately inhibition of tumor growth (Henderson and

Fingar 1987) (Chen et al. 1996) (Dolmans 2002).

Lastly, PDT has been shown to cause an immunological response resulting in tumor damage. Post-PDT treatment analysis in the early 1990’s showed an influx of leukocytes and lymphocytes into the treatment region indicating an immunological response. Researchers have found that murine vaccinations prepared from the lysate of tumor cells treated with PDT are more effective than those prepared from the lysates of tumor cells treated with ionizing or ultraviolet radiation (Gollnick et al. 2002).

Tumors exhibit several properties that increase the preferential uptake and retention of photosensitizers compared with surrounding tissues including: poor lymphatic drainage, lower pH, and leaky vasculature (Pass 1993).

1.8 Aluminum Phthalocyanine Disulfonate

Aluminum Phthalocyanine Disulfonate [AlPcS(2a)] is a second generation photosensitizer used for PDT. The structure is shown in Figure 8. The central metal ion determines the photochemical properties of phthalocyanines.

Figure 8. Aluminum Phthalocyanine Disulfonate structure (Hogset et al. 2004).

For this reason it is important to use a metal that has a high triplet yield and fluorescence yield: both, zinc and aluminum exhibit these properties (Ambroz et al. 1991). AlPcS(2a) exhibits several advantages over early photosensitizers used in PDT including water solubility and its high absorbance in the red part of the visible spectrum (675nm) makes it ideally suited for in vivo use due to the high penetrance of red light in biological tissues.

The absorption spectrum for AlPcS(2a) is shown in Figure 9.

Figure 9. AlPcS(2a) absorption spectrum (Prasmickaite et al. 2001).

Light of this wavelength is able to penetrate almost twice as deep into tissue as the 630 nm light used to activate photofrin II (PII). Another advantage involves the relatively low absorbance in other regions of the solar spectrum, minimizing the risk of photoreaction in patients after treatment (Chan et al. 1997).

Localization of the photosensitizer to the tumor, specific location inside the cell and the ability to induce tissue damage are important characterisitics of photosensitizers.

Studies have shown that, as the degree of sulphonation increases, the ability to localize to the tumor increases but the ability to induce necrosis decreases (Berg et al. 1989). The

19 two sulfonate groups on the adjecent phthalate rings lend to the amphiphicity of

AlPcS(2a) allowing it to primarily localize in the membranes of endocytic vesicles (Berg and Moan 1994). The uptake properties of AlPcS(2a) determined in vitro and the tumor necrosis properties from in vivo studies have made AlPcS(2a) a possible clinical alternative to Photofrin (Witjes et al. 1996)

Precautions must be taken when working with AlPcS(2a). Although it is a relatively stable photosensitizer, it is important to avoid light which could induce damage to the photosensitizer. Also, long periods of storage can cause aggregation which reduces the efficacy of the photosensitizer (Prasmickaite et al. 2001).

1.9 Photochemical Internalization (PCI)

PCI is a technology that enhances the efficacy of several different classes of molecules by allowing for site specific drug delivery. PCI is based on studies that showed when photosensitized cells were exposed to light the photosensitizer was released into the cytosol due to permeabilization of the vesicle (Figure 10). Lysosomal enzyme activities were also observed in substantial amounts in the cytosol after exposure to light demonstrating that other molecules within the lysosomes are released into the cytosol

(Berg and Moan 1994).

This delivery mechanism is possible without causing excess damage to the cell and minimally affecting the biological activity of the released molecule. The photochemical damage to the enveloped molecule varies with its location relative to the photosensitizer. The photosensitizer accumulates in the endocytic membrane and the O2 molecules (singlet molecular oxygen) that are produced from the photochemical reaction

20 have a very short range of action. The specific mechanism that causes the endocytic membrane to rupture after photochemical excitation is not known. Belzacq et al. (2001) and Zavodnik et al. (2002) have shown for mitochondrial and erythrocyte membranes respectively that damage to membrane proteins may contribute more than oxidation of membrane lipids to the mechanism causing membrane rupture.

