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Impact of Tumor VEGF Expression on the Response to Anti-Cancer Therapies

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Impact of Tumor VEGF Expression on the Response to Anti-Cancer Therapies
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NORRIS, CHRISTINA M. ( Author, Primary )
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2008

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Angiogenesis ( jstor )
Blood vessels ( jstor )
Cancer ( jstor )
Cell growth ( jstor )
Cell lines ( jstor )
Dosage ( jstor )
Hypoxia ( jstor )
In vitro fertilization ( jstor )
Receptors ( jstor )
Tumors ( jstor )

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University of Florida
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University of Florida
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Copyright Christina M. Norris. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2016

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1 IMPACT OF TUMOR VASCULAR ENDOTHELI AL GROWTH FACTOR EXPRESSION ON THE RESPONSE TO ANTI-CANCER THERAPIES By CHRISTINA M. NORRIS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by CHRISTINA M. NORRIS

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3 In loving memory of my gra ndmother, Anneliese Endrikat

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4 ACKNOWLEDGMENTS I would like to acknowledge my parents a nd, most importantly, my husband, Juan, for always encouraging me to follow my own path. Without their utmost support, this project would have been much more difficult. I would also like to extend my gratitude to my advisor, Dietmar Siemann, for believing in my abilities and constantly pushing me to higher levels. In addition, I would like to thank my committee members (Dr. Naser Chegini, Dr. Maria Grant, and Dr. Kathleen Shiverick) for their encouragement and advice. I would also like to acknowledge other labora tory members, especially, Dr. Wenyin Shi, Sharon Lepler, Chris Pampo, and Lori Rice, for th eir help with experimental techniques/design and invaluable advice. Finally, I wish to sa y thank you to W.D. Brazelle for his consistent willingness to preview any manuscript drafts and presentations. Without the input of all the people listed above, I am afraid this di ssertation may never have been written.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............10 CHAPTER 1 INTRODUCTION..................................................................................................................12 Tumor Microe nvironments.....................................................................................................12 Angiogenesis................................................................................................................... ........13 Angiogenic Cascade........................................................................................................13 Aniogenic Factors............................................................................................................14 VEGF........................................................................................................................... ....15 VEGF Receptors..............................................................................................................18 Pre-clinical studies...................................................................................................20 Clinical studies.........................................................................................................21 Vascular Targeting Therapies.................................................................................................21 Research Outline and Significance.........................................................................................22 2 CREATION OF CLONAL CELL LINES.............................................................................27 Introduction................................................................................................................... ..........27 Materials and Methods.......................................................................................................... .28 Generation of Stable Cell Lines by rAAV Infection.......................................................28 Angiogenesis Factor Expression.....................................................................................29 In Vitro Cell Growth.......................................................................................................29 Animals and Tumor Models............................................................................................29 In Vivo Growth Rate.......................................................................................................29 Results........................................................................................................................ .............30 Discussion..................................................................................................................... ..........31 3 PHYSIOLOGICAL CHARACTER IZATION OF TUMORS...............................................39 Introduction................................................................................................................... ..........39 Methods........................................................................................................................ ..........41 Intradermal Angiogenesis Assay.....................................................................................41 Vessel Density.................................................................................................................42 Assessment of Functional Vessels/Perfusion..................................................................42 Evidence of Hypoxia.......................................................................................................42

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6 Necrotic Fraction.............................................................................................................43 Results........................................................................................................................ .............43 Discussion..................................................................................................................... ..........45 4 RESPONSE OF CLONAL CELL LINES TO RADIATION................................................52 Introduction................................................................................................................... ..........52 Tumor Microenvironment...............................................................................................52 Radiation’s Mechanism of Action...................................................................................53 Fractionation Schedules...................................................................................................54 Repair.......................................................................................................................54 Repopulation............................................................................................................55 Redistribution...........................................................................................................55 Reoxygenation..........................................................................................................56 Role of VEGF in Radiation Response.............................................................................56 Materials and Methods.......................................................................................................... .58 Cell Culture................................................................................................................... ..58 In Vitro Clonogenic Cell Survival Assay........................................................................58 Animals and tumor models..............................................................................................59 Tumor Growth Delay Assay............................................................................................59 Tumor Dissociation.........................................................................................................59 Hypoxic Fraction.............................................................................................................60 Results........................................................................................................................ .............60 Discussion..................................................................................................................... ..........62 5 RESPONSE OF CLONAL CELL LINE S TO VASCULAR TARGETING THERAPIES...................................................................................................................... .....71 Introduction................................................................................................................... ..........71 Antiangiogenics...............................................................................................................72 Vascular Disrupting Agents............................................................................................74 Materials and Methods.......................................................................................................... .75 In Vitro Clonogenic Cell Survival Assay........................................................................75 Tumor growth delay assay...............................................................................................75 Results........................................................................................................................ .............75 Discussion..................................................................................................................... ..........77 Antiangiogenic Agents.......................................................................................................... .80 Vascular Disrupting Agents....................................................................................................80 6 SUMMARY........................................................................................................................ ....86 LIST OF REFERENCES............................................................................................................. ..93 BIOGRAPHICAL SKETCH.......................................................................................................108

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7 LIST OF TABLES Table page 1-1 Angiogenesis is governed by a variety of promoters and inhibitors, of which several are listed below............................................................................................................... ...25 5-1 Antiangiogenic agents and va scular disrupting agents are two subclasses that fall under the general heading of va scular targeting therapies. Representative agents and their mode of action are indi cated in the table below........................................................80

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8 LIST OF FIGURES Figure page 1-1 The steps of the angiogenic cascade invol ve vasodilation and increased permeability. Endothelial cells can then proliferate and mi grate towards the pro-angiogenic source. ............................................................................................................................... .............24 1-2 Schematic displaying the human VEGF-A isoforms following mRNA alternative splicing....................................................................................................................... ........26 2-1 Diagram of the rAAV vector that co des for the expression of the human VEGF165 gene and also contains a chicken -actin promoter and neomycin resistance gene...........34 2-2 Human VEGF expression levels as assayed by ELISA of the (A) HT29 and (B) SCCVII clonal cell lines....................................................................................................35 2-3 Cell growth curve of the HT29 (A) and S CCVII (B) clonal cell lines as a function of time after plating............................................................................................................. ...36 2-4 The number of days, post-injecti on, for tumors to appear (~ 200 mm3). 1 x 106 HT29 tumor cells (A) or 1 x 105 SCCVII tumor cells (B) were injected i.m. in the left hind leg of nude or C3H mice, respectively...............................................................................37 2-5 In vivo growth rate of the tumors deri ved from the parental and clonal cell lines............38 3-1 Angiogenic potential of the parental and clonal cell lines as assessed by the intradermal assay.............................................................................................................. .47 3-2 HT29 tumors (A) and SCCVII tumors (B) re sulting from the clonal and parental cell lines were stained for the CD31 antigen............................................................................48 3-3 HT29 parental and clonal cell tumors we re stained with for the hypoxia marker, EF5 (red), and had functional vessels la beled with Hoechst 33342 (blue)...............................49 3-4 SCCVII parental and clonal cell tumors were stained with for the hypoxia marker, EF5 (red), and had functional vessels labeled with Hoechst 33342 (blue)........................50 3-5 Paraffin sections of the HT29 tumors (A ) and SCCVII tumors (B) resulting from the clonal and parental cell li nes were H&E stained...............................................................51 4-1 Schematic showing the formation of DNA lesions following energy transfer from ionizing radiation. The biol ogical lesions may then be “fixed” with oxygen making it difficult for the cell to repair this damage [adapted from (Hall, 1994)].........................65 4-2 Survival curves for i onizing radiation under anoxic ( ) and normoxic ( ) conditions. The oxygen enhancement ration (OER) shown in this figure is 2.5..................................66

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9 4-3 Clonogenic cell survival curv e demonstrating that the in vitro sensitivity to single doses of radiation is similar among the pare ntal and clonal cell lines of the HT29 (A) and SCCVII (B) tumor types.............................................................................................67 4-4 Response to a single 10 Gy dose of radi ation of tumors from HT29 (A) and SCCVII (B) parental and clonal cell lines. Treatme nt was administered when the tumors reached an approximate size of 200 mm3..........................................................................68 4-5 Response to a fractionated schedule 2 Gy/day given Monday through Friday for 2 weeks for the HT29............................................................................................................69 4-6 Paired survival curves showing the cell surv ival of anoxic and normoxic tumors following irradiation wi th a single dose.............................................................................70 5-1 Schematic representing the different targ eting approaches of vascular targeting therapies...................................................................................................................... .......79 5-2 Clonogenic cell survival curv e demonstrating that the in vitro sensitivity to a range of ZD6474 doses is similar among the parent al and clonal cell lin es of the HT29 (A) and SCCVII (B) tumor types.............................................................................................81 5-3 Clonogenic cell survival curv e demonstrating that the in vitro sensitivity to a range of ZD6126 doses is similar among the parent al and clonal cell lin es of the HT29 (A) and SCCVII (B) tumor types.............................................................................................82 5-4 Ability of ZD6474 to modulate the angioge nic potential of the parental and high VEGF clonal cell line, as assesse d by the intradermal assay.............................................83 5-5 Response of tumors from HT29 (A) and SCCV II (B) parental and clonal cell lines to a treatment of 50 mg/kg ZD6474.......................................................................................84 5-6 Response of tumors from HT29 (A) and SCCV II (B) parental and clonal cell lines to a treatment of 100 mg/kg ZD6126.....................................................................................85

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10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IMPACT OF TUMOR VASCULAR ENDOTHELI AL GROWTH FACTOR EXPRESSION ON THE RESPONSE TO ANTI-CANCER THERAPIES By CHRISTINA M. NORRIS December 2006 Chair: Dietmar W. Siemann Major Department: Medical Scie nces-Physiology and Pharamcology Angiogenesis is a complex process that has be en implicated in a variety of pathological diseases, including cancer. It is widely accepted that tumors must elicit an angiogenic response for survival, growth, and metastases. Vascular endothelial growth f actor (VEGF) is an important pro-angiogenic factor that has be en found to be upregulated in a wide variety of tumor types. Clonal cell lines that express different levels of VEGF were created from two tumor types, a human colorectal carcinoma (HT29) and a murine squamous cell carcinoma (SCCVII), to investigate the relationship between this pro-angi ogenic factor and tumor response. Three clones were then chosen for each tumor model: the first clone expressed VEGF at a level comparable to the parental (non-infected) cell li ne, the second expressed VEGF at an intermediate level, and the third clone expressed VEGF at a high level. Th e in vitro growth kinetics of the clonal cell lines did not significantly differ from that of the parent al cell line. However, the in vivo growth rate of the HT29 tumor model increased with increas ing VEGF expression. P hysiological differences were also noted in both tumor models; the tumors arising from the clonal cell lines had distinctively different levels of vascularity, perfusion, and hypoxia. The clonal cell lines were then used to evalua te tumor response to ra diation and vascular targeting agents. In vitro, no significant differe nce was observed in the response to radiation,

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11 ZD6474, an antiangiogenic agent, and ZD6126, a vascular disrupti ng agent, between the clonal and parental cell lines. The results showed that tumors from the highest expressing VEGF clonal cell line demonstrated an enhan ced response to radiation. The HT29 tumors arising from the high expressing VEGF clonal cell line also responde d better to both vascul ar targeting agents. The SCCVII model did not appear to be sens itive to ZD6474, perhaps due to its high growth rate, and there was no difference in the growth delay among the clonal cell lines. There was a trend towards increased efficacy with increa sing VEGF expression when the SCCVII tumorbearing mice were treated with ZD6126. The resu lts from these studies suggest that VEGF expression may play an important role in tumor response to anti-cancer therapies.

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12 CHAPTER 1 INTRODUCTION Cancer is a disease that affects millions of pe ople in the United States alone. According to the American Cancer Society, it is predicted th at 2006 will see more than 1.3 million new cancer patients. The current modalitie s for its treatment consist of surgery, radiation therapy, and chemotherapy. However, all of these treatment options have drawbacks and despite the progress that has been made in the past few decades, approximately 40% of cancer patients will fail therapy (National Canc er Institute, 2006). Tumor Microenvironments It is thought that as a tumor grows, the growth of new blood vessels lags behind the expanding tumor (Tannock, 1970). In addition, the vessels that are present within the tumor are highly irregular, chaotic structur es that may contain dead ends and arteriovenous shunts (Brown, 1999). Together, the lack of an efficient vasc ulature creates tumor microenvironments where cells residing in these regions experience de creased nutrient delivery and impaired uptake of metabolic waste products (Vaupel et al , 1989). As a result, tumor cells in these microenvironments may exist in states of lowe red proliferation, or even quiescence (Tannock, 1968). The impact of these tumor microenvironments on conventional therapies has been shown to be negative. Many conventiona l chemotherapeutic agents target cancer cells due to their high rate of proliferation. Thus, fo r cells that are not undergoing mito sis, like those residing in the microenvironments, these agents have little effect . Another concern with cells located in these microregions is poor drug penetration resul ting in less tumor cell killing (Tannock, 2001). The existence of microenvironments can also affect the radiosensitivity of the cells residing in these regions. Sin ce radiation therapy is also more effective against rapidly

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13 proliferating cells, cells in these microregions ar e not as extensively affected as are tumor cells located in regions containing nor mal amounts of nutrients. In a ddition, cells surviving in areas of decreased oxygen tensions (hypoxia) may also not be as sensitive to radiation (Hockel & Vaupel, 2001). Hypoxic cells are typi cally found at a distance of 100-150 m from the nearest functional vessel in tumors (Horsman, 1998). Fo llowing exposure to radi ation, available oxygen can chemically react with the resulting cellula r biological lesions, thus causing the damage to become more difficult for the cell to repa ir (Brown, 1999). In cells where the oxygen concentration is very low this phenomenon occurs less frequen tly, allowing for much of the damage to be repaired (Vaupel, 2004). Angiogenesis Angiogenesis, the process of new blood vessel fo rmation from the existing vasculature, is a tightly regulated process that occurs mainly during development (Bellamy, 2002). In adults, it is instrumental in wound healing and reproduc tive functions in females (Folkman, 1995). Endothelial cells are usually quiescent in nor mal tissues, dividing approximately once every seven years. However, in a variety of pathologi cal disorders, including cancer, the growth rate of endothelial cells can be rapi dly accelerated, with divisions o ccurring as fast as once a week (Hobson & Denekamp, 1984). This “angiogenic switch” has been shown to be vital to the growth of a tumor beyond a diameter of approximately 1-2 mm (Bergers & Benjamin, 2003). Angiogenic Cascade The sequence of events in the angiogenic proces s is orchestrated by a variety of molecules; the full extent of this process is yet to be completely understood [Fig. 1-1]. It is believed that the earliest stages of the cascade can be defined by vasodilation and increased permeability (Bergers & Benjamin, 2003). Then, endothelial cells that have been stimul ated by a pro-angiogenic signal can proliferate and migrate towards the stimul us source (Papetti & Herman, 2002). Following

