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Spectral Imaging of Tumor Microvascular Oxygenation Changes after Administration of Vascular Disrupting Agent OXi4503

Permanent Link: http://ufdc.ufl.edu/UFE0022689/00001

Material Information

Title: Spectral Imaging of Tumor Microvascular Oxygenation Changes after Administration of Vascular Disrupting Agent OXi4503
Physical Description: 1 online resource (61 p.)
Language: english
Creator: Wankhede, Mamta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: chamber, disrupting, hemoglobin, hyperspectral, model, oxi4503, saturation, spectral, targeting, tumor, vasculature, vessel, window
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Vascular disrupting agent-induced alterations in tumor and normal tissue vasculature are essential parameters for the assessment of drug-efficacy and its effect on combinatorial cancer therapies. Time-based changes in tumor microvasculature structure and function after treatment with OXi4503 have been previously reported. For example, time-dependent decrease in tumor perfusion, increased tumor vascular permeability and substantial tumor microvascular damage has been reported after treatment with OXi4503 indicating efficacy of the drug. Furthermore, persistent tumor rim vessels and recovery of tumor perfusion at later time points after OXi4503 treatment have been observed implying incomplete tumor destruction. Vascular oxygenation information obtained from spectral imaging could give further insight into these observed phenomena. Hyperspectral imaging is quantitative method providing spatial maps of microvascular blood oxygenation in terms of hemoglobin saturation. This technique can be utilized to image blood oxygenation and create maps of hemoglobin saturation in blood vessels. The present work demonstrates the utility of hyperspectral imaging for investigating the structural alterations as well as oxygenation changes in the tumor vasculature and the normal tissue vasculature after repeat treatments with OXi4503. Highly metastatic mouse mammary adenocarcinoma cell line 4T1 tumors were grown (7-8 days old, 3-4 mm size) in athymic (nu/nu) female mouse dorsal skin flap window chambers and treated with OXi4503 (10 mg/kg, IP) three times at an interval of 48 hours. Hemoglobin saturation maps were obtained immediately prior and post-treatment, and also at 2, 4, 6, 8, 24, and 48 hours post-treatment. Immediately after each treatment the microvascular-structure started collapsing from the tumor core towards the periphery. Progressive hemorrhaging of some tumor core microvascular network was observed between 2 to 8 hours post-treatment, while the remaining tumor interior vessels were rendered non-functional. The tumor rim vessels and normal tissue vasculature remained structurally intact. Synchronized with the tumor microstructural breakdown, the hemoglobin saturation of the tumor vessels plummeted rapidly from core towards tumor periphery between 2 to 8 hours after drug administration. Tumor rim vessels remained oxygenated initially but a delayed deoxygenation occurred from 4 to 6 hour post-treatment. Onset of reoxygenation and vessel recovery was noticed near the rim at 8 hours. Rapid vascular regrowth of the viable vessels, their reoxygenation, and neovascularization occurred 24 to 48 hours post-treatment. Notably, transient fluctuations in hemoglobin saturation values of the tumor rim and normal vasculature were noticed predominantly at 4 hours after drug administration indicating drug impact on normal vasculature oxygenation and blood flow. To investigate whether the treatment response of OXi4503 was altered when used on a different tumor type, the experiment was repeated with Caki-1 renal carcinomas. It was observed that treatment of Caki-1 tumors progressed similar to that of Oxi4503, except that the recovery phase for Caki-1 tumor vasculature initiated earlier. These findings could be pivotal in time-scheduling the combinatorial cancer treatments such as chemotherapy and radiation therapy.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mamta Wankhede.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Sorg, Brian.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022689:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022689/00001

Material Information

Title: Spectral Imaging of Tumor Microvascular Oxygenation Changes after Administration of Vascular Disrupting Agent OXi4503
Physical Description: 1 online resource (61 p.)
Language: english
Creator: Wankhede, Mamta
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: chamber, disrupting, hemoglobin, hyperspectral, model, oxi4503, saturation, spectral, targeting, tumor, vasculature, vessel, window
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Vascular disrupting agent-induced alterations in tumor and normal tissue vasculature are essential parameters for the assessment of drug-efficacy and its effect on combinatorial cancer therapies. Time-based changes in tumor microvasculature structure and function after treatment with OXi4503 have been previously reported. For example, time-dependent decrease in tumor perfusion, increased tumor vascular permeability and substantial tumor microvascular damage has been reported after treatment with OXi4503 indicating efficacy of the drug. Furthermore, persistent tumor rim vessels and recovery of tumor perfusion at later time points after OXi4503 treatment have been observed implying incomplete tumor destruction. Vascular oxygenation information obtained from spectral imaging could give further insight into these observed phenomena. Hyperspectral imaging is quantitative method providing spatial maps of microvascular blood oxygenation in terms of hemoglobin saturation. This technique can be utilized to image blood oxygenation and create maps of hemoglobin saturation in blood vessels. The present work demonstrates the utility of hyperspectral imaging for investigating the structural alterations as well as oxygenation changes in the tumor vasculature and the normal tissue vasculature after repeat treatments with OXi4503. Highly metastatic mouse mammary adenocarcinoma cell line 4T1 tumors were grown (7-8 days old, 3-4 mm size) in athymic (nu/nu) female mouse dorsal skin flap window chambers and treated with OXi4503 (10 mg/kg, IP) three times at an interval of 48 hours. Hemoglobin saturation maps were obtained immediately prior and post-treatment, and also at 2, 4, 6, 8, 24, and 48 hours post-treatment. Immediately after each treatment the microvascular-structure started collapsing from the tumor core towards the periphery. Progressive hemorrhaging of some tumor core microvascular network was observed between 2 to 8 hours post-treatment, while the remaining tumor interior vessels were rendered non-functional. The tumor rim vessels and normal tissue vasculature remained structurally intact. Synchronized with the tumor microstructural breakdown, the hemoglobin saturation of the tumor vessels plummeted rapidly from core towards tumor periphery between 2 to 8 hours after drug administration. Tumor rim vessels remained oxygenated initially but a delayed deoxygenation occurred from 4 to 6 hour post-treatment. Onset of reoxygenation and vessel recovery was noticed near the rim at 8 hours. Rapid vascular regrowth of the viable vessels, their reoxygenation, and neovascularization occurred 24 to 48 hours post-treatment. Notably, transient fluctuations in hemoglobin saturation values of the tumor rim and normal vasculature were noticed predominantly at 4 hours after drug administration indicating drug impact on normal vasculature oxygenation and blood flow. To investigate whether the treatment response of OXi4503 was altered when used on a different tumor type, the experiment was repeated with Caki-1 renal carcinomas. It was observed that treatment of Caki-1 tumors progressed similar to that of Oxi4503, except that the recovery phase for Caki-1 tumor vasculature initiated earlier. These findings could be pivotal in time-scheduling the combinatorial cancer treatments such as chemotherapy and radiation therapy.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Mamta Wankhede.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Sorg, Brian.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022689:00001


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SPECTRAL IMAGING OF TUMOR MICRO VASCULAR OXYGENATION CHANGES AFTER ADMINISTRATION OF VASC ULAR DISRUPTING AGENT OXI4503 By MAMTA WANKHEDE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008 1