Figure 10. Photochemical internalization process. The drug “D” enters the cell by endocytosis. Light activation of the photosensitizer “S” disrupts the endosomal membrane releasing the drug into the cytosol thus preventing degredation in the lysosome. (Norum et al. 2009)

Another method of performing PCI has come to be known as “light first” PCI.

This is similar to the standard mode of PCI (Figure 10) with the alteration of performing the illumination and excitation of the photosensitizer before treatment with the molecule to be internalized. Again the mechanism of “light first” PCI is not completely understood but it has been hypothesized that new vesicles containing the macromolecule combine with vesicles that have been previously damaged by the photochemical reaction and the resultant vesicle has an imperfect structure that leaks the macromolecule into the cytosol

(Hogset et al. 2004). This mechanism is shown in Figure 11.

Figure 11. “Light first” PCI process. Light activation of the photosensitizer “S” disrutps endosomal membranes. Later, the drug “G” enters the cell by endocytosis and is released into the cytosol when previously damaged endosomes fuse with endosomes containing the drug. (Hogset et al. 2004)

Various studies have indicated the ability of PCI to induce biological effects due to the endosomal release of several different molecules including: ribozymes, plant protein toxins, oligodeoxynucleotides, immunotoxins, and genes using viral and non-viral vectors (Berg et al. 1999) (Selbo et al. 2000) (Hogset et al. 2000).

The optimal light dose for the PCI effect will vary depending upon the cell type.

Different cell types will exhibit varying levels of photosensitizer uptake, sensitivity to the cytotoxic effects produced by the photochemical reaction and different concentrations of the photosensitizer in the endosomal membranes (Hogset et al. 2004). In determining the optimal light dose, one must consider the desired end result. While increasing the light dose has been shown to increase the drug delivery effect, it also increases the cytotoxic effects of the photochemical treatment. For various cancer therapies in which the desired

22 goal is to kill all the cells in a specific area, PCI is able to produce a substantial effect at lower light doses than traditional PDT. This enables the therapeutic treatment of much deeper tissues than is possible with PDT. For treatments with a desired effect of treating target cells that are intermixed with normal cells and using a drug that acts on the target cells; the lower light doses used for PCI are able to aid the release of the drug while minimizing the cytotoxic effects of the photochemical treatment (Hogset et al. 2004).

Just as with any therapeutic modality, there are several advantages and disadvantages to PCI. Experiments have shown that PCI is effective at delivering molecules ranging from very small adenoviral particles to large macromolecules giving the technology great flexibility. By increasing the efficiency of specific drugs, PCI is able to maintain a similar biological effect while reducing the dose administered. This can significantly reduce the observed side effects of drug therapy. Another advantage of

PCI is its ability to damage non-dividing cells, such as resting malignant cells which directly affect the outcome of the cancer therapy. Traditional radiation therapy is far less efficient at killing quiescent cells. An obvious disadvantage is damage to the enveloped molecule as a result of the photochemical reaction. Lipid-like complexes will localize closer to the membrane containing the photosensitizer resulting in a higher probability of damage. The “light first” mode could be performed to induce the photochemical reactions prior to the introduction of the molecule thus potentially minimizing damage to molecules that localize near the photosensitizer.

1.10 Bleomycin

The chemotherapeutic agent used in this study is the glycopeptide antiobiotic bleomycin

(Figure 12). Although bleomycin (BLM) actually refers to a family of structurally similar compounds produced by various Streptomyces the chemotherapeutic forms consist mainly of bleomycin A2 and B2 (Barber et al. 1981). The several structures of bleomycin differ in the cationic side chain and at the bithiazole moiety.

In order to become activated and initiate DNA damage, the bleomycins require a reducing agent, a reduced metal and molecular oxygen. Several pathways have been proposed to explain the mechanism of damage to the DNA and they depend on the oxygen concentration (Strekowski and Wilson 2007). Hoehn et al. (2001) have proposed a double strand cleavage by a single BLM molecule.