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14 migration, the endothelial cells can adhere to one another, create a lumen, and become a functional vessel (Drake & Little , 1999). The final steps involve basement membrane formation and maturation of the blood vessels (Jain, 2003). Aniogenic Factors Angiogenesis is a balancing act between proand anti-angiogenic molecules [Table 1-1] (Grunstein et al , 1999). In order to stimulate angiogenesi s, the angiogenic scale must be tipped towards the pro-angiogenic side, and this can be caused by an incr ease in pro-angiogenic factor expression, decreased anti-angioge nic factor expression, or a co mbination of both. Endogenous anti-angiogenic factors include th rombospondin and a variety of inhi bitors derived from type IV collagen and type XVIII collagen, such as arrest in, endostatin, and tumstatin (Kalluri, 2003). The positive regulators include fibr oblast growth factor (FGF), pl acenta derived growth factor (PDGF), the angiopoietins (Ang), and vascul ar endothelial grow th factor (VEGF). bFGF. The fibroblast growth factors were di scovered in the 1970s and two similar molecules were found that shared a 55% identity : acidic and basic fibroblast growth factors (aFGF and bFGF) (Slavin, 1995). These proteins were found to be potent mitogens for endothelial cells as well as ot her cell types (Ferrara, 2002). Th e two molecules do not contain a conventional secretory signal peptide, but they do have a relatively high affinity for heparin (Cronauer et al , 2003). It is thought that these proteins ar e sequestered in the extracellular matrix (ECM) until their release from hepari n-sulfate proteoglycans (Vlodavsky et al , 1987). Since aFGF is primarily localized to neural tissues it is not thought to be a major player in tumor angiogenesis (Friesel & Maciag, 1995). However, bFGF has a ubiquitous distribution and has been implicated in tumor pathology (Czubayko et al , 1997). PDGF. PDGF is also a potent mitogen for bot h normal and tumor cells (Heldin, 2004). PDGF is a dimer that can consist in the follo wing forms: AA, BB, AB, CC, or DD (Fredriksson

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15 et al , 2004). The binding of this ligand to its receptor has been found to stimulate cell proliferation, cell migration, and angiogenesis (Ta llquist & Kazlauskas, 2004). The activation of the PDGF signaling pathway has also been shown to inhibit apoptotic pathways in some cells as well, leading to its classification as a survival factor (Claesson-Welsh, 1994). Angiopoietins. This family of extracellular ligands specifically binds to an endothelial cell specific receptor tyrosine ki nase termed Tie-2 (Ziegler et al , 1993). Although Ang-1 and Ang-2 are similar in structure (60% identity at the pe ptide level), these molecules elicit different responses after binding to the Tie-2 receptor (Tait & Jones, 2004). Ang-1 acts as an agonist and activates the Tie-2 signali ng pathways (Witzenbichler et al , 1998). Ang-2 acts mainly as an antagonist in the endothelium by blocking A ng-1 dependent activa tion (Maisonpierre et al , 1997). However, in some instances Ang-2 ma y act as an agonist as well (Stratmann et al , 1998). During angiogenesis, both molecules play importa nt roles. Ang-2 is thought to act at the beginning of the angiogenic cascade as a destabilizing signal that is seen prio r to vessel sprouting (Zhang et al , 2003). On the opposite end of the cascad e, Ang-1 acts as a maturation factor, promoting the recruitment of pericytes and smooth muscle cells (Suri et al , 1996). VEGF VEGF is considered to be the most importa nt pro-angiogenic signal in tumor pathology. However, VEGF is actually a family of molecule s, some of which are more important to tumor growth than others. The VEGF family is comprise d of seven structurally related growth factors: VEGF-A, VEGF-B, VEGF-C (VEGF-related protein), VEGF-D, VEGF-E, VEGF-F, and placenta growth factor (PlGF). These genes shar e a common eight cysteine residue motif which is found in the VEGF homology domain (Roy et al , 2006). PlGF. The PlGF gene shares 37% homology with VEGF-A and encodes for four different isoforms via alternative splicing. PlGF was firs t identified in the placenta, but it has since been

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16 found in the heart and lungs as well (Tjwa et al , 2003). PlGF-1 and PlGF-3 do not bind heparin and are therefore diffusible molecules, while Pl GF-2 and PlGF-4 have heparin binding domains. The results from in vitro studies regarding th e role of PlGF in angiogenesis have been inconsistent. Some studies have shown that Pl GF failed to promote angiogenesis after binding with VEGFR1 (Park et al , 1994) while the opposite has been found in other studies (Ziche et al , 1997;Nagy et al , 2003). Further studies of this molecule will be needed to elucidate its role. VEGF-E and VEGF-F. The newest family members, VEGF-E and VEGF-F, were discovered in the parapoxvirus genome (Lyttle et al , 1994) and snake venom (Junqueira, I et al , 2001), respectively. Both family members are able to bind to VEGFR2 and VEGF-E is also able to bind to Nrp-1 (Takahashi & Shibuya, 2005). VEGF-E ca n induce a strong angiogenic response in vivo, whereas VEGF-F a ppears to specifically block VEGF-A165 activity (Yamazaki et al , 2005). VEGF-D. VEGF-D has been indentified as a c-fo s-induced gene that contains a cysteine rich C-terminus domain that is similar to VEGF-C (Achen et al , 1998). The processed form of this glycoprotein can bind to eith er VEGFR-2 or VEGFR-3 (Lohela et al , 2003). Hence, this secreted protein has been shown to have angiog enic effects in vitro and in vivo in addition (Achen et al , 2001) to its role in lymphangiogenesis (Stacker et al , 2001). VEGF-C. VEGF-C is produced as a precursor protein that is pr oteolytically activated in the extracellular space to bind to th e receptors VEGFR-2 and VEGFR-3 (Joukov et al , 1997). VEGF-C is believed to be primarily a lympha ngiogenic growth fact or (mediated by binding VEGFR-3) (Enholm et al , 2001), but it has been shown to increase blood vessel permeability through its binding to VEGFR-2.

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17 VEGF-B. VEGF-B shares a 23% homology w ith VEGF-A. Hypoxia does not regulate expression of this family member since th e VEGF-B promoter region lacks a HRE (Enholm et al , 1997). Both isoforms are able to bind to VEGFR-1 and Nrp-1 (Tammela et al , 2005). VEGF-B is thought to be weakly angiogenic and it may play a role in inflammatory angiogenesis (Mould et al , 2003). VEGF-A. VEGF-A is considered to be the most important pro-angiog enic growth factors in tumor pathology. VEGF-A is an endothelial ce ll mitogen that is devoid of any appreciable mitogenic activity for other cell types (Ferrara, 1999). This protein is known to activate and stimulate the migration of endothelial cells as well as increase vascular permeability (Harmey & Bouchier-Hayes, 2002). VEGF-A has also been shown to enhance endot helial cell survival by upregulating the expression of the anti-apoptic molecule, Bcl-2 (Nor et al , 1999). VEGF-A has also shown the potential to m odulate the immune system. One group has suggested that VEGF-A may allow for enhanced tumor growth by avoiding induction of the immune system. An in vitro study showed that VEGF-A inhibited the growth of immature dendritic cells, but did not significantly affect mature cells (Gabrilovich et al , 1996). This was also seen in an in vivo system when VEGF-A infusion resulted in decreased dendritic cell development and changes in hematopoiet ic lineages, probably by blocking NFB signaling (Gabrilovich et al , 1999;Oyama et al , 1998). VEGF-A expression can be regulated by a numbe r of cytokines, including interleukin-1 (IL-1), IL-6, insulin-like growth factor-1, and PDGF (Stewart et al , 2001). However, tissue hypoxia appears to be the most potent in vivo st imulus; a hypoxia response element (HRE) that is found within the promoter of this gene allows VEGF-A expression to r eact quickly to changes

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18 in oxygen tension (Olenyuk et al , 2004). An increase in VE GF-A mRNA transcripts was detectable within one hour of cells be ing placed in a hypoxic environment (Stavri et al , 1995). The human VEGF-A gene contains eight exons, separated by seven in trons, with a coding region of approximately 14 kb (Houck et al , 1991). Due to alternativ e splicing, at least four isoforms are known to exist in varying ratios with the following base pair lengths: 121, 165, 188, and 206 (Tischer et al , 1991)[Fig. 1-2]. VEGF165 is the predominant species with VEGF121 and VEGF188 being detected in the majority of cells; VEGF206 is very rare. The 165, 188, and 206 isoforms are basic, heparin-bi nding glycoproteins while VEGF121 is slightly acidic and does not bind heparin (Houck et al , 1992). VEGF121 is also a freely diffusible protein while VEGF188 and VEGF206 are sequestered in the extracellular matrix (ECM). VEGF165 is bound to the cell surface and the ECM, as well as secreted (Cross et al , 2003). VEGF Receptors The effect that human VEGF exerts on cells is mediated by its interaction with the following receptors: VEGFR-1, VEGFR-2, VEGF R-3, Neuropilin-1 (Nrp-1), and Nrp-2. Historically, it was believed that these receptors were only present on endothelial cells, but it is now known that VEGF receptors also occur on bone marrow-derived cells, macrophages, and monocytes (Ferrara & Davis-Smyth, 1997). VE GFR-1 and VEGFR-2 are two related receptor tyrosine kinases (RTKs) that contain seven immunoglobin-like domains and a single transmembrane domain (Klagsbrun & D'Amore, 1996) . VEGFR-3 (flt-4) is in the same RTK family and is a receptor for only VEGF-C and VE GF-D. In addition to these receptors, some VEGF family members are also ab le to bind to a family of co-receptors named the neuropilins (Nrps) (Ferrara et al , 2003). Flt-1/VEGFR-1. The VEGFR1/Flt-1 receptor binds VE GF with an approximately 10-fold greater affinity than the VEGFR2/Flk-1 receptor , but is unable to generate a mitogenic response

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19 when stimulated (de Vries et al , 1992). The main role for this receptor appears to be in the production of tissue factor and monocyte migration. Disruption of the VEGFR1/Flt-1 receptor gene has been shown to be embryonic lethal due to endothelial cell overgrowth and general blood vessel disorganization (Fong et al , 1995). There is also a sol uble form of this receptor (sVEGFR-1) that lacks the transmembrane and in tracellular portions of th e receptor. sVEGFR-1 binds to VEGF-A with high affinity and it is ex pressed at considerable levels in the placenta during pregnancy (Clark et al , 1998). Since VEGFR-1 binds VEGF-A with a high affinity yet is weakly mitogenic, it has been suggested that th e main role of VEGFR1/Flt-1 may be to function as a negative regulator of VEGF activities (Zhu & Witte, 1999). Flk-1/VEGFR-2. VEGFR-2 looks to be the main tran sducer of VEGF signals that result in endothelial cell pro liferation, migration, differentiation, tu be formation, increased vascular permeability, and maintenance of vascular integrity (Waltenberger et al , 1994). The intracellular signaling events that mediate these downstream e ffects follow several different paths. Activation of a protein kinase C (PKC) in a Ras-dependent or Ras-independent ma nner leads to activation of the Erk pathway, resulting in ce llular proliferation (Takahashi et al , 1999;Meadows et al , 2001). VEGFR-2 can also activate PI3K that can then activate the cell survival pathway, Akt/PKB (Gerber et al , 1998). The Akt/PKB pathway can al so induce the nitric oxide (NO) pathway that is involved in vascular permeability (Dimmeler et al , 1999) [Fig]. Flt-4/VEGFR-3. The main role of flt-4/VEGFR3 is during lymphangiogenesis. Disruption of the VEGFR-3 gene is embryoni cally lethal; mouse embryos display fluid accumulation and cardiovascular failure that is ca used by deficiencies in vascular remodeling (Dumont et al , 1998). In adults, this receptor is expr essed on lymphatic endothelial cells and point mutations that inactive VEGFR-3 lead to chronic lymphadema (Karkkainen & Alitalo,

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20 2002). To a small extent, VEGFR-3 is expressed on quiescent vascular endot helial cells and its expression may be induced in proliferating cells (Witmer et al , 2002;Witmer et al , 2001). Neuropilins. Neuropilin-1 (Nrp-1) was or iginally identified on neuronal cells, but is now known to be expressed on endothelial and tumor cells as well (Soker et al , 1998). Nrp-1 lacks an intracellular domain and theref ore acts as a co-receptor to mediate VEGF signaling. Nrp-1 interacts with VEGFR-1 and VEGFR2 as an isoform specific (VEGF165) receptor (Fuh et al , 2000;Soker et al , 2002). Nrp-2 has been shown to bind VEGF165 and PlGF (Gluzman-Poltorak et al , 2000). Pre-clinical studies Due to the importance of VEGF expression on tu mor growth, there have been a variety of studies that have manipulated VEGF expression to examine the effects on tumor cells. For example, studies that have targeted VEGF-A (subsequently referred to simply as VEGF) via siRNA techniques have shown that tumor cells display unaffected in vitro growth rates (Wannenes et al , 2005). Yet, when these cells are placed in vivo, the tumors are slower to form (Guan et al , 2005) and they display a decr eased growth rate (Takei et al , 2004). Results from these studies also indicate that by decreasing VEGF expression, the resulting tumors display decreased number of blood vessels. Additional studies that have used an an tisense mRNA technique to downregulate the expression of VEGF also showed that the in vitro growth rate of the tumor cells was not affected (Hao et al , 2006;Riedel et al , 2003). However, endothelial cell proliferation and tubule formation was diminished when conditioned medium from stably transfected cells, secreting lesser amounts of VEGF, was adde d to endothelial cells (Kang et al , 2000;Shi & Siemann, 2002). In addition, the results from the in vivo portion of these studies showed that the