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2008 Mamta Wankhede 2

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To my Mom 3

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ACKNOWLEDGMENTS I thank my family, friends and colleagues for their love, friendship, and support. In this long journey, their undying faith gave me the st rength and motivation to keep going. I would like to thank my mentor Dr. Brian Sorg, who gave me every opportunity to excellence and growth. Finally, I thank the University of Florida, an amazing institution, for let ting me be part of a culturally and academically diverse and dynamic envi ronment. I am very proud to be a Gator. Go Gators! 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF FIGURES .........................................................................................................................7 ABSTRACT .....................................................................................................................................9 CHAPTER 1 BACKGROUD AND SIGNIFICANCE.................................................................................11 1.1 Physiological and Pa thological Angiogenesis ................................................................11 1.2 Abnormal Tumor Vasculature ........................................................................................12 1.3 Tumor Vasculature as a Therapeutic Target ...................................................................12 1.4 Vascular Targeting Agents .............................................................................................12 1.5 Small Molecule Vascular Disrup ting Agent OXi4503: Classification of VDAs ...........14 1.6 Goal.................................................................................................................................17 2 MATERIALS AND METHODS...........................................................................................18 2.1 Cell Culture .....................................................................................................................18 2.2 Animal Model and Imaging ............................................................................................20 2.3 Drug Preparation a nd Experimental Design ...................................................................20 2.4 Imaging System ..............................................................................................................21 2.5 Image Acquisition ...........................................................................................................23 2.6 Image Processing ............................................................................................................23 3 RESULTS...................................................................................................................... .........26 3.1 Treatment Results for the 4T1 Mouse Mammary Carcinoma Cell Line ........................26 3.1.1 Graphic Evidence.................................................................................................26 3.1.2 Quantification .......................................................................................................27 3.2 Control Results for the 4T1 Mouse Mammary Carcinoma Cell Line ............................29 3.2.1 Graphic Evidence.................................................................................................29 3.2.2 Quantification .......................................................................................................29 3.3 Treatment Results for the Caki-1 Renal Carcinoma Cell Line .......................................29 3.3.1 Graphic Evidence.................................................................................................30 3.3.2 Quantification .......................................................................................................31 3.4 Temporal Fluctuations in Hemogl obin Saturations Preand Post-Treatment ................31 3.5 Transient Fluctuations in Tumor Ri m and Normal Vasculature PostTreatment ..........32 4 DISCUSSION................................................................................................................... ......52 5 CONCLUSION................................................................................................................... ....57 5

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LIST OF REFERENCES ...............................................................................................................58 BIOGRAPHICAL SKETCH .........................................................................................................61 6

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LIST OF FIGURES Figure page 1-1 Mechanisms of action of vari ous vascular targeting agents. .............................................14 2-1 The window chamber model.. ............................................................................................22 2-2 Zeiss-based imaging microscope system. ..........................................................................22 2-3 Image processing method. .................................................................................................25 3-1 Brightfield images of OXi4503 treatmen t progression through the various imaging time points.. ........................................................................................................................33 3-2 Example of neovascularization observed between 8 to 48 hours post-treatment along the tumor rim. Vessels sprouting from periphery towards tumor core. Original tumor vessels grew more prominent afte r recovery. (4T1 mouse mammary adenocarcinoma). ...............................................................................................................34 3-3 Hemoglobin saturation (HbSat) maps of the brightfield images in Figure 3-1. ................35 3-4. Hemoglobin saturation maps of the images in Figure 3-2.. ...............................................36 3-5 Example of longitudinal fluctuations in vessel oxygenation af ter drug treatment. (4T1 mouse mammary adenocarcinoma). ..........................................................................37 3-6 Classification of core, periphery and normal areas.. ..........................................................38 3-7 Analysis of temporal fluctuations in core, periphery and normal vessels for tumor shown in Figure 3-1 (treatment mouse 1) and 3 other mice.. ............................................39 3-8 Combined averages of hemoglobin saturations in 4 mice from Figure 3-7. ......................40 3-9 Brightfield images of control mouse treated with carrier only (4T1 mouse adenocarcinoma). ...............................................................................................................41 3-10 Hemoglobin saturation maps of the images in Figure 3-9. ................................................42 3-11 Analysis of temporal fluctuations in co re, periphery and normal vessels of the tumor shown in Figure 3-9.. .........................................................................................................43 3-12 Brightfield images of OXi4503 treatmen t progression (Caki-1 renal carcinoma cell line). ...................................................................................................................................44 3-13 Example of neovascularization observed between 8 to 48 hours post-treatment along the tumor rim. (Caki-1 renal carcinoma cell line). .............................................................45 3-14 Hemoglobin saturation (HbSat) maps of the brightfield images in Figure 3-12. ..............46 7

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3-15 Hemoglobin saturation maps of the images in Figure 3-13.. .............................................47 3-16 Individual vessel analysis for th e Caki-1 tumor shown in Figure 3-12.. ...........................48 3-17 Analysis of temporal fluctuations in co re, periphery and normal vessels of the Caki-1 tumor in Figure 3-12.. ........................................................................................................49 3-18 Example of hemoglobin saturation fluctuat ions in the tumor vasculature before and after OXi4503 treatment.. ..................................................................................................50 3-19 Transient fluctuations in tumor rim and normal vasculature. ............................................51 8

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Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SPECTRAL IMAGING OF TUMOR MICRO VASCULAR OXYGENATION CHANGES AFTER ADMINISTRATION OF VASC ULAR DISRUPTING AGENT OXI4503 By Mamta Wankhede August 2008 Chair: Brian Sorg Major: Biomedical Engineering Vascular disrupting agent-indu ced alterations in tumor and normal tissue vasculature are essential parameters for the assessment of drugefficacy and its effect on combinatorial cancer therapies. Time-based changes in tumor microva sculature structure and function after treatment with OXi4503 have been previously reported. Fo r example, time-dependent decrease in tumor perfusion, increased tumor vascular permeability and substantial tumor microvascular damage has been reported after treatment with OXi4503 indicating efficacy of the drug. Furthermore, persistent tumor rim vessels and recovery of tumor perfusion at later time points after OXi4503 treatment have been observed implying incomp lete tumor destruction. Vascular oxygenation information obtained from spectral imaging coul d give further insigh t into these observed phenomena. Hyperspectral imaging is quant itative method providing spatial maps of microvascular blood oxygenation in terms of he moglobin saturation. This technique can be utilized to image blood oxygenation and create ma ps of hemoglobin saturation in blood vessels. The present work demonstrates the utility of hyperspectral imaging for investigating the structural alterations as well as oxygenation changes in the tumor vasculature and the normal tissue vasculature after rep eat treatments with OXi4503. 9