In the clinical setting BLM has been approved by the FDA for use alone or in combination with other drugs as palliative treatment of Hodgkin lymphoma, squamous cell carcinoma of the head, neck and cervix, testicular cancer and to treat malignant pleural effusion (NCI Drug Information). BLM is used extensively in the research setting in the development of treatments for various other cancers.

Figure 12. Structure of bleomycins. (Barber et al. 1981)

1.11 Hyperthermia

The combination of brain tumor treatment modalities, such as photodynamic therapy [PDT], and hyperthermia have shown increased tumor control in a number of in vitro and animal studies (Hirschberg et al. 2004).

Treatments under hyperthermic conditions are already in clinical use. Exposure to hyperthermic conditions can cause cell death, inhibit repair of sub-lethal damage and increase the sensitivity of hypoxic cells to radiation. The extent of the effects of hyperthermia on tumor cells depends on the temperature and length of time of exposure

(Hirschberg et al. 2004). Tumor permeability to macromolecules has been shown to increase in the presence of heating.

Studies have shown that hyperthermia can greatly affect the ability of proteins and, in some cases, it can inhibit protein synthesis (Fuhr 1974) (Henle and Leeper 1979).

One example of this is the enhancement of bleomycin treatments by hyperthermia.

Studies have demonstrated that this enhancement is not due to increased bleomycin uptake in heat treated cells (Braun and Hahn 1975). Bleomycin hydrolase is an enzyme that degrades bleomycin and is present in higher concentrations in bleomycin resistant cells (Miyaki et al. 1975). Typically, bleomycin survival curves are biphasic showing an initial induction of resistance which decays following the removal of the bleomycin.

Hyperthermia is able to alter the function of the bleomycin hydrolase and effectively remove the bleomycin resistant phase in the survival curve. Heat treated cells therefore have a reduced bleomycin inactivation ability (Lin et al. 1983). The enhancement of the bleomycin cytotoxic effect by hyperthermia is well documented in several in vitro and in vivo studies (Magin et al. 1979) (Meyn et al. 1979) (Miyaki et al. 1975).

1.12 Scope of work

The purpose of this thesis research is to investigate the effect of combined

AlPcS(2a) – mediated PCI + HT on multicellular tumor spheroids. Earlier studies have shown a synergistic effect of PCI and bleomycin over PDT or bleomycin alone. In all cases, treatment efficacy will be evaluated by comparing the growth kinetics and viability of multicellular tumor spheroids after HT, AlPcS(2a) – mediated PCI and AlPcS(2a) – mediated PCI + HT treatments.

Hypothesis – A combined AlPcS(2a) – mediated PCI of bleomcyin + HT treatment will show greater tumor control than would be expected from the addition of the individual effects from HT and AlPcS(2a) – mediated PCI of bleomcyin.

CHAPTER 2

MATERIALS AND METHODS 2.1 Cell Lines

Experimental trials were performed on the F98 and MCF-7 cell lines at the

University of Nevada, Las Vegas. Both cell lines were cultured in Dulbecco’s Modified

Eagle’s Medium [DMEM] (Life Technologies, Carlsbad, CA) with high glucose [1X] and supplemented with 10% heat inactivated-fetal bovine serum [FBS], 25mM HEPES buffer and 5 mL Pen-Strep (10,000 Units/mL Penicillin and 10,000 µg/mL

Streptomycin). During experimentation the cell lines were maintained at 37ºC, 95% humidity and 5% CO2 in T-25 BD Falcon tissue culture vented cap flasks. The cells were sub-cultured using the following protocol. The cellular confluency was determined using light microscopy. The culture medium was then discarded and the cells were washed by adding 5 mL phosphate buffered saline [PBS] (Life Technologies, Carlsbad, CA) to the flask and then discarding the solution. One mL of 0.25% Trypsin-EDTA solution was added to the flask and allowed to sit for 5 minutes. This allows enough time for the proteolytic enzyme to aid the detachment of the cells from the flask. After the cell layer dispersed and the cells detached, 4 mL of DMEM was added to the flask and the cells were aspirated by gently pipetting. Depending on the confluence of the original cell suspension, 0.5 mL to 1.0 mL aliquots of the sub-cultured cell suspension were added to a new culture flask with additional DMEM to bring the total solution volume to 5 mL.