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21 downregulation of VEGF suppressed in vivo tumor growth of hepatocellular carcinoma, renal cell carcinoma, and head and neck squamous cell carcinoma xenografts. The relationship between vascularity and tu mor response to radiation has also been studied in a pre-clinical setti ng. It was noted in the 1970s that the vasculature patterns within tumors were of great significance in determini ng the tumor response to radiation (Falk, 1978). Clinical studies The role of VEGF in clinical studies has not been entirely clear. Some studies have suggested that a high pre-treatment level of se rum and/or tumor VEGF expression is a prognostic indicator of poor survival (Salven et al , 1998;Chen et al , 1999;Karayiannakis et al , 2002). Other studies have shown the oppos ite relationship (Poncelet et al , 2004) or no relationship at all (West et al , 2005). Contradictory results ha ve also been seen when the issue of tumor vascularity and its impact on treatment outcome was addressed. Several studies that examined the relationship between vessel density and radia tion therapy have shown that decr eased intratumoral vascularity leads to a poor outcome (Nativ et al , 1998;Lauk et al , 1989) whereas others have shown that increased vascularity is associated with better tumor control (Kaanders et al , 2002). In addition, a large study involving head and neck tumors has shown a “U-shaped” response, where both increased and decreased intratumoral vascular density resulted in a poor outcome (Koukourakis et al , 2000). It is most likely these different obs ervations stem from the existence of several inter-study differences, including tumor types st udied, endpoints assessed, and vessel density determination methods. Vascular Targeting Therapies Given the close relationship be tween vasculature and tumor gr owth, as well as metastasis, it is not surprising to find the emer gence of a new class of agents that target this crucial network. Vascular targeting agents (VTA s) aim to exploit the inherent differences between tumor and

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22 normal vasculature to gain a therapeutic advantage (Siemann & Shi, 2003). For example, normal endothelial cells are ge nerally quiescent while tumor-associ ated endothelial cells are in a constant state of proliferation. There are cu rrently two subclasses of VTAs: antiangiogenic agents and vascular disrup ting agents (VDAs). To date, though, it is unknown which type of tumor may be most amenable to this new therapy: highly vascularized tumors or tumors w ith lesser degree of vascularity. It would not be surprising to find that these agents are more eff ective in highly vascular ized systems since the targets of these agents are the vessels: with more blood vessels present there would be more targets and, hence more damage done. On the other hand, it could also be argued that less vascular tumors may be injured more readily through the use of VTAs. After destroying just a few vessels this system could be critically damaged, whereas a highly angiogenic tumor may be able to recover faster due to its greater number of resources. Research Outline and Significance Tumor growth and metastasis are known to re ly on the growth of new blood vessels. Due to the extraordinary angiogenic demand by tumors, the growth of the tumor vasculature often lags behind that of the tumor. The intratumoral vessel structure can also be characterized as an inefficient network, full of abnormalities. As a result of the inadequate and nonuniform tumor vasculature, there exist tumor microenvironments where cells survive in lowered states of proliferation, due to poor nutrient levels and impair ed uptake of metabolic waste products. It has been found that cells existing in these microenvironments may be re fractory to conventional anticancer therapies, such as ra diation and chemotherapy. Angiogenesis is known to be a delicate balan ce between proand anti -angiogenic factors. For a tumor to grow beyond a small size, it mu st tip the balance and elicit an angiogenic response. Although a number of angiogenic molecu les have been reported, VEGF is considered

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23 to be the most important pro-angiogenic growth factor in tumor pat hophysiology. A variety of pre-clinical studies have shown th at, in general, decreasing the expr ession of this molecule leads to decreased tumor growth rates a nd intratumoral vascularity. Clin ical data have also not been entirely consistent: studies have shown a positiv e, negative, or no correlation between VEGF expression/tumor vascularity and patient survival . A number of crossstudy variables (i.e., genetic differences among tumor types) are likel y to blame for the contradictory results. Since tumor survival and metastasis have been linked to the establishment of a vasculature, vascular targeting agents have recently been introdu ced as a means to exploit this close relationship. Two different strategies have emerged by which to target the tumor vessels: antiangiogenics and vascular di srupting agents. Antiangiogenics aim to inhibit the growth of new blood vessels while VDAs target the existing va sculature within the tumors. It has not yet been established which type of tumor would most benefit from these new therapies: highly or poorly vascularized tumors. The goal of this project was to examin e the impact of VEGF expression/tumor vascularity on the impact of anti-cancer therap ies by employing a genetic approach in order to avoid the complications that are inherent in the inter-comparison of tumor types. The first section of this dissertation will focus on the creation of th e clonal cell lines and their in vitro and in vivo growth characteristics. The following section w ill analyze the resulting tumors for important physiological parameters. The final two sec tions will then determine what role VEGF expression plays in the response of tumors to anti-cancer therapie s such as radiation therapy and vascular targeting therapies.

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24 Figure 1-1. The steps of the angiogenic cascade involve vasodilation and increased permeability. Endothelial cells can then proliferate and mi grate towards the pro-angiogenic source. Following migration, the endothelial cells can adhere to one anothe r, create a lumen, and become a functional vessel. The fi nal steps involve basement membrane formation and maturation of the blood vessels. [Adapted from (Papetti & Herman, 2002), used with permission]

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25 Table 1-1. Angiogenesis is govern ed by a variety of promoters a nd inhibitors, of which several are listed below. Inhibitors Promoters Thrombospondin Fibroblast growth factors (aFGF and bFGF) Endostatin Transforming growth factor (TGF) Arrestin Platelet derive d growth factor (PDGF) Tumstatin Angiopoietins (Ang-1, Ang-2) N-terminal fragment of prolactin Vascul ar endothelial grow th factor (VEGF) Angiostatin Insulin growth factor-1 (IGF-1) Interleukins (IL-6, IL-8)

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26 Figure 1-2. Schematic displaying the human VE GF-A isoforms following mRNA alternative splicing.

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27 CHAPTER 2 CREATION OF CLONAL CELL LINES Introduction As briefly discussed in the prev ious chapter, it has been difficu lt to determine the effect of the level of tumor VEGF expression on outcome, bot h in preclinical and c linical studies. For example, some studies have suggested that a hi gh pre-treatment level of serum and/or tumor VEGF expression is a prognostic indi cator of poor survival (Salven et al , 1998;Chen et al , 1999;Karayiannakis et al , 2002). Other studies have shown the opposite relationship (Poncelet et al , 2004) or no relationship at all (West et al , 2005). It should be noted that making comparisons across a broad number of studies is complicated by differe nces in the genetic background of tumors, endpoint diffe rences, and variability in samp le collection techniques. Therefore, to avoid such complications in this study, a gene therapy technique that utilizes a recombinant adeno-associated virus (rAAV) to infect cultured cells was used. Many investigators have previously used rAAV to sa fely deliver gene products to mammalian cells (Xiao et al , 1996;Hermonat & Muzyczka, 1984). It is cu rrently believed that rAAV infection of cultured cells results in the integration of th e viral genome into the host cell genome (Church & Gilbert, 1984). By employing a high multiplicity of infection (MOI), the copy number of the viral genome can vary among the in fected cells, thus causing vari ous levels of gene expression (Walz & Schlehofer, 1992). The goal of this study was to cr eate clonal cell lines that expre ssed a range of VEGF levels and then examine their subsequent in vitro grow th rates and ability to form tumors in vivo. Clonal cell lines varying in humanVEGF expressi on were created from two tumor types: a human colon carcinoma (HT29) and a murine squamous cell carcinoma (SCCVII). The rAAV vector that was used, pT R-UF21, contained a chicken -actin promoter driving expression of the

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28 human VEGF165 gene as well as a neomycin resistance gene [Fig. 2-1] that allowed for the subsequent selection of infected cells. Following this selection process th e in vitro and in vivo growth characteristics of the cell lines were assessed. In additi on, the VEGF receptor status and expression pattern of other pro-a ngiogenic factors were examined to ensure that any changes among the clonal cell lines were due solely to changes in the expression of VEGF ligand. Materials and Methods Cell Culture HT29 colon carcinoma cells were grown in Dulbecco’s modified minimum essential medium (DMEM) (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Grand Island, NY), 1% peni cillin-streptomycin (Invitrogen, Grand Island, NY), and 2 mmol/L L-glutamine (Invitrogen, Gr and Island, NY). Murine SCCVII squamous cell carcinoma cells were grown in alpha minimal essential media ( -MEM) supplemented with 10% FBS (Invitrogen, Grand Island, NY), 1% peni cillin-streptomycin (Invitrogen, Grand Island, NY), and 2 mmol/L L-glutamine (In vitrogen, Grand Island, NY). Generation of Stable Cell Lines by rAAV Infection HT29 or SCCVII cells were infected with a recombinant adeno-associated virus (rAAV) containing the cassette for human VEGF165 as previously described for the human endostatin gene (Shi et al , 2002). Briefly, 1 x 104 HT29 or SCCVII cells were suspended in 50 L of serumand antibiotic-free medi um. A rAAV containing the VEGF165 gene and a neomycin resistance gene were then added at a multipli city of infection (MO I) of 10,000 and the mixture was incubated for 3 hr at 37C. Cells were th en grown in selection media that contained 1 mg/mL geneticin for 48 hr. Cells were plated at a low density in 60 mm dishes to obtain clones. Stable cell lines were maintained in the a ppropriate cell media with the addition of 500 g/mL geneticin.

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29 Angiogenesis Factor Expression HT29 or SCCVII cells were plated in 60 mm dishes at a density of 2 x 106 cells in 2 mL of cell culture media. Following a 24 hr incubatio n period, the cell culture supernatants were collected and analyzed by ELISA (R&D Systems, Minneapolis, MN) for the following factors: VEGF, PDGF, bFGF, Ang-1, and Ang-2. In Vitro Cell Growth HT29 or SCCVII cells were plated in 60 mm dishes at a density of 1 x 104 cells. At various times thereafter, cells were trypsiniz ed and counted using a hemocytometer. The average number of cells per plate (three plates per time point) was determined as a function of time after plating. Animals and Tumor Models Mice were injected with either 1 x 106 HT29 tumor cells or 1 x 105 SCCVII tumor cells intramuscularly (in a volume of 0.02 mL phosphat e buffered saline) into a single hind limb of 68 week-old female NCR nu/nu or C3H/HeJ mice (F rederick Cancer Research Facility, MD), respectively. The mice were maintained under specific-pathogen-free conditions (University of Florida Health Science Center Vivarium) with food and water provided ad libitum. In Vivo Growth Rate For both the HT29 and SCCVII models, tumor size was measured by passing the tumor bearing leg through a series of increasing diameter holes in an acrylic plate. The smallest diameter hole that the tumor-bearing leg could pass through was recorded and converted to a tumor volume using the following formula: tumor volume=1/6( d3)-100, where d is the hole diameter and 100 represents a vo lume correction factor determin ed for a mouse leg without a tumor. The times for the tumors to gr ow from appearance (approximately 200 mm3) to 1000 mm3 was recorded (n = 7-10 mice per group).

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30 Results HT29 and SCCVII cells were infected with a rAAV containing the gene for human VEGF165 and colonies were selected for their expression of human VEGF. The conditioned medium from clones was obtained and analy zed via ELISA to determine the secreted concentration of VEGF. Three clones were ul timately chosen for each tumor model: the first clone expresses VEGF at a level that is comparab le to the parental cell line, the second clone expresses VEGF at an intermediate level, and the third clone expresses VEGF at a high level [Fig. 2-2]. For the HT29 model the increase in VEGF expression is approximately 20 and 60 fold greater than the parental cell line for the intermediate and hi gh clone, respectively. Since many growth factor pathways are interconnected, it was conceivable that manipulating VEGF expression might also alter the expression of other angiogenic factors. The clonal cell lines therefore were also tested for the expression le vels of the pro-angiogenic factors bFGF, Ang-1, Ang-2, and PDGF (data not shown) . Neither the parental HT29 or SCCVII nor the clonal cell lines derived fr om either model were found to have significant paracrine expression of these growth factor s. In addition, it was possible that an autocrine feedback loop could exist if the tumor cells expressed the VE GF receptors. However, RT-PCR confirmed the lack of expression of VEGFR1 a nd VEGFR2 on the clonal and pare ntal tumor cell lines (data not shown). Studies assessing the inherent growth characteristics of th e parental (non-infected) and clonal cell lines showed that the elevation of VEGF levels did not significantly alter the growth rates in either the HT29 or SCCVII tumor mode ls, leaving the in vitro doubling times at approximately 24 and 15 hours, respectively [Fi g. 2-3]. The in vivo time to appearance (~200 mm3) and the subsequent growth rate (200 to 1000 mm3) of the tumors resulting from the parental and clonal cell lines were then determined. Figure 2-4A shows that the tumors arising

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31 from the midand high-level VEGF expressing cl ones of the HT29 cell line appear faster than the parental and low-level expre ssing clonal cell lines (10.5 versus 18 days). Tumors resulting from the highest expressing VEGF clone also grew significantly fa ster from 200 to 1000 mm3 than the other groups (10 days versus 18 days) [F ig. 2-5A]. This was not seen with the SCCVII tumors; there was no significant difference in the time to tumor appearance or the growth rate from 200 to 1000 mm3 among the different clones and the pare ntal cell line [Fig. 2-4B, 2-5B]. Discussion VEGF is known to be a potent pro-angiogenic growth factor and th e upregulation of its expression has been shown in a wide variety of tumors (Takekoshi et al , 2004;Tamura et al , 2004;Bowden et al , 2002). Although some clinical studies have demonstrated the potential use of VEGF as a prognostic indicator (Kyzas et al , 2005;Ferroni et al , 2005), not all studies agree with its use as a biomarker (Chung et al , 2006). This is perhaps not surprising given the wide range of factors, such as genetic differences among tumor types, which may influence the interpretation of clinical data. In the present study, stable clonal cell lines th at express different le vels of VEGF while possessing the same genetic backgrounds were ge nerated. Two tumor types, one a human colon carcinoma (HT29) and the other a murine squam ous carcinoma (SCCVII) were used. Clonal cell lines were established from both pa rental tumor types and were subsequently examined for their in vitro as well as in vivo growth characteristics. Manipulation of the VEGF expression levels did not significantly affect the in vitro growth rate. However, the in situ characteristics of tumo rs arising from the various clones were affected. Tumors derived from the HT29 clonal cell lines clearly showed that an increase in VEGF expression resulted in an earlier tumor appearance as well as an increased growth rate. This was not seen with the SCCVII clonal cell lines. A like ly explanation for the lack of an effect of

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32 VEGF expression on SCCVII growth may be that the inherently high in situ growth rate of the parental line of this model (2 day doubli ng time) minimizes the likelihood of further enhancement in the growth rate [Fig. 2-3B]. Three different scenarios were investigated to explain the observed in crease in the in vivo growth rate of the HT29 model. One possibili ty was that a positive autocrine feedback loop existed. However, RT-PCR analysis of the clona l and parental cell lines showed no expression of the VEGF receptors Flt-1 or Flk-1. A second possibility was that physiological characteristics of the tumors, namely vascular ity and perfusion, had changed with increased VEGF expression. Since VEGF is a potent angiog enic factor its upregul ation could lead to increased nutrient delivery and growth rate, as detailed in the next ch apter. It was also conceivable that manipulating VEGF levels had led to altered expression patterns in other proangiogenic factors, but ELISA a ssays indicated that there was negligible expression of bFGF, PDGF, Ang-1, and Ang-2 in the clonal and parent al cell lines. Therefor e, it appears that the increased growth rate observed in the HT29 m odel does not result from changes in expression patterns of other angiogenic factors nor the VE GF receptors, but from differences in tumor physiology. In summary, clonal cell lines were created by infecting two different tumor types with a rAAV that contained the human VEGF gene. Thr ee clones were then selected that expressed a range of VEGF levels for each tumor model. Th e infection process did not alter the in vitro growth characteristics of these cell lines nor di d it change the expres sion levels of other important pro-angiogenic factors. The in vivo growth rate did increase in the HT29 model following increased VEGF expression. Since these tumor cells do not express the VEGF

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33 receptors, it appears that the incr eased growth rate is due to ch anges in the physiology of the resulting tumors.