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Highly metastatic mouse mammary adenocarcino ma cell line 4T1 tumors were grown (7-8 days old, 3-4 mm size) in athym ic (nu/nu) female mouse dorsal skin flap window chambers and treated with OXi4503 (10 mg/kg, IP) three times at an interval of 48 hours. Hemoglobin saturation maps were obtained immediately prio r and post-treatment, and also at 2, 4, 6, 8, 24, and 48 hours post-treatment. Immediately after each treatment the microvascular-structure started collapsing from the tumor core towards the periphery. Progressive hemorrhaging of some tumor core microvascular network was observed between 2 to 8 hours post-treatment, while the remaining tumor interior vessels were rendere d non-functional. The tumor rim vessels and normal tissue vasculature remained structur ally intact. Synchronized with the tumor microstructural breakdown, the hemoglobin saturation of the tumor vessels plummeted rapidly from core towards tumor periphery between 2 to 8 hours after drug administration. Tumor rim vessels remained oxygenated initially but a de layed deoxygenation occurred from 4 to 6 hour post-treatment. Onset of reoxygenation and vessel recovery was noticed near the rim at 8 hours. Rapid vascular regrowth of the viable vessels, their reoxygenation, and neovascularization occurred 24 to 48 hours post-treatment. Notably, tr ansient fluctuations in hemoglobin saturation values of the tumor rim and normal vasculature we re noticed predominantly at 4 hours after drug administration indicating drug impact on norma l vasculature oxygenation and blood flow. To investigate whether th e treatment response of OXi4503 was a ltered when used on a different tumor type, the experiment was repeated with Caki-1 renal carcinomas. It was observed that treatment of Caki-1 tumors progr essed similar to that of Oxi4503, except that the recovery phase for Caki-1 tumor vasculature ini tiated earlier. These findings coul d be pivotal in time-scheduling the combinatorial cancer treatments such as chemotherapy and radiation therapy. 10

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CHAPTER 1 BACKGROUD AND SIGNIFICANCE 1.1 Physiological and Pathological Angiogenesis Angiogenesis is the sprouting of new vasc ulature from the pre-existing vessels. Physiological angiogenesis is observed during fe tal growth, wound healing process as well as in female reproductive system and gradually become s a rare event in healthy adults. Under normal physiological conditions, angiogenesis is a tightly controlled mechanism with the pro-angiogenic factors delicately balancing th e anti-angiogenic factors[1]. A functional vascular supply is es sential for solid tumors to be able to proliferate beyond a few millimeters in size. The rapidly growing tumor mass demands for fast growth of new vasculature to fulfill the excessive oxygen and nut rients requirements. One of the mechanisms known to be used by the tumors to initiate this pathological angiogenesis is known as the "Angiogenic switch". The angiogenic switch is considered to be turned on when the proangiogenic factors out-balance the anti-angiogenic factors. Nu merous factors are known to be responsible for turning on the angiogenic switch including hypoxia, hypoglycemia, oncogenic transformations, or on autocrine growth factor loops[2]. Oxygen deprivation, commonly known as hypoxia, induces angiogenesis in tumors via increase in the hypoxia-in ducible transcription factor-1 (HIF-1 ), which in turn up-regulates the vasc ular endothelial grow th factor (VEGF). VEGF is considered among one of the most impo rtant regulators of angiogenesis. Nutrient deprivation, also called as hypoglycemia, induces VEGF expression through a pathway independent of HIF. Growing tumor mass is co mmonly inflicted with hypoxic and hypoglycemic insults due to mismatch in demand and supply ra te. Angiogenesis and vasculogenesis can thus be called as inherent characteristics of the solid tumors. 11

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1.2 Abnormal Tumor Vasculature Even though pathological conditions such as tu mors trigger excessive vascular growth via mechanisms such as angiogenesis, this new vasc ulature differs from norma l tissue vasculature in several ways. In order to m eet the ever-growing nutrient a nd oxygen supply demands of the proliferating tumors, the vasculature growth tries to keep pace, but eventually fails. In most big tumors the core remains comparatively non-vasc ularized. Vasculature in the remaining tumor areas is often found to be lea ky, unorganized, and deformed resu lting in a vasculature system which is maximally stimulated yet minimally fu lfilling the metabolic needs of the host tumor mass[3]. Other vascular abnormalities include tem porary occlusions, rapidl y dividing endothelial cells, blind ends, and a reduction in pericytes[4]. This results in heterogeneously distributed hostile micro-milieus within the tumor mass characterized by parameters such as hypoxia, hypoglycemia, extra-cellular acidosis etc. These hos tile parameters maybe directly or indirectly responsible for selection of aggressive cells, tumor progression and acquired treatment resistance. 1.3 Tumor Vasculature as a Therapeutic Target Tumor vasculature plays essential role in tumor survival, tumor metastasis and progression, and in controlling the tumor microe nvironment, thereby influencing conventional therapies[5]. Targeting tumor va sculature could thus have devastating effects on the tumor. Based on these grounds, strategies targeting tumo r blood vessels have been actively developed over the years[6-10]. 1.4 Vascular Targeting Agents The idea of exploiting the tumor vasculature as a form of treatment has been around since mid-1800s. Innovative strategies including bacter ial inductions, use of lead colloids and arsenicals, colchicines were implemented thr ough 1900s. However, in 1982, Juliana Denekamp 12

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proposed to use the specificity of tumor endotheli al cells as a targeting approach. She suggested that the much higher prolifera tion rate of tumor endothelial cells compared to normal tissue could be specifically targeted to destroy the tumor vasculature. Recently this approach has gained momentum and a number of different agents are being developed selectively and speci fically targeting the tumor va sculature without damaging the normal vasculature[11-16]. Collectively these agen ts are known as Vascular Targeting Agents (VTAs). The VTAs can be broadly classified as an ti-angiogenic agents (AIs) and vascular disrupting agents (VDAs) depending upon their va scular targets. AIs aim at preventing the tumor-initiated angiogenic process, i.e. they cont rol or reduce the further proliferation of tumor vasculature by suppressing angiogenesis. VDAs target the estab lished tumor vasculature in solid tumors. They cause a rapid and selective damage in tumors, resulting in secondary tumor cell death due to ischemia[1]. VDAs are particularly suitable for treating large tumors which are typically resistant to conventi onal cytotoxic therapies[13, 17]. Complete tumor eradication with either AIs or VDAs acting alone is highly unlikely. In case of AIs, blocking one or some of the angiogenesis pathways is not sufficient, considering the complexities of pathways available for neo-vascularization[18, 19]. The viable rim problem associated with VDA treatment was first propos ed by William Woglom in as early as 1923[20]. Even though VDAs cause extensive tumor-core necros is, a thin layer of tumor periphery remains well fed by the surrounding normal vessels resu lting in a viable rim. The VDAs cannot overcome the viable rim problem resulting in incomplete treatment[13]. However, the combination of VDAs with many conventional and emerging therapeuti c strategies, including anti-angiogenics, has shown promise in pre-clinical models[21, 22]. 13

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Figure 1-1 gives the taxonomy and examples of vascular targeting agents. This method of classification allows description of the targets of each group and their ke y mechanisms of action. For example, Bevacizumab is an anti-angiogeni c agent that functions through inhibition of VEGF activity. Similarly, Combretastatin (CA4P) is a VDA that functions by binding to tubulin of proliferating endothelial cells. Figure 1-1. Mechanisms of action of va rious vascular targ eting agents[23]. 1.5 Small Molecule Vascular Disrupting Agent OXi4503: Classification of VDAs Vascular disrupting agents essentially refer to all the agents which aim at destroying the well established tumor microvasc ulature without damaging the normal vessels. These agents range from physical treatments such as hype rthermia or photodynamic therapy (PTD), to induction of cytokines such as tumor necrotic f actor (TNF) and interleukins, chemotherapeutics 14