The cells were sub-cultured twice weekly.

2.2 Multicellular Tumor Spheroid Formation

The multicellular tumor spheroids were formed based on a rapid generation technique (Ivascu and Kubbies 2006). Immediately after sub-culturing, 0.1 mL of the cell suspension was added to 9.9 mL of IsoFlow Sheath Fluid (Beckman Coulter Inc., Brea,

CA) in a Coulter cup and the number of cells was counted in the Model Z2 Beckman

Coulter Particle Count and Size Analyzer. The cell count was used to determine the cellular concentration in cells/mL. DMEM was added to dilute the concentration of the cellular suspension to 32,000 cells per mL. Then 0.2 mL of the cellular suspension

(~6400 cells) was placed in each well of an ultra-low attachment round bottom 96 well plate coated with matrigel. The plate was then centrifuged at 1,000 x g for 10 minutes in a Heraus Megafuge 16 (Thermo Scientific, Waltham, MA). The resulting spheroids were incubated at 37ºC, 95% humidity and 5% CO2 for 48 hours. This allowed enough time for the 3D formation of the spheroid.

2.3 Photodynamic Therapy

After 48 hours the spheroids were visually inspected and then transferred to 35 x

10 mm petri dishes for treatment. AlPcS(2a) was used as a photosensitizer for all experiments. The stock AlPcS(2a) solution was diluted in DMEM to 1 µg/mL and 2 mL of the diluted AlPcS(2a) solution was added to each treatment group. This was done in minimal lighting to prevent premature activation of the AlPcS(2a). The treatment groups were then incubated at 37ºC, 95% humidity and 5% CO2. After 18 hours the treatment groups were washed with DMEM to remove excess AlPcS(2a) and placed in new medium. The treatment groups were then incubated for 4 hours to allow the

29 internalization of the photosensitizer and minimize the damage induced in the plasma membrane.

2.4 Photochemical Internalization

After washing the treatment groups to remove excess AlPcS(2a), the PCI groups were then treated using BLM. The stock BLM solution was diluted in DMEM to 0.25

µg/mL and 2 mL of the diluted BLM solution was added to the petri dishes containing the PCI groups. After 4 hours of incubation at 37ºC, 95% humidity and 5% CO2, the PCI groups were washed with DMEM to remove excess BLM and placed in new medium.

2.5 Hyperthermia

All groups treated with HT were placed in an oven at 42ºC for 40 minutes. All non-HT groups were placed in the same oven for 40 minutes at 37ºC. The laser therapy was performed while the spheroids were in the oven.

Laser therapy was performed using a photodiode laser (670 nm) coupled to a 200

µm diameter optical fiber containing a microlens. PCI and PDT groups were treated with radiant exposures of 1, 1.5 and 2.5 J/cm2 @ an irradiance of 5 mW/cm2. The treated spheroids were then transferred into 48 well plates coated with 0.25 mL agarose-

(2X)DMEM solution and 0.75 mL DMEM.

2.6 Measurements

Spheroid size was determined by taking the average of two orthogonal diameter measurements using a light microscope with a calibrated micrometer on the eyepiece.

F98 spheroid growth was recorded twice weekly for one month resulting in nine measurements. MCF-7 spheroid growth was recorded twice weekly for 5 measurements.

This was due to the fact that after the MCF-7 spheroids reached a certain size they became non-spheroidal and measurements were no longer feasible. This is explained in more detail in the MCF-7 results section. After each measurement, the growth medium was renewed by removing 0.4 mL of medium from each well and replacing it with 0.4 mL fresh DMEM.

2.7 Statistical analysis

Data analysis was performed in Microsoft Excel. Three trials were performed for each treatment group consisting of approximately 16 spheroids. The standard error was used in creating error bars. Statistical significance was determined using the Student’s T- test. In order to determine the degree of interaction between the combined PCI+HT treatment compared with the individual PCI and HT treatments the following equation was used (Drewinko et al. 1976).