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34 Figure 2-1. Diagram of the rAAV vector that codes for the expression of the human VEGF165 gene and also contains a chicken -actin promoter and neomycin resistance gene.

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35 A B Figure 2-2. Human VEGF expre ssion levels as assayed by ELI SA of the (A) HT29 and (B) SCCVII clonal cell lines. Bars repr esent an average of two readings.

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36 A B Figure 2-3. Cell growth curve of the HT29 (A) and SCCVII (B) clona l cell lines as a function of time after plating. Symbols represent an av erage of 3 plates per time point. No significant difference in grow th rates was detected using the student’s T-test.

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37 A B Figure 2-4. The number of days, post-inj ection, for tumors to appear (~ 200 mm3). 1 x 106 HT29 tumor cells (A) or 1 x 105 SCCVII tumor cells (B) were injected i.m. in the left hind leg of nude or C3H mice, respectively. Asterisk (*) indicat es a p-value of less than 0.05 when groups were compared via th e Wilcoxon rank sum test to the parental group.

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38 A B Figure 2-5. In vivo growth rate of the tumors deri ved from the parental and clonal cell lines. 1 x 106 HT29 tumor cells (A) or 1 x 105 SCCVII tumor cells (B) we re injected i.m. into the left hind leg of nude or C3H mice, re spectively. The number of days from the time of tumor appearance (~ 200 mm3) until the tumor reached a size of 1000 mm3 was recorded. Asterisk (*) indicates a pvalue of less than 0.05 when groups were compared via the Wilcoxon rank sum test to the parental group.

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39 CHAPTER 3 PHYSIOLOGICAL CHARACTERIZATION OF TUMORS Introduction In the preceding chapter, clonal cell lines were created that differed only in the expression levels of VEGF; expression of other important pro-angiogenic factors remained unchanged and there was no expression of VEGF receptors on the tumor cells. The in vitro growth rate also remained unchanged among the clonal and parental cell lines. In vivo, it was found that the clonal cell lines maintained the ability to fo rm tumors. However, the HT29 tumor model displayed differences in the time to tumor a ppearance (the amount of time following tumor cell implantation to an approximate tumor size of 200 mm3 decreased with increasing VEGF expression) and demonstrated an in creased in vivo growth rate. VEGF is a potent pro-angiogenic molecule th at can be closely li nked to intratumoral vascularity and the in vivo growth rate of tumors ; studies that have used siRNA to target VEGF have noted that reducing the VE GF expression level resu lts in a decrease in intratumoral vessel density, increased areas of necrosis, and marked suppression of in vivo tumor growth (Takei et al , 2004;Guan et al , 2005;Wannenes et al , 2005). Therefore, the goa l of this chapter was to investigate the resulting tumor physiology of the clonal and parent al cell lines and the following physiological parameters were examined: abil ity to induce blood vessel growth, perfusion, evidence of hypoxia, and ar eas of tumor necrosis. Two strategies were undertaken to evaluate th e ability of the clonal cell lines to elicit an angiogenic response. The first approach was to use an intradermal assay that allows for the visualization of new blood vessel growth toward s tumor cell inoculates in vivo. The other strategy was to assess intratumoral vessel de nsity, the measure of blood vessel area to tumor area, in tumors resulting from the parental and cl onal cell lines. Endothelial cells were identified

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40 with the commonly used pan-e ndothelial cell marker CD31 (PECAM -1), which is expressed on newly formed and existing vasculature (Ilan & Madri, 2003). Since VEGF is a pro-angiogenic factor, it was expected that increased VEGF ex pression would lead to an increased angiogenic response that would be appa rent in both assays. Tumor perfusion is a measurement of the f unctional blood vessels wi thin a given area and this parameter is also expected to increase with increasing VEGF expression. However, blood vessels within a tumor are highly chaotic struct ures that have been shown, by vascular casting techniques, to have blind ends and arteriovenous shunts (Grunt et al , 1985;Shah-Yukich & Nelson, 1988). Sluggish and highly i rregular blood flow is expected in such irregular vessels. Indeed, it was confirmed that bl ood flow does fluctuate within tumo r vessels in previous studies where two different diffusible dyes that marked pa tent vessels were injected intravenously a few minutes apart. The results from these studies show ed that some of the vessels were labeled with one dye or the other (not both) , thus implying that vessels can open and close in a small time frame (Chaplin et al , 1989;Chaplin et al , 1987). Taken together, th e results of these two approaches demonstrate that it is possible for a highly vascularized tumor to contain a significant proportion of non-functional blood vessels. In order to assess functional ve ssels in the tumors derived fr om the parental and clonal cell lines the diffusible dye, Hoechst 33342, was use d. Tumor bearing mice were injected with a solution of Hoechst 33342 dye and then killed one minute later, followed by removal of the tumor. Hoechst 33342 is a dye that binds to double stranded DNA in the minor groove (Smith et al , 1988) and by allowing only a short time for th e dye to diffuse out from blood vessels it is possible to visualize the patent blood vessels of a tumor (Olive et al , 1985). Hoechst 33342 is readily visible in frozen tumor s ections under a fluorescent microscope.

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41 Although it is assumed that areas of high vasc ularity imply a well-oxygenated tissue, this may not always be the case (Ziemer et al , 2001). Areas of hypoxia within a tumor can be visualized with the aid of a bio-reductive agen t that is preferentially metabolized under hypoxic conditions. EF5 is a nitroimiadazole-based ag ent whose nitro group undergoes hypoxiadependent metabolism by cellular n itroreductive enzymes. During this metabolic process, one of the intermediates produced is highly reactive and can form stable adducts with cellular macromolecules, such as proteins, th iol residues, and nucleic acids (Koch et al , 2001). These adducts can then be detected in tumor secti ons with a monoclonal antibody conjugated to a flourophore (Lord et al , 1993). Previous studies using this agent have shown that, in general, there is an inverse correlation between the location of blood vesse ls and areas of hypoxia (Evans et al , 2001). Finally, necrotic fraction, the m easure of the area of necrosis as compared to the total area of the tumor, can also be affected by change s in vascularity. Typi cally, the oxygen diffusion distance determines the area of viable tumor tis sue and thus, the areas of necrosis. Large intervessel distances, or low ve ssel density, would lead to a gr eater amount of poorly supplied areas and, hence tumor necrosis. With an incr ease in vessel density, leading to decreased intervessel distances, the extent of tumor necr osis arising from highl y expressing VEGF tumor cells is expected to decrease. This fracti on can be obtained by imaging paraffin embedded sections that have been H & E stained with a morphometric microscope (Solesvik et al , 1982). Methods Intradermal Angiogenesis Assay 1 X 105 HT29 or SCCVII cells were injected intradermally in a volume of 10 L of PBS at four sites on the ventral surface of a female nude mouse. The addition of one drop of a 0.4% trypan blue solution to the cellular mixture wa s used for light coloring to allow for the

PAGE 42

42 subsequent location of the injec tion sites. Three days post-inje ction the mice were killed. The skin was separated from the underlying muscle and the number of vessels around each injection site was counted using a dissection microsco pe. Scoring of each site was done at 5X magnification and only vessels that were readily visible and to uching the edge of the tumor inoculates were included. Data from each tr eatment group (n=5) were pooled for statistical analysis via the Wilcoxon rank sum test. Vessel Density Frozen sections of tumors arising from each of the clonal cell lines were cut on a cryostat, air-dried, and fixed in acetone/m ethanol at 4C for 10 min. Tu mor microvessels were stained using a mouse monoclonal antibody to the CD31 (PECAM-1) antigen fou nd on endothelial cells (Beckman Coulter, Brea, CA), applied overnight at 4C at a dilution of 1:50. A secondary antibody conjugated with Cy3 (Jackson ImmunoRes earch Laboratories, Inc., West Grove, PA) was applied for 1 hour at room temperature. The staining was followed by standard washing and then slides were allowed to air-d ry prior to storage at 4C. Assessment of Functional Vessels/Perfusion Hoechst 33342 was dissolved in sterile sali ne just prior to use and was injected intravenously at a dose of 40 mg/kg. One minute later the tumor bearing mice were killed and the tumors were removed and snap frozen in liqu id nitrogen. Frozen sections of a thickness of 20 m were cut and the Hoechst 33342 dye was im aged with the aid of a morphometric microscope. Evidence of Hypoxia EF5 (provided by Dr. Cameron Koch , University of Pennsylvania, Philadelphia, PA) was prepared in a 5 mg/ml solution consisting of 40% propylene glyc ol, 50% warm saline, and 10% ethanol. Each mouse was injected intravenously with 0.2 ml of this so lution and one hour later

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43 mice were killed via cervical dislocation. Th e tumor was excised and snap frozen in liguid nitrogen. Frozen sections of the tumo rs were then cut to a thickness of 20 m. The sections were fixed in 4% para-formaldehyde for one hour at 4C. These sections were blocked overnight at 4C in PBS containing 0.3% Tween-20, 1.5% albumin, 20% skim milk, and 5% normal mouse serum. Sections were incubated with co ld Cy3-conjugated Elk351 antibody (provided by Dr. Cameron Koch) for six hours at 4C. Pictures were obtained using a morphometric microscope. Necrotic Fraction HT29 or SCCVII tumors were fixed in form alin, embedded in paraffin, sectioned, and H&E stained by the University of Florida molecula r pathology core facil ity. Pictures of the entire tumor section were taken using a morpho metric microscope and these pictures were analyzed using the software program ImageJ (NIH). The necrotic fraction was obtained by dividing the area of necrosis by the total tumor section area. Results Previous studies using siRNA techniques targeting VEGF have shown that a greater level of VEGF expression leads to a higher vessel dens ity in a tumor (Takei et al , 2004) and a decrease in tumor necrosis (Guan et al , 2005;Takei et al , 2004). The aforementioned studies found that by targeting VEGF with a siRNA appro ach, the tumor cells behaved the same in vitro, yet the resulting tumors displayed different physio logical characteristics. Since VEGF has been shown to be a potent angiogenic growth factor for a variety of tumor types, it is not altogether surprising to find that manipulati ng this pro-angiogenic factor w ould lead to observable changes in vessel density and othe r physiological factors. It was anticipated that the ability of the parental and clonal cell lines to initiate angiogenesis could vary. To determine the abil ity of the tumor cells to induce blood vessel growth, small inoculates of the cells were injected intradermally on the ventral surface of mice

PAGE 44

44 and the number of vessels induced by the various clonal cell lines was determined. The results showed that the tumor cell inoculates correspond ing to the higher VEGF expressing clonal cell line could induce a 2-3 fold greater number of blood vessels than the parental cell line [Fig. 3-1]. The ability of the tumor cell lines to indu ce an angiogenic respons e was also quantified immunohistochemically. The CD31 an tigen, a pan-endothelial marker, was used to visualize the blood vessels present within frozen sections obtai ned from the tumors arising from the clonal and parental cell lines. The entire tumor sections were photographe d and vessel density was then calculated with the aid of an image analysis program. As expected, the tumors resulting from the higher VEGF expressing clonal cell line demonstrat ed a significant increase in vessel density when compared to tumors of the parent al line in both tumor types [Fig.3-2]. Dual staining of frozen sections was then performed to assess areas of perfusion and hypoxia within the tumors derived from the parental and clonal cell lines. Patent blood vessels were identified using the diffusible Hoechst 3334 2 dye and areas of perfusion were recognized with the bio-reductive marker EF5. As can be readily seen in figures 3-3 and 3-4, there is a substantial increase in perfusion and decreased areas of hypoxia in those tumors resulting from the high expressing VEGF clonal cell lines as compared to the parental line. As a result of increased vascularity/perfusi on, it was expected that those tumors derived from high expressing VEGF clonal cell lines woul d demonstrate decreased amounts of necrosis. Indeed, the HT29 tumor model dem onstrated a reduction in tumor n ecrosis from ~20% in control HT29 xenografts to <10% in tumors establishe d from the v2-8 clone [Fig 3-5A]. However, increasing vascularity did not affect the ~4 % ne crotic fraction associated with SCCVII tumors [Fig 3-5B].