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such as Vinca alkaloids and arseni c trioxide (ATO), antibodies, peptides, and growth factors that selectively bind to tumor vessels and ultimately destroy them[5]. Small-molecule vascular targeting agents are rapidly becoming the most popular VDAs[5]. As evident from Table 1-1, majority of VDAs in c linical trials use inhibi tion of tubulin assembly as their mechanism of action[1]. Table 1-1. Small-molecule VDAs in active clinical development. Adopted from[1]. Compound Mechanism Current development status Tumor blood flow reductions in patients Company CA4P (Zybrestat) Tubulin depolymerizing agent Phase II and III clinical trials ongoing Yes OXiGENE, Inc. DMXAA (ASA404) TNF induction Phase II clinical trials completed Yes Antisoma/Novar tis MN-029 (Denebulin) Tubulin depolymerizing agent Phase I clinical trials completed Yes Medicinova AVE8062 (AC7700) Tubulin depolymerizing agent Phase I clinical trials ongoing Yes Sanofi-Aventis ZD6126 Tubulin depolymerizing agent Phase I clinical trials completed Yes Angiogene Pharmaceuticals Ltd OXi4503 Tubulin depolymerizing agent Phase I clinical trials ongoing Studies ongoing OXiGENE, Inc NPI2358 Tubulin depolymerizing agent Phase I clinical trials ongoing Studies ongoing Nereus Pharmaceuticals CYT997 Tubulin depolymerizing agent Phase I clinical trials ongoing Studies ongoing Cytopia MPC-6827 Tubulin depolymerizing agent Phase I clinical trials ongoing Studies ongoing Myriad Pharmaceuticals There are two major sub-categ ories of small molecule VDAs. The first sub-category utilizes the cytokine induction capacity of agents like flavone acetic acid (FAA) and its derivative 5, 6-dimethylxanthenone-4-acetic ac id (DMXAA). The second sub-category utilizes the tubulin-binding capability of agents such as Combretastatin A4 Phosphate (CA4P), the phosphate prodrug of N-acetyl-colchinol (ZD6126), Ave8062, NPI2358, MN-029, and OXi4503. 15

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OXi4503 is the diphosphate prodrug of Combreta statin A1 (CA1), which along with its predecessor Combretastatin A4 Phosphate (CA4P) is isolated from the South African tree Combretum Caffrum and is considered to be amo ng the most potent tubulin binding agents[24]. When used as a single agent against the murine breast adenocarcinoma CaNT the murine colon tumor MAC29, the human adenocarcinoma MDA-MB-231 and the murine myocardial endothelioma MHEC5-T, OXi4503 exhibits a stronge r antivascular and antitumor effect than CA4P[24]. Oxi4503 has been shown to retard tumor growth in a dose-dependent manner and improved survival in murine model of colorect al liver metastases[25]. CA4P, the analogue of OXi4503, has been shown to enhance the effects of radiation, hyperthermia, chemotherapy, and radio-immunotherapy[4]. Studies have shown that this second gene ration vascular disr upting agent OXi4503 not only possesses significant antivascular effects in solid tumors but also follows a similar treatment-response -trend after s ubsequent repeat treatments[4]. OXi4503 preferentially binds to the -tubulin subunits inside the dividing endothelial cells, thereby preventing the formation of microtubul es. The cytoskeleton of the proliferating endothelial cells gets disrupted which in turn results in e ndothelial cell shap e changes. The endothelial cells round up, de tach from the vascular wall, and fina lly the vascular wall collapses. This vascular collapse and the subsequent th rombus formation blocks the vessel blood supply resulting in tumor cell death and extensive necrosis due to lack of oxygen, nutrition, and metabolic waste removal[4, 26]. While experimental evidence has confirmed the selective tumor vessel targeting and immediate microvascular destruc tion capacity of OXi4503, the viabl e rim problem is still not overcome even after using the maximum tolera ble doses (MTD) of th e drug[25]. Since the 16

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peripheral tumor cells obtain nutrition mainly from the surrounding normal vessels, these few peripheral tumor cells survive resulting in incomplete tumo r destruction. Consequently, a combination therapy of OXi4503 with traditional anticance r therapies is suggested for complete tumor eradication[4]. 1.6 Goal The present work aimed at using spectral imaging technique for wide-field regional assessment of oxygen transport at microvessel le vel after repeat trea tment with vascular disrupting agent OXi4503. Hyperspectral imaging was used to create sp atial maps of vessel oxyge nations in terms of hemoglobin saturation (HbSat) to gi ve a quantitative, direct and r eal time evidence of structural and functional changes in tumor as well as norma l vasculature after rep eat treatments with OXi4503. 17

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CHAPTER 2 MATERIALS AND METHODS 2.1 Cell Culture Highly metastatic 4T1-GFP mouse mammary adenocarcinoma cell line was used. 4T1GFP constitutively expressed green fluorescent protein (GFP). The cells were donated by Mark W. Dewhirst (Duke University Medical Center Durham, NC). This cell line made a good candidate for the current study due to several reasons. It enabled us to have a syngeneic cancer model since 4T1 cell line originally comes fr om a spontaneously arising BALB/c mammary tumor and its metastatic properties have been extensively characterized[27]. Also problems associated with growing human derived tumor cell lines such as slow growth, inefficient metastasis etc were eliminated. The cells were cultured in 75 cm 2 flasks (NUNC Inc., catalogue # 12565350) as a monolayer in a growth media comprising of DMEM (Cellgro Inc.,1X, 4.5 g/l glucose, Lglutamine and sodium pyruvate, catalogue # 10-0 13-CV), 10% fetal bovine serum (Biowhittaker Inc., catalogue # 14-501F), 1% L-glutamine (Clonogen Inc., catalogue # 25-0050-CL), and 1% penicillin streptomycin (Clonogen Inc., catalogue # 30-002-CL). For long term storage of the cells in liquid Nitrogen, freeze media was prepar ed by adding 10% DMSO (Fisher Scientific Inc., catalogue # D128-500) to the growth media. The cells were stored in -80 C overnight tight sealed in Styrofoam box, and transferred into -270 C liquid nitrogen stor age the next day. This temperature gradient helped the cells to better ad just to the thermal shoc k. As per need, the cells vials were thawed out from the liquid nitrogen frozen stock. The cells were passaged a couple of times before using them in vivo so as to ensu re total recovery from thermal shock and normal growth rate. 18