(Eq. 1)

The numerator includes the product of the surviving fractions (SF) of the individual treatments separately and the denominator includes the surviving fraction of the combined treatment. A value of α = 1 indicates an additive effect. A value of α < 1 or α

> 1 indicates an antagonistic or synergistic effect, respectively.

CHAPTER 3

RESULTS

3.1 F98 Spheroid Viability

F98 rat glioma spheroid viability is shown in Figure 13. No cytotoxic effects were observed for spheroids exposed to temperatures of 42ºC suggesting that higher temperatures are required to produce hyperthermic effects in this cell line. The data illustrates the importance of radiant exposure on survival: increased radiant exposures resulted in decreased viability. For example, PCI spheroids (42ºC) exposed to 1 and 2.5 J cm-2 resulted in viabilities of 82 and 61%, respectively. The advantage of combining PCI

+ HT is evident from the data. For example, overall viabilities for the PCI + HT and PCI

(37oC) groups were 61 and 87%, respectively for a radiant exposure of 2.5 J cm-2. An alpha coefficient of 1.43 ± 0.04 at 30 days post-treatment indicates a synergistic effect between PCI and HT (42oC). The significant difference in viability between PDT + HT and PCI + HT groups (89 vs. 60%) suggests that the observed effect was due to PCI.

Figure 13. F98 spheroid viability as a function of days post-treatment. Each data point corresponds to the mean of three experiments (16 spheroids per experiment) and error bars denote standard errors.

3.2 F98 Spheroid Growth Kinetics

As illustrated in Figure 14, the growth kinetics of spheroids subjected to 42ºC were not statistically different from controls: HT treatment resulted in only a 5% reduction in volume at the conclusion of the 30-day observation period. The data presented in Figure 15 show that there were no statistically significant differences in growth kinetics between PDT and PCI at low radiant exposures. This is in good agreement with results obtained from human ACBT spheroids and therefore provides the rationale for using higher exposures in this cell line. A slight, but statistically significant

PCI effect was observed beyond 19 days post-treatment (Figure 16). The data presented in Figure 17, show that the combined PCI + HT treatment produced a statistically significant growth delay compared to: (1) either treatment modality alone, and (2) PDT +

HT. The combined PCI+HT treatment at a radiant exposure of 2.5 J/cm2 and a

33 temperature of 42 ºC resulted in a 31% reduction in size at 30 days versus control and

HT-only and reductions of 26 and 28% compared to PCI-only and PDT+HT groups.

Figure 14. Growth kinetics of F98 spheroids subjected to HT. Each data point represents the mean of three experiments and error bars denote standard errors. The HT effect was not statistically significant, p > 0.05.

Figure 15. Growth kinetics of F98 spheroids. Each data point corresponds to the mean of three experiments and error bars denote standard errors. The effects were not statistically significant, p>0.05 for both PDT vs. PCI at 1 and 1.5 J cm-2.

Figure 16. Growth kinetics of F98 spheroids subjected to PDT and PCI at physiological temperatures. Each data point corresponds to the mean of three experiments and error bars denote standard errors. The PCI effect was statistically significant, p ≤ 0.05.

Figure 17. Growth kinetics of F98 spheroids subjected to combination treatments. Each data point corresponds to the mean of three experiments and error bars denote standard errors. The effects were statistically significant, p < 0.01 and p < 0.001 for 37C PCI vs. 42C PCI and 42C PDT vs. 42C PCI respectively.

3.3 MCF-7 Spheroid Viability

The MCF-7 spheroids elongated and became non-spheroidal at approximately 2.5 weeks and measurements were therefore terminated. Greater cortical cell proliferation formed regionalized buds that lengthened in the spheroids. Eventually this resulted in a luminal arrangement similar to those seen in breast glands (Amaral et al. 2010). Figures

18 and 19 show the process of spheroid formation with cortical budding and the elongation of the spheroid, respectively.