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45 Discussion In the preceding chapter, clonal cell lines were created that ex pressed increasing levels of VEGF. Although the in vitro growth rate rema ined unchanged among the clonal and parental cell lines, HT29 tumors derived from the high VEGF expressing clonal cell line demonstrated an increased in vivo growth rate. Previous studies have shown th at by manipulating cellular VEGF expression, the resulting tumors display different physiological characteris tics. For example, studies that have used siRNA to target VEGF have noted th at reductions in cellular VEGF expression results in a decrease in intratumoral vessel density a nd marked suppression of in vivo tumor growth (Takei et al , 2004;Guan et al , 2005;Wannenes et al , 2005). Therefore, a number of physiological parameters were evaluated in th e tumors resulting from the clonal and parental cell lines to determine the e ffects of increased VEGF expression on tumor physiology. The intradermal assay and the calculated vessel density demonstrated that an increase in the cellular expression levels of VEGF is relate d to an increase in the vessel density of the resulting tumor. This observati on is not altogether su rprising since VEGF is a potent angiogenic factor. Taken together, the highe r level of intratumoral vessel density that was seen with the increased cellular VEGF expression may be the expl anation for the increased in vivo growth rate that has already been noted in th e preceding chapter [Fig. 2-5]. Increasing vascularity should result in an incr ease in tumor perfusion and a decrease in areas of hypoxia. Hoechst 33342 is a dye that re adily diffuses from the blood vessels and binds to double stranded DNA in the minor groove (Smith et al , 1988). By allowing only a short time for the dye to diffuse out from blood vessels it is possible to visualize the patent blood vessels of a tumor (Olive et al , 1985). When functional blood vessels were identified with the Hoechst 33342 dye, there was clearly an obser vable increase in perfusion in both tumor models. With the

PAGE 46

46 increased perfusion, it was not surprising to fi nd a decrease in the evidence of hypoxia, as indicated by EF5 staining [Fig. 3-4]. An increase in vessel density within a tumor shoul d result in increase d nutrient delivery and enhanced survival of tumor cells. As exp ected, the increased vascularity associated with tumors derived from the high VEGF expressing HT 29 clonal cell line demonstrated a decrease in the extent of necrosis in the xenografts approxima tely 2-fold as compared to parental tumors [Fig. 3-5]. Such a reduction in necrosis wa s not seen in SCCVII tumors of varying vessel density, probably because the very low level of n ecrosis present even in the parental tumors of this cell line (~4 %) would make it difficult to de monstrate any further re duction in the necrotic fraction. In conclusion, the results of this study have shown that cl onal cell lines expressing various levels of the pro-angiogenic factor VEGF range in their ability to elicit an angiogenic response; tumors from high expressing VEGF clonal cell lines were able to induce more blood vessel growth and as a result th e tumors were more vascularized. The tumors from the high expressing clonal cell line also displayed increased perfusio n and decreased areas of hypoxia and necrosis. Tumor vascularity affects the distribution of nutri ents such as oxygen as well as the delivery of chemotherapeutic agents and the inadequate a nd non-uniform vascular ne twork within a growing tumor has been linked to the failure of many an ti-cancer therapies (Tannock, 2001). As detailed in this chapter and the preceding chapter, the ge netic approach used here to establish clonal tumor cells may be applied to directly examin e the relationship between angiogenic factors, tumor vascularity and treatment outcome w ithout the confounding difficulties typically associated with inter tumor comparisons.

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47 A B Figure 3-1. Angiogenic potential of the parental and clonal cell lines as assessed by the intradermal assay. 1 x 105 HT29 tumor cells (A) or SCCVII cells (B) were injected at 4 sites on the ventral surface of a nude mice. 72 hours later the mice were killed and the skin flap containing the inoculati on sites was excised. The number of blood vessels intersecting each inoculate was c ounted and data points for each group were pooled. Asterisk (*) indicates a p-va lue of less than 0.05 when groups were compared via the Wilcoxon rank sum test to the parental group.

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48 A B Figure 3-2. HT29 tumors (A) and SCCVII tumors (B ) resulting from the clonal and parental cell lines were stained for the CD31 antigen. Vessel density was obtained by dividing the CD31-positive area by the total tumor area. Value indicated by a horizontal line is the median value of group data points. As terisk (*) indicates a p-value of less than 0.05 when data points were compared to control data points as computed by the Wilcoxon rank sum test.

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49 Figure 3-3. HT29 parental and cl onal cell tumors were stained with for the hypoxia marker, EF5 (red), and had functional vessels labeled with Hoechst 33342 (blue). Entire tumor sections were imaged at 5X magnificati on. A) HT29-parental, B) v1-8, C) v1-3, and D) v2-8.

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50 Figure 3-4. SCCVII parental and clonal cell tumors were stained with for the hypoxia marker, EF5 (red), and had functional vessels labe led with Hoechst 33342 (blue). Entire tumor sections were imaged at 5X magnifi cation. A) SCCVII-parental, B) v1-2, C) v1-9, and D) v2-7.

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51 A B Figure. 3-5. Paraffin sections of the HT29 tumors (A) and SCCVII tumors (B) resulting from the clonal and parental cell lines were H&E st ained. The necrotic fraction was obtained by dividing the area of necrosis by the total tumor section area. Asterisk (*) indicates a p-value of less than 0.05 we re data points were compared to control data points via the Wilcoxon rank sum test.

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52 CHAPTER 4 RESPONSE OF CLONAL C ELL LINES TO RADIATION Introduction The use of ionizing radiation has been a me thod of cancer treatment for over 100 years and more than half of cancer patients today will receiv e radiation therapy as part of their treatment plan (Owen et al , 1992). Despite its widespread use, radi otherapy is not always curative and the failure to control tumors in patients can be attribut ed to a wide variety of factors, one of which is the presence of tumor microenvi ronments. As mentioned in th e introductory chapter, one of these factors, the existence of inadequate a nd non-uniform blood vessel networks, is known to create tumor microenvironments in which cells can be resistant to conventional anti-cancer therapies, such as radiation (D urand, 1991). The studies in this chapter will uti lize clonal cell lines to better elucidate the role of VEGF, a ke y regulator of tumor vasculature and hence tumor microenvironments, in the radiation response of tumors. Tumor Microenvironment As a tumor grows, it is believed that the growth of new blood ve ssels lags behind the expanding tumor (Tannock, 1968). In its constant effort to gr ow this network the tumor develops a vessel structure that is characterized by dead-ends, leaky vessels, and arteriovenous shunts (Brown, 1999). Aberrant vessels may lead to oxygen deficiency, termed hypoxia that is typically defined as 10 mm Hg and can be either a chronic or acute condition (Hockel & Vaupel, 2001). Areas of chronic hypoxia ar e diffusion-limited, typically oc curring at distances of 100150 m from the nearest functiona l vessel which corresponds to the limit of oxygen diffusion (Horsman, 1998). Acute areas of hypoxia are perf usion-limited; acute hypoxia is a consequence of intermittent cessation of bl ood flow that renders normally well-oxygenated areas suddenly hypoxic (Lanzen et al , 2006).

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53 Radiation’s Mechanism of Action Ionizing radiation exists as moving energy pack ets termed photons and it is these packets of energy that are deposited into a substrate during irradiation. The chief manner in which energy deposition occurs, in the most commonly used energy range for cancer treatments, is via the Compton effect, a mechanism where the collisio n of a high energy photon and an atom’s outer orbital shell results in the removal of an el ectron (Tannock & Hill, 1998) . Since electrons are small particles, they can easily be deflected a nd, thus, their travel through the tumor tissue is random. The energy from electrons is then either absorbed direc tly or indirectly (via energy transfer) by cellular molecules (Barcellos-Hoff et al , 2005). Due to the cell being in an aqueous environment, the main energy-absorbing molecule in tissue is water. Upon the radiolysis of water, reactive chemical species are formed that can then damage biological molecules [Fig. 4-1] (Walden & Farzaneh, 1990). The response of mammalian cells to ionizing ra diation is to halt their progression through the cell cycle, allowing for DNA repair of sublethal damage (SLD) (Shiloh, 2003). Radiation that results in single strand break s is usually not as important as double strand breaks due to the logistics of a cell’s DNA repair mechanisms; double strand breaks are more difficult for the cell to repair due to the loss of the DNA template (Prise et al , 2005). Incomplete repair may result in chromosomal aberrations that can then result in cell death in s ubsequent early mitotic divisions (Withers, 1992). The significance of double strand br eaks is illustrated in the fact that of the thousands of DNA lesions induced by radiation, typically less than 50 double strand breaks will occur, yet following radiation th ese double strand br eaks correlate most with cell survival (Tannock & Hill, 1998). Oxygen is known to enhance the cellular sensitiv ity to radiation. It has been found that anoxic cells typically require 2.53 times the dose of radiation needed for normoxic cells to

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54 produce the same amount of cell kill (Harrison & Blackwell, 2004) [Fig. 4-2]. The oxygen enhancement ration (OER) is a result of the abilit y of available oxygen to chemically react with radiation-induced biological le sions. Although the exact mechanism is uncertain, oxygen present during the time of irradiation can “fix” DNA da mage, causing increased difficulty for cellular repair. In areas of decreased oxygen concentr ations, the oxygen fixation phenomenon occurs less frequently and thus hypoxic cells incur less damage following irradiation (A.H.W.Nias, 1988). Fractionation Schedules In the clinic, patients are more likely to receive radiation therapy on a fractionated schedule. Fractionation is a proc ess where radiotherapy is given in small doses (typically 1.5-2 Gy) for a period of several weeks, usually five times per week. There are several reasons for utilizing fractionation when treati ng patients and these biological fa ctors are often referred to as the 4 R’s of radiotherapy: re pair, repopulation, redistribut ion, and reoxygenation (Withers, 1992). Repair One of the most important principles of frac tionation is that of re pair. Sub-lethal DNA damage can generally be repaired within cells over the course of a few hours. However, the extent of repair among normal, slower-growing cells is typically greater than the repair that malignant cells are capable of perfor ming before undergoing mitosis (Bernier et al , 2004). Since cell killing via radiation is logarithmic, the difference in cellular repair is amplified exponentially. Therefore, small differences in survival following radiation may have an enormous impact on treatment outcome.

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55 Repopulation In both tumor and normal tissue, surviving ce lls may proliferate dur ing the course of fractionation. In addition, some tissues, in response to accumulated damage, respond by increasing their rate of prolifer ation as a result of a homeostatic response. This phenomenon is most important in early-responding tissues that are generally highly proliferative, such as mucosa and skin, and is of little consequence in thos e late-responding tissues (eg. kidney, lung). In general, repopulation rates for tu mors are lower than the rates of the early-responding tissues and this response allows the normal tissues to tole rate increased radiation doses (Tannock & Hill, 1998). Redistribution As may be expected, there are significant diffe rences in the sensitivity to radiation during different phases of the cell cycle. For example, the S-phase of the cell cycle is radioresistant while the G2-M phase is radiosensitive; th e relative lack of radiosen sitivity during th e S-phase is thought to result from the presence of a greate r number of repair enzymes during DNA synthesis (Sinclair, 1968). It is the vari ations in radiosensitivity during the cell cycle that can lead to a greater proportion of cells dying during the most sensitive phases of the cell cycle while cells that are in a more resistant pha se have a better chance of surv iving. It was believed that radiotherapy could be tailored to take advantage of the re distribution phenomenon; properly timed doses of radiation could have resulted in the cell population becoming synchronized, possibly leading to a larger por tion of cells in a sensitive ce ll cycle phase for subsequent fractions. In reality, however, the small radi ation dose given in each fraction minimizes the differences in radiosensitivity among the cell cy cles and the heterogeneity of the tumor cell population conspire against this approach (Denekamp, 1986).

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56 Reoxygenation Another important radiobiological principle may be that of reoxygenation. This is the only radiobiological principle that is applicable to tumors alone si nce it is based on the presence of hypoxia. As previously discusse d, hypoxic cells are more resistan t to radiotherapy than their normoxic peers [Fig. 4-2]. When multiple doses of radiation are given, those cells that are normoxic are preferentially killed. The remaini ng cells, many of which will be hypoxic, may then be able to gain access to oxygen (Hall, 1994). It is thought that reoxygenation may occur as a result of reduced oxygen consumption by radiati on-damaged cells, redistributed blood flow due to decreased tissue tensi on, or a decrease in the average dist ance to the neares t functional vessel following the removal of damaged cells (Kallman, 1988). However, there is still some controversy surrounding the mode of reoxygenation in pre-clinical studies and it may be tumor type dependent. Although the timing and degr ee of reoxygenation is di fficult to predict for a particular tumor, its impact may be seen duri ng a subsequent radiation fraction when formerly hypoxic cells that have been reoxygenated may then be killed. Thus, fractionation tends to mitigate the negative effects of hypoxia. Reoxygen ation has not been extensively studied in human tumors since it is quite unpleasant fo r the patient to obtain multiple oxygen tension measurements. Therefore, the clinical imp lications of this prin ciple are unclear. Role of VEGF in Radiation Response Angiogenesis is a critical pr ocess that a tumor must manipul ate in order to grow beyond a small size (Folkman, 2002). This process of new blood vessel formation from the existing vasculature is a tightly regulated process of endogenous factors that can either promote or inhibit new vessel growth (Sridhar & Shepherd, 2003). Of the many different growth factors known, VEGF is considered to be a key regulator in the growth of a wide variety of tumor types (Ferrara, 1999).

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57 A variety of pre-clini cal studies have examined the inte raction between tu mor angiogenesis and radiation effect, including the anti-apoptotic properties of angioge nic factors, such as VEGF. Growth delay studies that have evaluated the response of tumo rs to ionizing radiation while blocking VEGF signaling has shown an enhanced response compared to radiation alone (Gorski et al , 1999;Winkler et al , 2004;Gupta et al , 2002). The mechanism by which VEGF blockage enhances radiation response of tumors remains largely unknown. Some studies have shown that antiangiogenic agents may incr ease tumor oxygenation through a pr ocess termed “normalization of the tumor vasculature.” The resulting incr ease in oxygenation following treatment with these agents would make tumor cells more sensitive to subsequent radiati on (Jain, 2005). However, not all studies agree with this assessment: st udies have shown an increase, decrease, and no change in tumor oxygenation following antiangi ogenic therapy (Siemann & Horsman, 2004). Another hypothesis is that by blocking a survival signal fo r tumor endothelial cells the endothelium becomes radiosensitized. Thus, the ab ility of tumor-associated endothelial cells to survive radiation treatment is severely dimini shed, compromising the in tegrity of the vessels feeding the tumor. This chain of events would le ad to the observed increase in tumor response to radiation (Gupta et al , 2002). There have also been some clinical studies suggesting a link between VEGF expression/tumor vascularity and th e response to radiation therapy. Several studies that examined the relationship between vessel density and ra diation therapy have shown that decreased intratumoral vascularity lead s to a poor outcome (Nativ et al , 1998;Lauk et al , 1989) whereas others have shown that increased vascularity is associated with better tumor control (Kaanders et al , 2002). In addition, a large st udy involving head and neck tumo rs has shown a “U-shaped” response, where both increased a nd decreased intratumoral vascul ar density resulted in a poor

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58 outcome (Koukourakis et al , 2000). Comparing results across such studies is made difficult by a number of confounding factors, including differen ces in the genetic b ackground of the tumors being studied and various vessel vi sualization techniques. By util izing the clonal cell lines that differ only in the expression of VEGF, it was possible to avoid thes e variabilities. The goal of this set of experiments was to use the clonal cell lines to evaluate the relationship between VEGF expression/tumor vasc ularity and radiation. The clonal cell lines were first examined for their inhe rent sensitivity to radiation. Subsequent studies examined the response of the clonal cell lines to radiation given as both a si ngle dose and on a fractionated schedule. Materials and Methods Cell Culture Murine SCCVII squamous cell carcinoma cells were grown in alpha minimal essential media ( -MEM) supplemented with 10% FBS (Invitr ogen, Grand Island, NY), 1% penicillinstreptomycin (Invitrogen, Grand Island, NY), a nd 2 mmol/L L-glutamine (Invitrogen, Grand Island, NY). As reported in Chapter 1, stable clon al cell lines were created via infection with a recombinant adeno-asscoiated virus and were maintained in culture in -MEM medium with the addition of 500 g/mL geneticin. In Vitro Clonogenic Cell Survival Assay HT29 and SCCVII cells were eith er treated with a single dose of radiation or kept as a control group. The cells were then plated at three different concen trations. After colony formation the plates were stained and fixed with a crystal violet and methanol solution. The number of colonies, where a co lony consists of at least 50 ce lls, for each group was counted using a dissecting microscope and a survival curve was generated.