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To obtain a single cell suspension, the cells were washed with sterile PBS (Hyclone Inc., 1X, 0.0067M (PO 4 ) with calcium and magnesium, catal ogue # SH30256.01) and treated with 0.05% Trypsin/EDTA (Clonogen Inc.,1X, 0.25% Tr ypsin/2.2 mM EDTA in HBSS, catalogue # 25-053-CL) for about 3 minutes. After 3 minutes th e growth media was added to deactivate the Trypsin/EDTA. This single cell suspension was ce ntrifuged for 5 minutes at 1000 rpm to obtain a cell pellet. After discarding th e supernatant the cell pellet was re-suspended in plain DMEM. Hemacytometer was used to count the number of cells per milliliter of volume. For counting purpose a 10% diluted sample was prepared in a microcentrifuge tube comprising of 10% cell suspension, 10% Trypan blue (Clonogen Inc.,0.4% w/v in normal saline, catalogue # 25-900CL), and 80% DMEM. A 10 L volume of this prepared sample was inserted on ea ch side of the Hemacytometer and total number of cells was co unted over a total of 10 squares (dimension of each square = 1mm x 1mm). The following formula was used to give the number of cells per milliliter of media. Caki-1, human derived renal carcinoma ce lls were kindly provided by Dietmar W. Siemann (University of Florida, Shands Cancer Center, Gainesville, FL). Tumor grafts were initiated by inoculating 2 x 10 5 to 5 x 10 5 tumor cells into a single hind limb of the donor mice. When the tumors were about 200mm 3 in size, the tumors were excised and a single cell suspension was obtained in DMEM. About 1 x 10 6 cells were used to implant the tumor in the experimental mice. 19

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2.2 Animal Model and Imaging All in vivo procedures were conducted in accordance with the protocol approved by University of Florida Institutional Animal Ca re and Use Committee. Athymic (nu/nu) female mice weighing at least 21g (Charles River Labor atories, Raleigh, NC) were used. A titanium window chamber was surgically im planted on the dorsal skin flap of the mice. During surgery mice were anesthetized by an intraperitoneal inj ection of a freshly prepared mix of Ketamine (100mg/kg; Phoenix Scientific, Inc., St. Jo seph, MO) and Xylazine (10mg/kg; Phoenix Scientific, Inc., St. Joseph, MO). Tumor was in troduced in the window chamber during surgery by injecting a 10 L single cell suspension (in plain DMEM) containing approximately 5 3 to 10 3 tumor cells in the dorsal skin flap prior to placing the 12mm di ameter #2 round glass coverslip (Erie Scientific, Portsmouth NH) over th e exposed skin. After surgery animals were housed in the environmental chamber maintained at 33C and 50% humidity with free access to food and water and standard 12-hour light/dark cycles. The window chamber implanted on a nude m ouse is depicted in Figure 2-1. During imaging sessions animals were placed on a heating pad attached to the microscope stage. A custom-built Teflon plate holder se cured the window chamber under the microscope objective. The holder was secured to the stage mount enabling the user to position the window chamber under the microscope objective using the standard manual control of the microscope stage unit. During imaging the animals were gas an esthetized via nose cone using Isoflurane (11.5%) in medical air. The imaging se t-up is depicted in Figure 2-2. 2.3 Drug Preparation a nd Experimental Design OXi4503 (Combretastatin A-1 trans-stilbene) was provided by Dietmar W. Siemann (University of Florida, Shands Cancer Center Gainesville, FL). A drug dosage of 10 mg/kg was given intraperitoneal at a con centration of 0.01 ml/g of mouse body weight. The tumor survival 20

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fraction was found to be least at this dosage rate[4]. The drug bei ng light-sensitive was protected from light and suspended in sterile saline and one drop per ml of 5% sodium carbonate solution before injection. The drug was prepared fr esh every day, although a 48 hour usage of the prepared drug is acceptable. C ontrol groups received an equivale nt volume of sterile saline and 5% sodium carbonate solution. Since the drug is supposed to act on only tumor vasculature, treatment of a mouse without tumor group wa s omitted from experiment. The tumor were allowed to grow for 7-8 days and the treatment was started with the tumor size about 3-4 mm. Spectral imaging was performed immediately prior to and post-treatment, and also at 2, 4, 6, 8, 24, and 48 hours post-treatment. After 48h imaging time point the mouse was re-treated and the same imaging schedule was maintained. This wa s repeated at least th rice on each of the experimental mice. In all 7 mice bearing 4T1 tumors were treated with OXi4503 and 3 mice were carrier-only controls. For Caki-1 tumors the OXi4503 treatment was performed on 2 mice. 2.4 Imaging System A Zeiss microscope (Carl Zeiss, Inc., Thor nwood, NY) was used as the imaging platform (Figure 2-2). For transillumina tion of window chambers, a 100 W tungsten halogen lamp was used. Images were obtained at 1380 x 1035 pixels and 12 bit dynamic range using a CCD camera thermoelectrically cooled to -20C (DVC Co mpany, Austin Texas; Model # 1412AM-T2-FW). The long working distance objectives used were 2.5x and 5x fluars, 10x EC Plan-NeoFluar, and a 20x LD-Plan-NeoFluar (Carl Zeiss, Inc., Thornwood, NY). H yperspectral images were obtained via band-limited optical filtering using a C-mounted liqui d crystal tunable filter (LCTF) (CRI, Inc., Woburn, MA) with a 400-720nm tr ansmission range and a 10 nm nominal bandwidth, placed in front of the camera. Images were saved as 16-bit TIF files. Fluorescent images for 4T1-GFP were taken using a FITC filter set (Carl Zei ss, Inc., Thornwood, NY; Excitation range: 450-490nm; Em ission range: 515-565nm) at 520nm. 21

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a b Figure 2-1. The window chamber model. Figure 21a shows the window chamber implanted on a healthy nude mouse. Figure 2-1b demonstr ates the 4T1 tumor growing at the implantation site in the window chamber indi cated by the dashed circle, several days after implantation. a b Figure 2-2. The Imaging system. The Zeiss-based imaging microscope system is shown in Figure2-2a. Figure 2-2b gives a close view of the mouse being imaged, placed on the heating pad and breathing through the nose cone. 22

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2.5 Image Acquisition LabVIEW8 (National Instrume nts Corp., Austin, TX) was used to prepare a custom designed virtual instrument for controlling the tuning of the LCTF filter and operation of the CCD camera. The software enabled automated image acquisition using the specifications for camera exposure time and gain for each filter wa velength. Since the LCTF filter transmits less at lower wavelengths and more at higher wavelength s the exposure time for the camera had to be controlled such that the full dynamic range of the camera was utilized. The minimum exposure time used was 400ms whereas the maximum exposure time used was 1400ms, resulting in a typical acquisition time of a pproximately 16ms for image ac quisition, filter tuning, image transfer, and saving images on external hard drive. One hemoglobin saturation image set comprised of 16 images acquired in the wavelength range of 500-575nm with an interval of 5nm. 2.6 Image Processing Image processing was performed using Matlab software (The Mathworks, Inc., Natick, MA). The hyperspectral images acquired using th e CCD camera and LCTF filter were converted into double-precision arrays for mathematical anal ysis. The mathematical analysis was based on Ross Shonats method to solve for hemoglobin saturation (HbSat) using linear least squares regression[28]. The model equa tion used is the following: .Eqn(1) Where, OD = optical density (absorbance) at wavelength i (no units), eHbO2 = extinction coefficient of oxyhemoglobin at wavelength i (1/(M.cm)), 23