Figure 18. MCF-7 spheroid formation. (Amaral et al. 2010)

Figure 19. MCF-7 spheroid elongation. (Amaral et al. 2010)

MCF-7 spheroid viability is shown in Figure 20. No cytotoxic effects were observed for spheroids exposed to temperatures of 42oC suggesting that higher temperatures are required to produce hyperthermic effects in this cell line. Increased radiant exposures resulted in decreased viability. For example, PCI spheroids (42oC) exposed to 1 and 2.5 J cm-2 resulted in viabilities of 97 and 82%, respectively. Again, the advantage of combining PCI + HT is evident from the data. For example, overall viabilities for the PCI + HT and PCI (37oC) groups were 82 and 93%, respectively. The calculated alpha coefficient 1.14 ± 0.01 indicates only a slight synergistic effect between

PCI and HT (42oC). In comparison, a higher degree of synergism was observed for the

F98 spheroids (α = 1.31 ± 0.02 at 16 days). The significant difference in viability between PDT + HT and PCI + HT groups (98 vs. 82%) suggests that the observed effect was due to PCI.

Figure 20. MCF-7 spheroid viability as a function of days post-treatment. Each data point corresponds to the mean of three experiments (16 spheroids per experiment) and error bars denote standard errors.

3.4 MCF-7 Spheroid Growth Kinetics

The degree of growth impairment of surviving MCF-7 spheroids was sensitively dependent on the treatment administered. As shown in Figure 21, temperatures of 42ºC did not result in statistically significant growth impairment. The data is in good agreement with those obtained for the F98 spheroids (Figure 14) and suggest that neither cell line is susceptible to HT at this temperature. Although a statistically significant difference in growth kinetics was observed between PDT and PCI groups at low radiant

38 exposures (Figure 22), the two exposures did not result in significant growth differences within either PDT- or PCI-exposed spheroids. The highest radiant exposure used (2.5 J cm-2) resulted in the greatest growth inhibition (Figure 23). This data also show a statistically significant difference in the growth kinetics of PCI and PDT treated spheroids at 37ºC. The effects of combined PCI and HT are illustrated in Figure 23. The

PCI treatment at a radiant exposure of 2.5 J cm-2 resulted in a 25% reduction in size at 16 days versus control. In comparison the HT treatment resulted in a 4% reduction in size 16 days post-treatment (Figure 21). The combined PCI+HT treatment at a radiant exposure of 2.5 J cm-2 and a temperature of 42 ºC resulted in a 31% reduction in size at 16 days versus control. A statistically significant difference between PCI + HT and PDT + HT combination treatments was observed.

Figure 21. Growth kinetics of MCF-7 spheroids subjected to HT. Each data point represents the mean of three experiments and error bars denote standard errors. The HT effect was not statistically significant, p > 0.05.

Figure 22. Growth kinetics of MCF-7 spheroids. Each data point represents the mean of three experiments and error bars denote standard errors. The effects were statiscally significant, p<0.001 for both PDT vs. PCI at 1 and 1.5 J cm-2.

Figure 23. Growth kinetics of MCF-7 Spheroids subjected to PDT and PCI at physiological temperatures. Each data point corresponds to the mean of three experiments and error bars denote standard errors. The PCI effect was statistically significant, p < 0.05.

Figure 24. Growth kinetics of MCF-7 spheroids subjected to combination treatments. Each data point represents the mean of three experiments and error bars denote standard errors. The effects were statistically significant, p < 0.05 and p < 0.01 for 37C PCI vs. 42C PCI and 42C PDT vs. 42C PCI respectively.