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59 Animals and tumor models Mice were injected with 1 x 106 HT29 or 1 x 105 SCCVII tumor cells intramuscularly (in a volume of 0.02 mL PBS) into a single hind limb of 6-8 week-old female nude or C3H/HeJ mice (Frederick Cancer Research Facility, MD), respectively. The mice were maintained under specific-pathogen-free conditions (U niversity of Florida Health Sc ience Center Vivarium) with food and water provided ad libitu m. Tumor size was measured by passing the tumor bearing leg through a series of increasing diameter holes in an acrylic plate. The smallest diameter hole that the tumor-bearing leg could pass through was recorded and convert ed to a tumor volume using the following formula: tumor volume=1/6( d3)-100, where d is the hole diameter and 100 represents a volume correction factor dete rmined for a mouse leg without a tumor. Tumor Growth Delay Assay Upon reaching an approximate tumor size of 200 mm3, tumor-bearing mice were separated into treatment and control groups. Treatment groups received radia tion on the following fractionation schedules: 2 Gy/day, 5 days a week for a period of 2 weeks and 3 Gy/day for 5 consecutive days for the HT29 and SCCVII models, respectively. Irradia tions were performed using a 6MV Clinac 600c linear accelerator (Varian Oncology Systems, Palo Alto, CA) operating at a dose rate of 4 Gy/min on restrained, una nesthetized mice. Tumor Dissociation Following resection, tumors were mechanically dissociated until uniform small chunks remained. HT29 tumor chunks were incubated at 37C for 1 hour in the following enzyme cocktail: pronase, collagenase, and DNAse. SC CVII tumor chunks were incubated at 37C for 1 hour in 1 mg/mL protease that was dissolved in -MEM media.

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60 Hypoxic Fraction Anoxic and normoxic mice were irradiated at various doses when tumor size reached approximately 500 mm3. Tumors were dissociated in the appropriate enzymatic cocktail and then plated following the protocol for an in vitro clonogenic cell surviv al assay. Paired cell survival curves were generated for the a noxic and normoxic conditi ons. The hypoxic fraction was estimated by calculating the ra tio of survival obtained by ai r-breathing conditions to the survival obtained under anoxic conditions at a dose where the survival curves are parallel. Results As was discussed in the previous two chap ters, stable clonal cell lines of the SCCVII model were created via infection with a rAAV that contained the human gene for VEGF in order to examine the effect of increased VEGF expre ssion on the physiological ch aracteristics of tumor cells. It was shown that the clonal cell lines ma intained similar in vitro growth rates as the parental (non-infected) cell line [Fig. 2-3]. However, the tumors resulting from the high expressing clonal cell lines did show a significant increa se in tumor vascularity as compared to the parental cell line, for both the HT29 and SCCVII tumor models [Fig. 3-2]. In order to evaluate the effect of the increas ed vascularity on the response to radiation, an in vitro clonogenic cell survival assay was us ed to ensure that the modulation of VEGF expression did not significantly alte r the inherent radiosensitivity of the clonal cell lines. As expected, the results of this in vitro assay indicated that ther e was no significant difference in survival among the parental and clonal cell lines when a single dose of radiation was administered [Fig. 4-3]. Since there was no change in the inherent cellula r response to radiation, any alterations in tumor radi osensitivity should be a product of the tumor’s physiology. Previous experiments had demonstrated that a measurable growth delay could be obtained for the HT29 tumors with a radiation dose of 10Gy (Brazelle et al , 2006). Thus, a single dose of

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61 radiation (10 Gy) was first used to evaluate th e response of the tumors resulting from increased VEGF expression. Tumors in the treatment groups were treated when the median of the group was approximately 200 mm3 and the growth delay was calcula ted by determining the difference in time needed for tumors to grow from approximately 200 to 1000 mm3 between the control and treated group. Tumors arising from the high expressing VEGF clonal cell lines showed a significant increase in the growth delay for bot h the HT29 and SCCVII tumor types; the growth delay increased from 10 to 19 days (parental versus high VEGF clone) in the HT29 model and from 2 to 16 days in the SCCVII model [Fig. 4-4]. In the clinic patients normally receive radia tion therapy on a fractiona ted schedule that is typically given as a few Gy per day for an exte nded period of time. Due to the significant increase in the growth delay that was observed after a single dose of ra diation was given, it was conceivable that this increased radiosensitivity would carry over to a fractionated dose setting. Since the HT29 and SCCVII tumor types display great differences in their in vivo growth rate [Fig. 2-5], differing fractionati on schedules were employed for the two models. The HT29 parental and clonal cell lines received 2 Gy/day given Monday through Friday for 2 consecutive weeks, but no significant increase in the grow th delay was observed [Fig. 4-5A]. The SCCVII tumor model, though, did show an increased growth delay among the parental and high expressing VEGF clonal cell line (1 to 11 days) when the tumors we re irradiated with a dose of 3 Gy/day for 5 consecutive days [Fig. 4-5B]. It has already been shown that increased VE GF expression leads to increased perfusion and decreased areas of hypoxia th rough immunohistochemical tec hniques [Figs. 3-3, 3-4]. However, it is also possible to estimate the hypox ic fraction of a tumor by analyzing in vivo paired survival curves over a range of radiation doses. By ex amining the radiation doses where

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62 the survival curves are parallel, the estimated hypoxic fraction of the SC CVII parental cell line was calculated to be 8%. The hypoxic fraction fo r the tumors derived from the high expressing VEGF cell line was approximately 4% [Fig. 4-6]. Discussion Radiation therapy is a common modality in use today for the treatment of cancer. It is estimated that approximately 70% of patients that are ultimately cu red of their disease received radiation treatments (DeVita et al , 1997). However, radiation ther apy has its failures and it has been known for decades that hypoxia can play a role in these failures. Tumors possess abnormal vasculature that can lead to de leterious microenvironments cont aining low levels of nutrients, such as oxygen. VEGF is one of the most important pro-angi ogenic factors and ha s been found to be upregulated in a wide variety of tumor types. Since abnormal tumor vasculature can result in intratumoral regions of poor oxygen delivery an d negatively impact tissue response to radiation, there have been a variety of studies that have tried to address the role of VEGF in the outcome of radiation treatment. However, the results from these studies have been inconclusive. Since the clonal cell lines differ only in VEGF expression, it was possible to evaluate the relationship between VEGF expression and ra diation response of tumors. In vitro clonogenic cell survival assays in re sponse to a range of radiation doses have shown that VEGF expression does not alter the radi osensitivity of tumor cells in an in vitro setting for both the HT29 and SCCVII tumor mode ls [Fig.4-3]. However, tumors arising from cells that display different levels of VEGF expression do demonstrate different radioresponsiveness to a single dos e of radiation; as VEGF expr ession increases the resulting tumors are more responsive to radiation, as a ssessed by tumor growth delay assays, in both the HT29 (from 10 to 19 days) and SCCVII (from 2 to 16 days) tumor models [Fig. 4-2].

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63 The most likely cause of the increased res ponse to radiation is differences in tumor oxygenation. It has already been shown that upregulation of VEGF results in increased vascularity in the tumors derived from the high expressing VEGF clonal cell line as compared to the parental line [Fig. 3-2]. Although it is genera lly assumed that regions of high vascularity are well oxygenated, this may not always be the case (Ziemer et al , 2001). By using a marker of hypoxia, EF5, our results have demonstrated that increased vascularity do es result in decreased evidence of hypoxia in both tumor models [Figs. 3-3 and 3-4]. Another method for analyzing the extent of hypoxia within a tumor is to generate paired survival curves under normoxic and hypoxic conditions. The hypoxic fraction obtained with this method for the SCCVII model (8% in the parental tumor reduced to 4% in the hi gh expressing VEGF tumor) further verified that increasing VEGF expression (resulting in increased perfusion) leads to decreased tumor hypoxia. Therefore, it appears that the changes in tumor physiology resulting from increased VEGF expression, mainly the increased perfusion and d ecreased tumor hypoxia, are responsible for the observed increase in radiation re sponse in the tumors derived from the high expressing VEGF clonal cell line. In the clinic, patients generally receive radi ation treatment on a fr actionation schedule and not as a single dose. As the single dose grow th delays showed an im pressive increase in the response to radiation with increa sing VEGF expression, growth dela y assays were again used to assess the responsiveness to ra diation given on a fractionated schedule. The SCCVII tumor model showed that tumors derived from the high expressing VEGF cell line were more sensitive to radiation given as small frac tions (growth delay increased fr om 1 to 11 days), but the HT29 tumor model showed no difference in radiosensitivity when a fr actionation schedule was used. One possible explanation for the lack of differe nce in the HT29 tumor model is the previously

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64 discussed principle of reoxygenation. Reoxygenation can be an important factor in some tumor types and a fractionation schedule can downplay the effects of differing hypoxia levels among the clonal cell lines. It is likely that the fr actionation schedule negate d the increased perfusion and decreased hypoxia observed in HT29 tumors resulting from the high expressing VEGF clonal cell line. In summary, increased levels of VEGF expre ssion do not alter the i nherent radiosensitivity of tumor cell lines. However, in vivo responses to radiation showed that tumors arising from high expressing VEGF clonal cell lines were more responsive to a single dose of radiation. Fractionated scheduling was not as conclusive: the SCCVII tumor m odel again demonstrated that increased VEGF expression led to an increase in radioresponsivene ss while the HT29 tumor model no longer showed this relationship. The likel y cause of the enhanced radiation sensitivity in these models is the change in tumor physiolo gy (eg. increased perfusion and decreased areas of hypoxia) that is induced by VEGF expression. These data suggest that tumor VEGF expression/tumor vascularity is an important factor in dete rmining radiation efficacy.

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65 Figure 4-1. Schematic showing the formation of DNA lesions following energy transfer from ionizing radiation. The biol ogical lesions may then be “fixed” with oxygen making it difficult for the cell to repair this damage [adapted from (Hall, 1994).

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66 Figure 4-2. Survival curves fo r ionizing radiation under anoxic ( ) and normoxic ( ) conditions. The oxygen enhancement ration (OER) shown in this figure is 2.5.

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67 A B Figure 4-3. Clonogenic cell survival curve demonstr ating that the in vitr o sensitivity to single doses of radiation is similar among the pare ntal and clonal cell lines of the HT29 (A) and SCCVII (B) tumor types.

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68 A B Figure 4-4. Response to a single 10 Gy dose of radiation of tu mors from HT29 (A) and SCCVII (B) parental and clonal cell lines. Treatme nt was administered when the tumors reached an approximate size of 200 mm3. Asterisk (*) indicates a p-value of less than 0.05 when the growth delay of each group was compared via the Wilcoxon rank sum test to the parental group growth delay.

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69 A B Figure 4-5. Response to a frac tionated schedule 2 Gy/day given Monday through Friday for 2 weeks for the HT29 (A) parental cell lines and 3 Gy/day for 5 consecutive days) for the SCCVII (B) parental and clonal cell lines . Treatment was administered when the tumors reached an approximate size of 200 mm3. Asterisk (*) indicates a p-value of less than 0.05 when the growth delay of each group was compared via the Wilcoxon rank sum test to the parental group growth delay.

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70 A B Figure 4-6. Paired survival cu rves showing the cell survival of anoxic and normoxic tumors following irradiation with a single dose. A hypoxic fraction of approximately 8% was calculated for the SCCVII parental tumo rs (A) and this decreased to 4% for tumors derived from the high expressing (v27) clone (B). Each point indicates the mean of 3 different tumors per group a nd the standard deviation is indicated.