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[HbO2] = concentration of oxyhe moglobin (M) which is also the hemoglobin saturation in this form of the equation, eHbR = extinction coefficien t of deoxyhemoglobin at wavelength i (1/(M.cm)), L = pathlength (cm), LS = scattering term (dimensionless here sin ce it is the product of the pathlength and the scattering). In this equation, the scat tering is handled in the LS term. A rearrangement of the OD func tion results in the following: .Eqn(2) Where, HbSat = hemoglobin saturation (= [HbO2]/([HbO2] + [HbR])), and HbTotal = [HbO2] + [HbR]. If the following substitutions are made, a = HbSat*L*HbTotal, b = L*HbTotal, c = LS, eDelta(i) = eHbO2(i)-eHbR(i), then the following equation results: .Eqn(3) The function solves this equation for a, b, and c by linear least squares regression, and then solves for HbSat by performing a/b. The image processing flow diagram is presented in Figure 2-3. 24

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Hemo g lobin Saturation linear least s q uares HbO2 and HbR ratio determined b y the li g ht 5nm intervals 500-575nm 16 images Figure 2-3. Flow diagram depicting the im age processing method to obtain hemoglobin saturation map from the stack of 16 images obtained via spectral imaging. Further details regarding the imaging system and image processing can be obtained from Sorg et al[29]. 25

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CHAPTER 3 RESULTS 3.1 Treatment Results for the 4T1 Mouse Mammary Carcinoma Cell Line 3.1.1 Graphic Evidence There were structural and func tional changes in the tumor microvasculature after treatment with OXi4503 while the normal vasculature remain ed intact throughout the treatment. Tumor periphery vessels also persisted. Immediately af ter each treatment the mi crovascular structure started collapsing from the tumor core toward s the periphery. In Figure 3-1, the brightfield images clearly demonstrate the onset of struct ural breakdown in the tu mor core 2 hours after treatment of the example mouse. The vessels progressively ruptured from core of the tumor towards the periphery from 2 hours to about 8 hours after treatment. After 8 hours the hemorrhaging stopped and the tumor vasculature r ecuperated. The vessel networks which were completely destroyed could not be recovere d by the tumor and remained permanently nonfunctional thereafter. Al though, some of the vessels which had disappeared in the earlier time points from 2 hours to 8 hours post-treatment rea ppeared 24 hours onwards, indicating that those vessels were temporarily occlude d and lost function but were not completely destroyed. From 24 to 48 hours after treatment there was rapid recovery in the tumor vasculature. Some of the original tumor vessels reappeared along with the so me new vessel growth sprouting from the tumor rim towards the core (Figure 3-2). Synchronized with the structur al alterations in the tumor microvasculature, there were significant changes in the oxygenati on levels of the tumor microvessels as shown in Figure 3-3. The hemoglobin saturation of the tumor vesse ls plummeted rapidly from core towards periphery between 2 to 8 hours after drug administration. Tumor periphery remained well oxygenated initially but from 4 to 8 hours after treatment the deoxygenation diffused from core 26

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towards periphery and the tumor rim also e xperienced deoxygenation. From 8 hours onwards initiation of vessels recovery and reoxygenation was observed near the tumor rim as shown in Figure 3-4. Some of the original vessels reappeared well oxygenated and the undamaged persistent vessel oxygenations also improved gradually after 8 hour s. Rapid vascular regrowth, reoxygenation of the viable vesse ls, and neovascularization was observed at 24 to 48 hours posttreatment with the neovascularized vasculature having high oxygen saturation. The normal vasculature oxygenation fluctuations in the close periphery of th e tumor were not as pronounced. 3.1.2 Quantification Along with direct graphic evidence of the patte rn of changes in stru cture and function of the tumor microvasculature, it was deem ed important to quantify the results. First of all, the longitudinal fluctuations in vessel oxygenations were quantified in the example treatment mouse. As shown in Figure 3-5a, a candidate vessel was randomly selected such that it was emerging from the normal tissue and penetrating into the tumor reaching almost up to the tumor core. Figure 3-5b sh ows a zoomed in brightfield im age of the same vessel. Five regions of interest (ROIs) were chosen on this vessel starting from core towards periphery with ROI 1 to ROI 4 being inside the tumor and ROI 5 belonging to th e normal tissue region. Hemoglobin saturations were obt ained at these ROIs throughout the different imaging time points and plotted as depicted in Figure 3-5c. It was observed th at from 2 hours to 4 hours posttreatment there was a steep fall in the HbSat valu es of the tumor region (ROIs 1 to 4). At 6 hours they try to recover. ROI 4 which was closer to the periphery was able to recover consistently from 6 hours onwards, whereas ROI 1, 2 and 3 again dropped at 8 hours and then steadily recovered. Interestingly, the norma l region ROI 5 did not indicate ma jor fluctuations at any point throughout the treatment. 27

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For further analysis, classification of tu mor core, periphery and normal areas was performed by manually selecting the respective areas on the images as shown in Figure 3-6. The tumor core included almost the entire tumor nodule area visible in the 2-D image except for a thin outer rim which was defined as tumor periphe ry. Periphery was consistently chosen to be between 250500 m. Any region outside the tumor periphe ry was considered as normal tissue area. Hemoglobin saturation values for all the vessels in the core, all the vessels in the periphery, as well as all the vessels in the normal area we re obtained. Figure 3-7 show s the plot of HbSats of these 3 regions for 4 different mice. The co re vessels saturation dr opped rapidly after drug treatment till 6 to 8 hours and then gradually rec overed. The results were very consistent in all the treatments with the core always undergoing the most drastic cha nges in HbSat values compared to normal and periphery vessels. Interestingly, while th e periphery and normal vessels remained relatively well oxygenated initially, there was a delayed de oxygenation of these regions at about 6 hours post-trea tment followed by quick recovery at later time points. Notably, the tumor and periphery vessel hemoglobin satu rations followed up with each other pretty closely. This data for 4 mice was combined into one pl ot by taking an average of HbSats of the core, periphery and normal vessels for the 4 mice at each time point. As seen in Figure 3-8a, on an average all three regions demonstrate oxygenation changes wh ich match up with the pattern of changes occurring in individual mice. The core vessels bore the most damage in oxygenation levels up to 8 hours and then gr adually recovered. The normal a nd peripheral vessels showed high correlation, remained oxygenated initiall y but experienced de-oxygenation at around 6 28

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hours, recovering rapidly afterward. The standard error was calcula ted for the 4 mice for each of the three regions (Figure 3-8b). 3.2 Control Results for the 4T1 Mouse Mammary Carcinoma Cell Line 3.2.1 Graphic Evidence The controls consisted of mice tr eated with the carrier only (sterile saline and 5% sodium carbonate solution) containing no OXi4503, and imag ed along with the treatment mice through the same time points. The brightfield images for one example mouse are shown in Figure 3-9. As expected, no structural damage was observed at any of the ti me points after the carrier only treatment. The tumor vasculature con tinued to persist and proliferate. Also, there was no effect of the carrier only treatment on the function of the tumor vasculature as indicated by Figur e 3-10. There were hardly any fl uctuations in the oxygenation levels of either the tumor or normal vasculature from immediately post-treatment up to 8 hours signifying that the carrier had no impact. At 24 hours there was a random reduction in the core vessels oxygenation which shows signs of recovery in the 48 hours time point. 3.2.2 Quantification Once again the tumor core vessels, periphery vessels and the normal vasculature were quantitatively investigated for temporal hemoglobin saturation changes af ter treatment with the carrier. The results are displayed in Figure 311. The plot of HbSat versus the imaging time points agreed with the graphic observations and further confirme d the non-effectiveness of the carrier only treatment, leading us to believe th at the trend of events observed to be occurring after OXi4503 treatment were truly caused by the drug only. 3.3 Treatment Results for the Caki-1 Renal Carcinoma Cell Line To investigate whether the OXi4503 treatment response varied with the tumor type, the experiment was repeated with a different tu mor cell line. Xenografted human derived Caki-1 29