CHAPTER 4

DISCUSSION

Photochemical internalization is a novel technology that is well documented and has been shown to enhance the effect of chemotherapeutics, cytotoxic targeting conjugates, gene-based therapeutics and nano-medicines by aiding the endosomal release of the macromolecule into the cytosol. Advantages of PCI include preferential accumulation of photosensitizers in malignant tissues, confinement of the treatment to tissues illuminated by light of a specific wavelength and the ability to deliver a wide range of macromolecules to specific targets. PCI has also been used to overcome induced drug resistance in therapeutic regimes and to avoid resistance mechanisms in multi-drug resistant (MDR) cancer cells (Selbo et al. 2006)

Clinical trials involving Amphinex-mediated PCI of bleomycin have shown complete clinical regression and high tumor to normal tissue specificity for patients with local recurrence of advanced/metastatic, cutaneous or sub-cutaneous malignancies (Selbo et al. 2010). This is most likely due to the accumulation of the photosensitizer in the endosomal membranes and the subsequent photochemical damage to those membranes releasing the sequestered bleomycin .

The optimization of PCI involves several factors including: choice of photosensitizer and macromolecule, light irradiance and radiant exposure. Different cell lines also have varying sensitivity to bleomycin.

The parameters used in this study were previously optimized for the ACBT human glioma cell line. While both studies showed a synergistic effect for the combined

PCI and HT treatment at 2.5 J cm-2 and 42ºC, a higher degree of synergism was observed

42 in the treatment of the ACBT spheroids. This is likely due to the fact that the treatment parameters were optimized for this cell line. The ACBT human glioma cells and F98 rat glioma cells reacted in a similar manner to low levels of radiant exposure (< 1.5 J cm-2)

(Figure 15) while the MCF-7 spheroids varied in this respect (Figure 22).

Comparing the relative damage from the PCI effect in the F98 and MCF-7 spheroids we see a much larger PCI effect on the MCF-7 spheroids. From the current experiments, the MCF-7 spheroids were observed to form larger periphery regions of proliferating cells. Bleomycin studies have shown that the mechanism of DNA degradation is metal ion and oxygen dependent. These dependencies result in greater cytotoxicity toward oxygenated cells compared with hypoxic cells (Teicher et al. 1981), thus causing a greater PCI effect in the MCF-7 spheroids due to a higher ratio of oxygenated cells to hypoxic cells.

The relative damage from the HT effect was not statistically significant in either cell line. The observed heat resistance is likely due to endogenous heat shock proteins

(Hsp’s). Heat induces the accumulation of Hsp’s in the cytoplasm. These proteins aid in the prevention of denaturation through various mechanisms including re-folding proteins that have undergone conformal changes. Research has shown that cells with higher expression of Hsp70 demonstrated increased cellular proliferation. This is probably due to the effective shortening of G0/G1 and S phases of the cell cycle by Hsp70 (Barnes et al.

2001). The MCF-7 spheroids increased in size by a factor of 10 in approximately 9 days compared with the F98 spheroids which had a similar increase in approximately 23 days possibly indicating higher levels of Hsp70’s in the MCF-7 spheroids.

The combined PCI+HT treatment showed a similar percent volume reduction of approximately 31% for both the F98 and MCF-7 spheroids versus control. For both cell lines the treatment demonstrated a synergistic effect that is supported by previous studies.

There are several processes that most likely contribute to this synergistic effect.

First, when cells are subjected to hyperthermia it induces the accumulation of

Hsp70’s. The accumulation of Hsp70’s allows the cell to continue to function by re- folding heat damaged proteins. One particular protein that relates to this study is the

Mre11 protein. Mre11 is a subunit of the MRN complex that is involved in signaling pathways for DNA double strand break (DSB) repair. This type of damage is produced by bleomycin. Hyperthermia denatures Mre11 proteins which then translocate from the nucleic region to the cytosol in an effort to associate with the Hsp70’s and re-fold

(Dynlacht et al. 2011). Treatment with bleomycin and hyperthermia (40ºC) in murine L cells has been shown to suppress the accumulation of Hsp70’s by up to 38% compared to

Hsp70’s levels induced by hyperthermia alone (Jin et al. 2002). In this situation the damaged Mre11 proteins are less readily repaired due to a suppression of Hsp70’s, and cannot initiate the DSB repair pathways. Thus, the cytotoxicity of the bleomcyin is enhanced by hyperthermia.