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71 CHAPTER 5 RESPONSE OF CLONAL CELL LINES TO VASCULAR TARGETING THERAPIES Introduction Angiogenesis, the formation of new blood vessels , is known to be a cr itical process in the growth and spread of a tumor. For a tumor to grow beyond approximately 1 mm in diameter, the formation of a vascular system is required (Folkman, 2002). In addi tion to providing the growing tumor with nutrients neces sary for cell survival, the estab lished vasculature may be used as a route for metastatic spread. It is also the presence of this rapidly expanding, tumor vessel network that may lead to the de velopment of microenvironments in which cells are refractory to conventional therapies (Vaupel et al , 1989). Due to the importance of the vasculature for tumor growth and spread, it is not su rprising to find ongoing efforts to target this ve ssel network. The premise for the development of vascular ta rgeting agents (VTAs) was that if one could damage or inhibit the growth of the vasculature, then the enormous numb er of tumor cells that depend on that network for nutrients would be a ffected as well (Denekam p, 1999). In addition to directly targeting the tumor “supply line,” these agents ha ve several advantages over conventional chemotherapeutic agents. The first advantage is that VTAs do not have the same drug delivery problems as chemotherapeutic agen ts since their target is the more readily accessible endothelial cell. Also, endothelial cells are more genetically stable than the tumor cells to which they supply nutrients. Therefore, th ese targets are less likely to acquire resistance to VTAs (Hicklin et al , 2001). Finally, the doses at which thes e agents are effective appear to be less toxic than the conventi onal chemotherapeutic drugs that are currently used. Although these agents offer an exciting new st rategy in anti-cancer therapy, it is not yet clear which tumor types will be most amenable to their use. In the case of antiangiogenic therapy, there have been reports of physicians as suming that only well vascularized tumors will

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72 be sensitive to this type of agent. Conseque ntly, some clinical trials examining the use of antiangiogenic agents have had restrictions placed on the tu mor types allowed, including pretreatment biopsies for vessel density in order to exclude patients whose vessel density is deemed too low (Kerbel & Folkman, 2002). As discussed in the first chapter, arguments can be made for either high or low vascularized tumors being mo re sensitive to these agents. For example, highly vascularized tumors present more target s (blood vessels) than le ss vascularized tumors and thus, the use of VTAs would cause more da mage in these tumors. On the other hand, less vascularized tumors may be more dependent on thei r vessels and following a critical loss of just a few vessels, this system may be most compromised. In the effort to target the tumor vasculature, two distinct classes of vascular targeting agents have already been defined: the antiangi ogenic agents and the vasc ular disrupting agents (Siemann et al , 2005). These two classes of agents are aimed at different aspects of the tumor vasculature. Antiangiogenic agents inhib it the process of new blood vessel formation (angiogenesis), while vascular di srupting agents damage existing vasculature [Fig.5-1]. Antiangiogenics Antiangiogenic methods have been developed th at target many different aspects of the angiogenic cascade. As discusse d in the first chapter, the pro cess of angiogenesis is a highly orchestrated event that involves a number of steps. Briefly, th e basement membrane must first be degraded so that prolifer ating endothelial cells may migr ate towards the pro-angiogenic signal. After the endothelial cells join and form a functional vessel, speci alized cells are then recruited to stabilize the na scent vessels, leading to vessel maturation (Jain, 2003). Although a wide range of angioge nic inhibitors have been id entified, inhibition of VEGF appears to be most promising given its crucia l role in tumor angiogenesis. Indeed, many different avenues have been explored in th e quest to target VEGF signaling, including

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73 monoclonal antibodies to ligands (Kim et al , 1993) or receptors (Prewett et al , 1999), ribozymes (Weng et al , 2005), and small molecule inhibitors (Mendel et al , 2003) [Table 5-1]. Perhaps the most recognized antiangiogenic agent is bevacizumab (Avastin). This monoclonal antibody to the human form of VEGF has been the first F DA-approved agent in this class; Avastin has shown a clinically significant increase in patien t median survival when combined with standard chemotherapy versus chemotherapy alone (Hurwitz et al , 2004). Another antiangiogenic agent that inhib its VEGF signaling is ZD6474. ZD6474 [N-(4bromo-2-fluorophenyl)-6-methoxy-7[(1-methylpiperidin-4-yl)met hoxy]quinazolin-4-amine] is an orally available low molecular weight inhib itor of VEGFR2, with additional activity against the epidermal growth factor receptor (EGFR), (Wedge et al , 2002). It is believe d that this agent works by adopting a kinase binding conformation wher e the adenine-binding si te of the kinase is occupied by the quinazoline ring and the aniline portion of the molecule fits into a hydrophobic pocket on the enzyme. Preclinical studies have shown that in nude mi ce with breast (MCF-7) xenografts the half-life of this agen t was approximately 25 hours (Gustafson et al , 2006). In addition, administration of ZD6474 has shown dos e dependent tumor growth inhibition in a variety of xenograft models and can inhi bit the growth of metastases (Drevs et al , 2004). ZD6474 is currently in clinical tr ials and appeared to be well tolerated in Phase I clinical trials (Sridhar & Shepherd, 2003). A randomized phase II trial evaluating the combination of ZD6474 with chemotherapy for the treatment of non-small cell lung cancer (NSCLC) found that the estimated time to progression had been increased to approximately 19 weeks with the combination of ZD6474 and docetaxel from 12 weeks for docetaxel alone (Herbst et al , 2005). Additional phase II studies are ongo ing and it is expected that ZD6474 will enter phase III trials soon.

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74 Vascular Disrupting Agents VDAs are the second subclass of agents that make up the VTAs. VDAs exploit the differences between normal and tumor vasculatur e; tumor vasculature is highly irregular and contains a large proportion of imma ture vessels while the normal vasculature is well-organized (Jordan & Wilson, 2004). As a re sult of these structural differe nces, the tumor vasculature is highly susceptible to damage caused by VDAs wh ereas the normal vasculature remains mostly untouched (Prise et al , 2002;Davis et al , 2002). There are a variety of approaches that fa ll under the VDA subclass, including biological response modifiers, ligand-based approaches, an d low molecular weight agents. The most extensively studied approach of VDAs include two classes of low molecular weight agents that have been shown to induce rapid vascular shut down within solid tumors: agents related to flavone acetic acid (FAA) and tubulin-binding ag ents. DMXAA is the lead agent in the FAA group and it appears to work by partial dissolving ac tin filaments and inducing expression of TNF Tozer et al , . The tubulin-binding agents incl ude the colchicines, the vinka alkaloids, and the combretastatins. These ag ents exploit the non-e quilibrium dynamics of microtubules that causes the constant, rapid as sembly and disassembly of tubulin subunits (Jordan & Wilson, 2004). By bindi ng tubulin, these agents lead to cell shape changes in newly formed endothelial cells, which in turn init iates a cascade of events, concluding in vessel blockage. Consequently, the tumor cells that ar e dependent on these affected blood vessels will die, due to ischemia (Siemann et al , 2004). ZD6126 [N-acetylcolchinol-O-phosphate] is a vascular disrupting agent that shows structural similarity to colchicine and is a comp etitive inhibitor of the binding of colchicine to tubulin (Blakey et al , 2002). Pre-clinical st udies have shown that treatment with ZD6126 can

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75 inhibit endothelial cell tu bule formation and, as a consequenc e, induce G2/M cell cycle delay (Hoang et al , 2006). In vivo, this agent can cause a drama tic decrease in vascular patency within a few hours that can persist for at le ast 24 hours (Siemann & Rojiani, 2002). Materials and Methods In Vitro Clonogenic Cell Survival Assay HT29 or SCCVII cells were either treated or kept as a control group. The cells were then plated at three different concentrat ions. Ten to fourteen days late r, plates were stained and fixed with a crystal violet and meth anol solution. The number of colonies for each group, where a colony consists of at least 50 cells, was counted using a dissecting microscope and a survival curve was generated. Tumor growth delay assay Upon reaching a size of approximately 200 mm3, the HT29 xenografts or the SCCVII tumors, were randomly assigned to treatment or control groups. Treatment was then administered as daily dosing of ZD6474 or dos ing on days 1, 3, and 5 for ZD6126. The time for the tumor to grow from 200 to 1000 mm3 for the various groups was recorded. Results among groups were compared via the Wilcoxon rank sum test. Results The parental and clonal cell lines were first examined for their inherent sensitivity to a range of doses of the antiangiogenic agent, ZD6474, and the vascular disrupting agent, ZD6126. Cells were exposed to each agent individually for a period of 24 hours and then in vitro clonogenic cell survival assays were used to de termine if these two agents elicited different responses among the tumor cell lines. As can be seen in figures 5-2 and 5-3 there is no appreciable difference in the in v itro cell survival curves among the clonal and parental cell lines. In addition, it can also be seen th at both of these agents have lit tle effect on tumor cell survival.

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76 VEGF is a potent pro-angiogenic growth factor that is produced in various levels among the clonal cell lines. A previous experiment has shown that the tumor cells’ ability to induce new blood vessel growth increases with increased VEGF expression [Fig. 3-1]. However, it was unknown whether ZD6474 would be equally effec tive in blocking VEGF signaling in both low and high VEGF expressing cell lines. To determine if there was a difference in response to this agent, an intradermal assay was used. 50 mg/kg ZD6474 was given by gavage for four consecutive days, beginning with the day prior to tumo r cell inoculation. Both the parental and high expressing VEGF groups of the HT29 tu mor model showed a significant decline (approximately 2-fold) in the number of blood ve ssels growing towards the tumor inoculates [Fig. 5-4A]. The high expressing VEGF group for the SCCVII tumor model also showed a similar decrease in new blood vessel growth [Fig. 5-4B]. A previous study examining the effect of ZD6474 on tumor growth using various xenograft models showed that a si gnificant increase in the growth delay could be measured when a dose of 50 mg/kg was used (Wedge et al , 2002). In order to test th e response of the clonal cell lines to this agent, a similar growth delay as say was used. Treatment with 50 mg/kg of ZD6474 on days 1-5 and 8-12 resulted in a significant increase in the grow th delay with increasing VEGF expression (4 days for the parental cell line ve rsus 10 days for the high expressing VEGF clone) [Fig. 5-5A]. Due to its high growth rate, SCCV II tumors only received treatment on days 1-5; no significant growth delay was observed for the pare ntal or clonal cell li nes [Fig. 5-5B]. The tumors derived from the clonal cell lines were also evaluated with ZD6126. Previous studies have shown that the HT29 tumor model was not sensitive to this agent. A growth delay assay confirmed this result, but interestingly the high expressing VEGF tumor group did show a significant growth delay (9 days) when tr eated with 100 mg/kg ZD6126 Monday, Wednesday,

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77 and Friday for a period of two weeks [Fig. 56A]. Once again, the SCCVII tumor model only received treatment for one week, due to its hi gh in vivo growth rate, yet there was a trend towards increased response with increasing VEGF expression [Fig. 5-6B]. Discussion Vascular targeting agents are a relatively ne w class of agents that aim to exploit the relationship between the tumor-asso ciated vasculature and the grow th and spread of tumors. Since these agents target the s upport system of the tumor cells it is possible to inflict damage on many tumor cells by only affecting a few vessels . The current VTAs are less toxic than conventional chemotherapeutic agents and they ha ve easily accessible targ ets – endothelial cells. ZD6474 is an antiangiogenic agen t that targets pro-angiogeni c signaling by inhibiting the VEGF and EGF receptors. This agent has been f ound to be well tolerated in clinical trials and has demonstrated tumor growth inhibition in a wide variety of xenograft tumors (Wedge et al , 2002). ZD6126 is a representative VDA that se lectively targets proliferating, immature endothelial by binding to tubulin (Blakey et al , 2002). Considering that both agents target different aspects of endothelia l cells, tumor cell survival s hould not be affected following treatment with either agent. As expected, both tumor cell lines were not responsive to either agent in an in vitro setting. In vivo studies showed that the HT29 tumo rs derived from the high expressing VEGF clonal cell line were more responsive to ZD6474; this was not seen with the SCCVII tumor model. The most likely cause of this difference is the increased growth rate demonstrated by the SCCVII tumors [Fig.2-5] that did not allow for mo re that one week of treatment. In addition, there is a growing amount of evidence that sugg ests that antiangiogenic agents may be more useful in targeting smaller tumors or early-stage cancers (Lozonschi et al , 1999;Yoon et al ,

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78 1999). Therefore, the rapid growth of SCCVII tumo rs leaves a smaller window of time in which ZD6474 treatment would be most beneficial. Surprisingly, ZD6126 treatment resulted in a measurable growth delay in the HT29 tumors arising from the high VEGF expressing cl onal cell line [Fig. 5-6]. Previous studies had demonstrated that treatment with this VDA was not effective agains t the parental cell line (also observed in this experiment) for unknown reason s. The SCCVII tumors, despite receiving only one week of treatment, also appeared to trend towards an increased grow th delay with increasing VEGF expression levels. Although VTAs have been shown to slow or even inhibit tumor growth, the current consensus is that these agents will be of greater use when combined with conventional therapies, such as radiation and chemotherapy (Siemann, 2004) . The rationale for combining treatment is based on enhanced antitumor efficacy, non-overlappi ng toxicities, and spa tial cooperation (Steel & Peckham, 1979). There have already been a number of studies that have reported on the enhanced effect when the combination of ZD6474 and radiation was used (Brazelle et al , 2006;Damiano et al , 2005;Williams et al , 2004) and the combination of ZD6126 with radiation (Hoang et al , 2006;Horsman & Murata, 2003;Siemann & Rojiani, 2002).

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79 A B Figure 5-1. Schematic representi ng the different targeting appro aches of vascular targeting therapies. Antiangiogenic therapy (A) ai ms to inhibit the formation of new blood vessel growth. One antiangioge nic strategy is to target the pro-anigogenic factor VEGF, typically resulting in delayed tumo r growth. Vascular di srupting agents (B) target the existing tumor vessels, causing rapid blood vessel collapse that leads to the formation of a central necrotic region.

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80 Table 5-1. Antiangiogenic agents and vascular disrupting agents are two subclasses that fall under the general heading of va scular targeting therapies. Representative agents and their mode of action are indi cated in the table below. Vascular Targeting Therapies Antiangiogenic Agents (inhibit angiogenesis) Vascular Disrupting Agents (target existing vasculature) Avastin Cytokine modulator DMXAA Monoclonal antibodies DC101 ZD6126 ZD6474 Tubulin-binding agents CA4DP Small molecule kinase inhibitors SU11248 Ribozyme Angiozyme

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81 A B Figure 5-2. Clonogenic cell survival curve demonstrating that the in vitro sensitivity to a range of ZD6474 doses is similar among the parent al and clonal cell lin es of the HT29 (A) and SCCVII (B) tumor types.

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82 A B Figure 5-3. Clonogenic cell survival curve demonstrating that the in vitro sensitivity to a range of ZD6126 doses is similar among the parent al and clonal cell lin es of the HT29 (A) and SCCVII (B) tumor types.

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83 A B Figure 5-4. Ability of ZD6474 to modulate the a ngiogenic potential of the parental and high VEGF clonal cell line, as assessed by the intradermal assay. 1 x 105 HT29 tumor cells (A) or SCCVII cells (B) were injected at 4 sites on the ventral surface of a nude mice. 72 hours later the mice were killed and the sk in flap containing the inoculation sites was excised. The number of blood vessels intersecting each inoculate was counted and data points for each group were pooled. Asterisk (*) indicates a p-value of less than 0.05 when groups were compared via th e Wilcoxon rank sum test to the parental group.

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84 A B Figure 5-5. Response of tumors from HT29 (A) and SCCVII (B) parental and clonal cell lines to a treatment of 50 mg/kg ZD6474. Treatment was administered when the tumors reached an approximate size of 200 mm3. Asterisk (*) indicates a p-value of less than 0.05 when the growth delay of each group was compared via the Wilcoxon rank sum test to the parental group growth delay.