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renal carcinomas were dissociated from the host m ouse, implanted into the experimental mouse window chamber and the whole experiment was repeated. 3.3.1 Graphic Evidence Similar structural alterations were recorded with drug-treated Caki-1 tumors as were observed after the treatment of 4T1 mouse mamm ary adenocarcinomas, except for an earlier recovery initiation. The treatment response of the Caki-1 cell line is shown in Figures 3-12. The tumor vasculature was progressively and severely damaged between 2 hours to nearly 6 hours post-treatment starting from core towards tumor periphery. The tumor ri m vessels were never completely destroyed. From 6 hours to 48 hours time point rapid rec overy and neovascula rization was observed at the tumor periphery (Figure 313). For the 4T1 tumors, the recovery phase started from about 8 hours post-treatment. Some of the original tumor vessels reappeared and also new vessel networks were noticed to be developing. The normal vasculature persisted unharmed throughout the treatment. Hemoglobin saturation maps of the Caki-1 tu mor treatment are given in Figure 3-14. As can be seen from these images, the HbSat values of the tumor vasculature undergo significant alterations accompanying the structural changes. Similar trends as observed in the case of 4T1 tumors were noticed after Oxi4503 treatment of Caki-1 tumors, excep t that the oxygenation recovered earlier. From 2 hours to 6 hours post-treatment, the oxygenation of the tumor vasculature reduced rapidly from core towards periphery. By 4 hours, the tumor rim as well as normal vasculature was already highly de-oxyge nated. The delayed de-oxygenation of the tumor rim and normal vessels (even if there) could not be captured. The reoxygen ation of the remaining vasculature and recovery star ted from 6 hours time point. 30

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Figure 3-15 shows the zoomed-in images of th e tumor area demonstrating rapid recovery and reoxygenation of original vessels along with the appearance of highly oxygenated neovasculature. 3.3.2 Quantification Individual tumor vessel oxygenation changes were quantified for the Caki-1 renal carcinoma cell line as shown in Figure 3-16. Four regions of interest were randomly selected inside the tumor as indicated by the white rectan gles in Figure 3-16a. From the graph in Figure 3-16b, we can see that all the four vessel oxygenations plummete d rapidly 2 hours post-treatment till 4 hours time point. From 6 hours all the vess els oxygenations recovered consistently to the maximum value at 24 hours and stabilized throu gh the next 24 hours till the 48 hour time point. Notably, the Caki-1 tumor vasculature oxygenations r ecovered faster as compared to that of the 4T1 tumors. Similar to the 4T1 tumor analysis, hemoglobin saturation values for all the vessels in the core, all the vessels in the pe riphery, and all the vessels in the normal area were obtained as shown in Figure 3-17. Overall, the oxygenation decr eased in the tumor core, periphery as well as normal vasculature after treatment till 4 hours, after which the satu rations consistently increased. By 24 hours maximum recovery was made and then the oxygenation levels stabilized around the values before treatment. 3.4 Temporal Fluctuations in Hemoglobin Saturations Preand Post-OXi4503 Treatment To study the temporal fluctuations in the hemoglobin saturations of the tumor, peripheral and normal vessels before and after treatment, the following study was performed. After the 3rd treatment with OXi4503, the 4T1 tumor was allowed to recover up to 48 hours and treated again. The mouse was imaged fo r 20 minutes before the 4th treatme nt and for 60 minutes immediately 31

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after treatment. The images were obtained ev ery 30 seconds, allowing us a high temporal resolution for closely obser ving the oxygenation change s after the treatment. As shown in Figure 3-18a, regions of inte rest were selected in the normal vessels, peripheral vessel, as well as core vessel. As s een in Figure 3-18b, immediat ely after the treatment there was a slight downshift in th e oxygenations of all 4 ROIs. This could be related to the fact that the tumor vasculature does not possess high vaso-response capabiliti es with respect to sudden blood volume changes. After about 21 mi nutes after treatment the normal vein, core venule, and peripheral venule underwent a rapi d reduction in oxygenation, indicating the drug action. The normal artery oxygenation remained least affected during this imaging session. 3.5 Transient Fluctuations in Tumor Ri m and Normal Vasculature PostTreatment Figure 3-19 shows hemoglobin saturation maps of several tumors at 4 hour post treatment time point. It was observed that predominantly after 4 hours post treatment, the tumor periphery vessels as well as normal vasculature experi enced de-oxygenation along with the tumor core vasculature. The oxygenation levels of the periphery and normal ve ssels returned to their base values at the later time points. Such information could be critical in te rms of time-scheduling of other combinatorial treatments su ch as radiation and chemotherapy. 32

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Figure 3-1. Brightfield images of OXi4503 treatment progression through the various imaging time points for one experimental mouse. The scale bar on each image indicates 500 m in length. (4T1 mouse mammary adenocarcinoma). 33

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Figure 3-2. Example of neovascul arization observed between 8 to 48 hours post-treatment along the tumor rim. Vessels sprouting from periphery towards tumor core. Original tumor vessels grew more prominent afte r recovery. (4T1 mouse mammary adenocarcinoma). 34

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Figure 3-3. Hemoglobin saturation (HbSat) maps of the brightfield images in Figure 3-1 obtained using a linear least square regression model. 35

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Figure 3-4. Hemoglobin saturation maps of the images in Figure 3-2. Recovery and reoxygenation observed from 8 to 48 hours pos t-treatment. Neovascularized vessels were highly oxygenated. 36

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Figure 3-5. Example of longitudina l fluctuations in vessel oxygena tion after drug treatment. The white rectangular areas in Figure 3-5b indicate the ROIs where the HbSat was obtained. The plot of the HbSat values of the 5 ROIs through the various time points is given in Figure 3-5c. (4T1 mouse mammary adenocarcinoma). 37

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Figure 3-6. Classification of core, periphery and normal areas. Periphery was chosen between 250-500 m on the rim of the solid tumor nodule. A ll the vasculature beyond tumor periphery was considered as normal vasculature. 38

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Figure 3-7. Analysis of temporal fluctuations in core, periphery and normal vessels for tumor shown in Figure 3-1 (treatment mouse 1) a nd 3 other mice. In each graph individual line represents mean hemoglobin saturation in all the vessels in the respective area over time. 39

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Figure 3-8. Combined averages of hemoglobin sa turations in 4 mice from Figure 3-7 are shown in Figure 3-8a. Figure 3-8b displays the same data as in Figure 3-8a in a bar chart format to include the standard er ror in their hemoglobin saturations. 40

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Figure 3-9. Brightfield images of control mouse treated with carrier only (sterile saline and 5% sodium carbonate solution) through the various imaging time points. The scale bar on each image indicates 500 m in length. (4T1 mouse mammary adenocarcinoma). 41