Second, studies have shown that bleomycin treatments traditionally result in greater cytotoxicity to oxygenated cells compared to hypoxic cells. Teicher et al. have shown that this preferential cytotoxicity is absent in hyperthermic treatments (42-43ºC).

Evidence supports that this non-preferential cytotoxicity is due to the ability of the bleomycin-iron complex to cleave DNA by an oxygen independent mechanism (Ajmera et al. 1986). It has also been proposed that the damage produced by the oxygen

44 independent mechanism is normally repaired at standard temperatures but becomes fixed by the heat treatment (Meyn et al. 1979).

Lastly, as discussed earlier, hyperthermia denatures the bleomycin hydrolase enzyme hindering the bleomycin inactivation ability of the cell.

The reduced ability of the cells to repair DSB’s and the heat–induced damage fixation and resistance reduction likely work in concert to produce the observed synergistic effect.

This is evidenced by the present results. The combined treatments resulted in a higher degree of synergism at 16 days for the F98 spheroids (alpha coefficient of 1.31 ±

0.02 compared to 1.14 ± 0.01 for the MCF-7 spheroids). With a smaller ratio of oxygenated to hypoxic cells the F98 spheroids showed a smaller PCI effect while the

MCF-7 spheroids with a larger ratio of oxygenated to hypoxic cells, exhibited a larger

PCI effect due to the preferential cytotoxicity towards oxygenated cells at standard temperatures. When the temperature was increased to 42ºC the preferential cytotoxicity disappeared and both F98 and MCF-7 spheroids exhibited a similar effect.

Although the combined PCI+HT treatment had an overall similar effect on the

F98 and MCF-7 spheroids, the proliferation gradient likely affected the extent and mechanism of the damage from the individual treatments.

CHAPTER 5

CONCLUSIONS

The overall objective of this work was to investigate the effect of a combined PCI and HT treatment on multicellular spheroids of two different cell lines. The hyperthermic treatment (42ºC) showed no decrease in spheroid viability for either cell line. There were no significant differences between PCI and PDT treatments at physiological temperatures and radiant exposures < 1.5 J cm-2 for F98 rat glioma spheroids. This is in agreement with the previous study of ACBT human glioma spheroids. The MCF-7 spheroids, however, showed a significant difference between PCI and PDT treatments at physiological and hyperthermic temperatures (37 and 42ºC) for all radiant exposures (1,

1.5 and 2.5 Jcm-2). The combined PCI and HT effect was shown to be statistically significant and synergistic for both cell lines, with a greater degree of synergism present in the F98 cell line (alpha coefficient of 1.42 ± 0.04 vs. 1.14 ± 0.01).

Further studies can be performed to better understand the processes that result in the observed synergism. Experiments that investigate the accumulation of Hsp’s and bleomycin hydrolase specific to these cell lines could lead to a better understanding of their resistances. With a better understanding of the underlying mechanisms, treatment parameters can then be optimized to the specific cell resulting in the greatest tumor control. In vivo studies can be performed to evaluate the effectiveness of the treatment and understand the effects of vasculature on the treatment. Treatment sequence is another area of possible exploration for these specific cell lines. Sequence optimization could be

46 performed by investigating the differences in cytotoxic effects of “light first” PCI vs.

“light after” PCI or hyperthermia prior to or after BLM administration.

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VITA

Graduate College University of Nevada, Las Vegas

Aaron Andersen

Local Address: 1050 Whitney Ranch Drive Apt. 1511 Henderson, NV 89014

Degrees: Bachelor of Science, Physics Utah State University 2010

Dissertation/Thesis Title: AlPcS(2a) – Mediated Photochemical Internalization of

Bleomycin and Concurrent Hyperthermia of Multicellular Tumor Spheroids.

Dissertation/Thesis Examination Committee:

Chairperson, Dr. Steen J. Madsen, Ph.D

Committee Member, Dr. Ralf Sudowe, Ph.D

Committee Member, Dr. Gary Cerefice, Ph.D

Graduate Faculty Representative, Daniel L. Young, DPT