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85 A B Figure 5-6. Response of tumors from HT29 (A) and SCCVII (B) parental and clonal cell lines to a treatment of 100 mg/kg ZD6126. Treatment was administered when the tumors reached an approximate size of 200 mm3. Asterisk (*) indicates a p-value of less than 0.05 when the growth delay of each group was compared via the Wilcoxon rank sum test to the parental group growth delay.

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86 CHAPTER 6 SUMMARY Tumors must elicit an angioge nic response if they are to grow beyond a very small size. Once a tumor vasculature has been established, the tumor may continue to grow and it may also use these vessels as a route for metastatic spread . However, the tumor vessels that develop are highly disorganized and lead to the development of microregi ons within the tumor where poor nutrient delivery and impaired upta ke of metabolic waste products ar e present. As a result, cells residing within these areas are less likely to be proliferating and have d ecreased sensitivities to conventional therapies, such as radiat ion and chemotherapeutic agents. Angiogenesis, the growth of new blood vessels from the existing vasculature, is known to be a highly orchestrated proce ss in which there is a delicate balance between proand antiangiogenic molecules. Although th ere have been a number of angiogenic molecules identified, VEGF is considered to be the most important pro-angiogenic molecule in tumor pathology. Due to the importance of VEGF, there have been a number of pre-c linical and clinical studies that have tried to elucidate the relatio nship between VEGF expression/tumor vascularity and patient outcome. Unfortunately , the results from these studies were not in agreement: some studies claim that VEGF is a poor prognostic indicator while othe r studies profess the opposite or state that there is no relationship at all. In addition, studies that addressed the link between VEGF expression and patient outcome following therapy, such as radiation, were also inconclusive. The most probable explanation for these inequities is that inter-study comparisons can be complicated by differences in tumor type and methods used to analyze study parameters, such as VEGF expression and blood vessel visualiz ation and quantitation. Th erefore, this project centered on the idea that creating clonal cell lines that varied only in the expression of VEGF

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87 would bypass these difficulties and would be advantageous when examining tumor VEGF expression and its relationship to treatment outcome. In chapter 2, a rAAV was used to infect two different tumor types, HT29 and SCCVII, with a viral plasmid containing the human gene for VEGF165. Recombinant AAVs have been used by a variety of investigators to safely deliver gene products to various mammalian cell lines. Generally, following infection with a rAAV the viral genome is randomly inserted into the host cellular genome. Thus, by employing a high MO I, it is possible to obtain clonal cell lines that vary in the expression of the delivered ge ne product. Following the selection of cells that had been infected with the viral plasmid, c onditioned medium was screened via ELISA to quantitate the amount of secreted VEGF produ ced by each of the clones. Ultimately, three clones from each tumor type were chosen for furt her study: one that expressed VEGF at a level that was comparable to the pare ntal (non-infected) cell line, a s econd clone that expressed VEGF at an intermediate level, and a third clone that expressed VEGF at a high level. Since many growth factor signaling pathways are interconnected, ELISA s were also used to confirm that increased VEGF expression did not alter the expression of bFGF, PDGF, Ang-1, and Ang-2. In addition, RT-PCR was performed on th e clonal and parental cell lines to verify the lack of expression of VEGFR1 and VEGFR2 since expression of these receptors could result in an autocrine feedback loop. Ov erall, these findings suggested that any subsequent differences that were found among the parental and clonal cel l lines would only be due to the different amounts of VEGF ligand produced by the clonal cell lines. The in vitro growth characteristics of the cl onal cell lines were then assessed using a growth curve assay. There were no significant alterations found in the in vitro growth rate of the clonal cell lines compared to the respective parent al cell lines for both tu mor types. The tumor

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88 cells were then implanted into mice and it was found that the clonal cell lines maintained the ability to form tumors. However, the results showed that the HT29 tumor model had an increased growth rate and the tumors appeared earlier with increased VEGF expression. The SCCVII model did not display any di fferences in the time to app earance or the in vivo growth rate. Previous studies have shown that the modulati on of VEGF expression can result in altered tumor growth rates and physiol ogical characteristics. In order to assess the physiological changes that result from increased VEGF expr ession the following parameters were assessed in the clonal and parental cell lines: ability to elicit an angiogenic respons e, perfusion, areas of hypoxia, and evidence of necrosis. Increased VEGF expression resulted in an incr eased ability of the tumor cells to elicit an angiogenic response. Results from the intrader mal assay showed that the high expression VEGF clones induced 2-3 fold greater number of blood vessels than the corresponding parental cell lines. Vessel density was also shown to in crease among the clonal cell lines with increasing VEGF expression in the HT29 tumor model. Tu mor perfusion and hypoxia were also expected to differ among the clonal cell li nes. Perfusion was measured with the fluorescent DNA-binding dye Hoechst 33342 and areas of hypoxia were visual ized using an antibo dy to the bio-reductive molecule EF5. A substantial increase in tumo r perfusion and decrease in hypoxia was observed in the tumors derived from the high expressing VE GF clonal cell line as compared to the parental line. Areas of necrosis within a tumor typically o ccur at the oxygen diffusion limit. In tumors where there is a low vessel density the occurrence of necrosis is expected to be greater than in well vascularized tumors. Indeed, this was obs erved in the HT29 tumor model: increased VEGF

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89 expression/tumor vascularity resulte d in decreased evidence of necrosis. However, this was not the case for the SCCVII model and the relatively low basal level of necrosis present in the parental cell line can probably explain these resu lts (i.e. a further decreas e in necrosis would be difficult to detect). The inadequate and nonuniform vasculature that is generally associated with tumors has been linked with the failure of many anti-cance r therapies. Since the observed changes in the physiological characteristics of th e tumor cell lines were substan tial, it was conceivable that these differences could impact the response of th e tumors derived from the clonal cell lines to anti-cancer therapies. Therefor e, the clonal cell lines were used to assess the impact of VEGF expression/tumor vascularity on the response to radiation and vascular targeting agents. Chapter 4 examined the relationship between VEGF expression and tumor response to radiation. In vitro data colle cted concluded that there was no significant difference in the inherent sensitivity among the clonal and parent al cell lines. However, the in vivo data demonstrated that highly vascul arized tumors responded significantly better to a single dose of radiation. Given the increased tumor perfusion and decreased areas of hypoxia, this result was not altogether surprising. It has been known fo r decades that hypoxia can confer resistance to a variety of tissue types. Typically, most cancer patients will receive sm all, daily (Monday through Friday) doses of radiation for several weeks. Si nce a single dose of radiation resu lted in a significant increase in radiation response, a fr actionated schedule was also investig ated. Again, an increased response to radiation was evident in th e SCCVII tumor model when a dose of 3 Gy/day was given for 5 consecutive days. There did not appear to be a significant difference between HT29 tumors derived from the high expressing VEGF clonal cell lin e and tumors arising from the parental cell

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90 line when a dose of 2 Gy/day was given for 10 days. A likely explanation for this finding involves the principle of reoxygenation. When tumors are irradiated aerobic cells are preferentially killed, leaving a remaining contin gent of hypoxic cells. In some tumor types these hypoxic cells undergo a process by which they ar e able to gain access to oxygen supplies. During subsequent irradiations the newly-aerobic cells (formally hypoxic) can then be killed. Thus, when giving multiple fractions of radiatio n, the negative effects of hypoxia are mitigated. In chapter 5 the impact of VEGF expression on tumor response to vascul ar targeting agents was examined. In vitro clonogenic cell survival assays demonstrated that there was no significant difference among the clonal and parent al cell lines to the antiangiogenic agent, ZD6474, or the vascular disrupting agent, ZD6126. In addition, these agents had little impact on the in vitro survival of the tumor cell lines, wh ich was not surprising gi ven the fact that both agents target endothelial (not tumor) cells. Initially, an intradermal assay was used to determine the efficacy of ZD6474 against the parental and high expressing VEGF clonal cell lines. In both tumor models, 50 mg/kg ZD6474 was able to decrease the number of blood vesse ls growing towards the tumor cell inoculates by approximately 2-fold. Further studies demonstrat ed a significantly increased growth delay with increasing VEGF expression when HT29 tumor-beari ng mice were treated with daily doses of 50 mg/kg ZD6474. This enhanced response with in creasing VEGF expression was not observed in the SCCVII model and is probably due to the high in vivo growth rate of this tumor model. Previous studies have shown that, fo r unknown reasons, the HT29 tumor model is effectively resistant to ZD6126. Interestingly, a significant grow th delay was observed in the tumors arising from the high expressing VEGF cl onal cell line while tumors derived from the

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91 parental cell line did not show any growth dela y. The SCCVII tumor model also showed a trend towards increased response to ZD6126 with increasing VEGF expression. The work comprising this dissert ation leads to several interesting questions. First, the mechanism behind the difference in basal vascul arity between the two tumor types, SCCVII and HT29, is unknown. SCCVII is a murine line and it is conceivable that the pa rental cells already express high levels of mouse VEGF. If this were the case then mouse VEGF expression may contribute significantly to the resultant tumor vasc ularity; in fact it might be the predominant factor determining the extent of tumor vascularit y even for tumor cells expressing varying levels of human VEGF. This would also explain why th e vascular differences were less apparent for tumors derived from the various SCCVII clones th an those of the HT29 clones. Unfortunately it is not possible to resolve this issue since the mouse VEGF expression levels of the SCCVII cells were not measured. In addition, while VEGF, bFGF, PDGF, and the angi opoietins are critical pro-angiogenic factors in tumor pathogenesis, th is list is by no means exhaustive. Evaluating the expression levels of other factors, such as the interleukins and IGF-1, may yield clues as to the large difference between the two tumor types as well. The behavior of the HT29 tumor model in regard s to the efficacy of the vascular disrupting agent ZD6126 also raises questions. Tumors ar ising from the HT29 parental cell line do not display a growth delay when ZD6126 is admini stered for a period of two weeks (100 mg/kg given Monday, Wednesday, Friday). However, tumors arising from the high VEGF clonal cell line demonstrate a 9-day growth delay in respons e to ZD6126. It may be interesting to explore why the parental line does not appear to be aff ected by this agent, yet a high expressing VEGF line is significantly affected.

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92 Finally, the extent of benefit in combining vascular targeti ng therapies with conventional therapies in these models also remains to be addressed. Studies have shown that combining VTAs with conventional therapy, such as radiati on, generally has an additiv e (potentially greater than additive) effect. However, it is not known if these observati ons will be consistent with the high expressing VEGF clonal cell line s. Overall, the results from this body of work suggest that VEGF expression/tumor vascularity may be an important factor to consid er in patient treatment.

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94 Chen,C.A., Cheng,W.F., Lee,C.N., Chen,T.M ., Kung,C.C., Hsieh,F.J., & Hsieh,C.Y. (1999) Serum vascular endothelial gr owth factor in epithelial ova rian neoplasms: correlation with patient survival. Gynecol.Oncol. , 74 , 235-240. Chung,G.G., Yoon,H.H., Zerkowsk i,M.P., Ghosh,S., Thomas,L., Harigopal,M., Charette,L.A., Salem,R.R., Camp,R.L., Rimm,D.L., & Burt ness,B.A. (2006) Vasc ular endothelial growth factor, FLT-1, and FLK-1 analysis in a pancreatic cancer tissue microarray. Cancer , 106 , 1677-1684. Church,G.M. & Gilbert,W. (1984) Genomic sequencing. Proc.Natl.Acad.Sci.U.S.A , 81 , 19911995. Claesson-Welsh,L. (1994) Platelet-derived growth factor re ceptor signals. J.Biol.Chem. , 269 , 32023-32026. Clark,D.E., Smith,S.K., He,Y., Day,K.A., Li cence,D.R., Corps,A.N., Lammoglia,R., & Charnock-Jones,D.S. (1998) A vascular endothe lial growth factor antagonist is produced by the human placenta and released into the maternal circulation. Biol.Reprod. , 59 , 15401548. Cronauer,M.V., Schulz,W.A., Seifert,H.H., Ac kermann,R., & Burchardt,M. (2003) Fibroblast growth factors and their receptors in urol ogical cancers: basic research and clinical implications. Eur.Urol. , 43 , 309-319. Cross,M.J., Dixelius,J., Matsumoto,T., & Cl aesson-Welsh,L. (2003) VEGF-receptor signal transduction. Trends Biochem.Sci. , 28 , 488-494. Czubayko,F., Liaudet-Coopman,E.D., Aigner,A., Tuveson,A.T., Berchem,G.J., & Wellstein,A. (1997) A secreted FGF-binding protein can se rve as the angiogenic switch in human cancer. Nat.Med. , 3 , 1137-1140. Damiano,V., Melisi,D., Bianco,C., Raben,D., Caputo,R., Fontanini,G., Bianco,R., Ryan,A., Bianco,A.R., De Placido,S., Ciardiello,F., & Tortora,G. (2005) Cooperative antitumor effect of multitargeted kina se inhibitor ZD6474 and ionizing radiation in glioblastoma. Clin.Cancer Res. , 11 , 5639-5644. Davis,P.D., Dougherty,G.J., Blakey,D.C., Galbraith,S.M., Tozer,G.M., Holder,A.L., Naylor,M.A., Nolan,J., Stratford,M.R., Chaplin,D.J., & Hill,S.A. (2002) ZD6126: a novel vascular-targeting agent that causes selective destruc tion of tumor vasculature. Cancer Res , 62 , 7247-7253. de Vries,C., Escobedo,J.A., Ueno,H., Houck,K ., Ferrara,N., & Williams,L.T. (1992) The fmslike tyrosine kinase, a receptor for vascular endothelial growth factor. Science , 255 , 989-991. Denekamp,J. (1986) Cell kine tics and radiation biology. Int.J.Radiat.Biol.Relat Stud.Phys.Chem.Med. , 49 , 357-380.

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108 BIOGRAPHICAL SKETCH Christina Michele Norris was born on December 22, 1979, in Fairfax, Virginia. She received her high school diploma in June 1997 from Osbourn Park High School in Manassas, Virginia and subsequently enrolled in the Univers ity of Florida. Christin a received her Bachelor of Science degree in biochemistry with highes t honors (minors in mathematics and zoology) in May 2001. She then worked for one year in a bi ochemistry laboratory before enrolling in the University of Florida’s graduate interdiscip linary program. In May 2003, Christina joined the laboratory of Dietmar W. Siemann and began pursui ng her doctoral studies in the department of pharmacology and experimental therapeutics. Fo llowing graduation, she hope s to continue work in translational oncology research.