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Figure 3-10. Hemoglobin saturation maps of the images in Figure 3-9. 42

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Figure 3-11. Analysis of temporal fluctuations in core, periphery and norma l vessels of the tumor shown in Figure 3-9. In each graph indi vidual lines represent mean hemoglobin saturation in all the vessels in the respective area over time. 43

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Figure 3-12. Brightfield images of OXi4503 treatment progressi on through the various imaging time points for one experimental mouse. The scale bar on each image indicates 500 m in length. (Caki-1 renal carcinoma cell line). 44

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Figure 3-13. Example of neovascularization observed between 8 to 48 hours post-treatment along the tumor rim. Original tumor vesse ls recovered along with new sprouting vessels forming dense network. Vessels spro uting from periphery towards tumor core. (Caki-1 renal carcinoma cell line). 45

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Figure 3-14. Hemoglobin saturation (HbSat) maps of the brightfield images in Figure 3-12. 46

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Figure 3-15. Hemoglobin saturation maps of the images in Figure 3-13. Recovery and reoxygenation observed from 8 to 48 hours pos t-treatment. Neovascularized vessels were highly oxygenated. 47

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Figure 3-16. Individual vessel analysis for the Caki-1 tumor shown in Figure 3-12. The white rectangular areas in Figure 3-16a represent the locations where HbSat values were obtained. 48

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Figure 3-17. Analysis of temporal fluctuations in core, periphery and norma l vessels of the Caki1 tumor in Figure 3-12. In each graph individual line represents mean hemoglobin saturation in all the vessels in the respective area over time. 49

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Figure 3-18. Example of hemoglobin saturation fluc tuations in the tumor vasculature before and after OXi4503 treatment. Figure 3-18a show s the 4 ROIs where the HbSats were obtained. Figure 3-18b gives the HbSat tempor al fluctuations before and after OXi4503 Tx4 on day 13 after 4T1 tumor impl antation The tumor vasculature was allowed to recover for 48 hours after Tx3 and then treated again. Imaging was performed every 30 seconds for 20 minutes before Tx4, and for 60 minutes immediately after Tx4. 50

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10A mouse4 4hAfter Tx2 4T1 tumor 10A mouse4 4hAfter Tx3 4T1 tumor 10B mouse2 4hAfter Tx2 4T1 tumor 10B mouse4 4hAfter Tx1 4T1 tumor 10B mouse4 4hAfter Tx2 4T1 tumor Group13 mouse3 4h After Tx1 Caki 1 tumor Figure 3-19. Transient fluctuations in tu mor rim and normal vasculature predominantly observed at 4 hours after treatment in both tumor types. 51

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CHAPTER 4 DISCUSSION An intact microvasculature is essential for surv ival, progression and metastasis of a tumor. Vascular disrupting agents such as OXi4503 seek to specifically destr oy the established tumor vasculature, occlude blood flow and cause tumor necrosis. Member of the Combretastatin family, OXi4503 is an analogue of combretastat in A4 phosphate (CA4P), and is considered among the most potent VDAs[24] having some cyto toxic activity, in addition to its vascular disrupting effects[25]. Time-dependent decrease in tumor perfusion, increased tumor vascular permeability, tumor microvascular damage, persistent tumor rim vessels and recovery of tumor perfusion at later time points after treatment are some of the hallmark characteristics of OXi4503[4, 25]. Detailed investigati ons regarding such VDAinduced structural and functional changes in tumor and normal tissue vasculature are required for complete assessment of drugefficacy and to study its effect on co mbinatorial cancer therapies. Our hypothesis was that spectral imaging, a quan titative method providing spatial maps of microvascular blood oxygenation in terms of he moglobin saturation coul d prove to be an effective tool to give further insight into th ese observed phenomena. And using this technique consequential information regarding structural ch anges over time as well as functional alterations in terms of oxygenation change s after drug treatment could be obtained. The present work demonstrates the utility of hypers pectral imaging for investigating the structural alterations and oxygenation changes in the tumor vasculature an d the normal tissue vasculature after repeat treatments with OXi4503. Two different tumor types, 4T1 mouse ma mmary adenocarcinoma and Caki-1 renal carcinomas, were treated with 10mg/kg of OXi45 03 every 48 hours, repeating the treatment at least 3 times. Hyperspectral im aging was performed immediatel y prior, immediately after, and 52

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also at 2, 4, 6, 8, 24 and 48 hours post-treatment to give quantitative, direct and real time information regarding the treatment progression. Hyperspectral imaging enabled us to perform serial measurements on the same animal over a span of multiple treatments, giving us an advantage over several other techniques whic h require sacrificing of animals through the experimental time points. The results obtaine d for both the tumor types were consistent throughout the multiple treatments. OXi4503 was found to be a fast acting drug in both the tumor types, acting within a few minutes after administration. There were signif icant structural and functional alterations observed post-treatment. The normal tissue vasculature and the tumor periphery vessels persisted structurally unharmed throughout the treatm ents, although the oxygenation changes were experienced from higher to lower degree by the tumor core, periphery and normal vessels. For both the tumor types the structur al and functional changes in th e tumor vasculature followed similar trend. Except that the recovery phase for the Caki-1 tumors seemed to be initializing a bit earlier as compared to that for the 4T1 tumors. Immediately after treatment there was pr ogressive tumor microstructural breakdown, diffusing from tumor core towards periphery, later followed by a rec overy phase progressing from tumor periphery towards the core. Interestin gly, during recovery some of the original tumor vessels which had disappeared in the earlier time points reappear ed, indicating that those vessel structures were not completely destroyed but were only tempor arily shut down and later reperfusion made them reappear. Vessel networks completely destroyed by the treatment could not be recovered and remained non-functional. Nota bly, the amount of vascular damage caused in the tumor core varied from treatment to treatment. The maturity level of the treated vasculature could have a role in this phenomenon. OXi4503 acts by binding to the tubulin subunits and, in 53

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proliferating endothelial cells, cau ses depolymerization of microtubu les, resulting in detachment and apoptosis[30]. Hence, even as tumors are generally characterized by vascular abnormalities including a rapidly dividing endothelial population[4], it could be a plausible reason that tumors having more proliferating endothelial cells suffered more damage after treatment with OXi4503. New vascular growth was observed developing at the tumor rim during the recovery phase, forming dense network and aggressively progressing towards the tumor core. The oxygenation changes in both the tumor types also appeared to follow similar pattern, though oxygenation recovery was in itiated earlier in Caki-1 tumors. Hemoglobin saturation values for tumor vessels plummeted rapidly af ter treatment progressively from core towards periphery. The periphery and normal vasculature also experienced changes in oxygenation levels which were found to be tightly correlated with each other. For the 4T 1 tumors, a delayed deoxygenation could be observed, demonstrating how the de-oxygenation progressed from tumor core into the tumor periphery and normal vascul ature. When the recovery phase started, the normal vessels along with the tumor periphery re covered first. And the recovery progressed towards the tumor core. The de-oxygenation of the tumor periphery and normal vessels could negatively influence the conventional cancer ther apies. Notably, transient fluctuations in hemoglobin saturation values of the tumor rim and normal vasculature were noticed predominantly at 4 hours after drug administra tion indicating drug impact on normal vasculature oxygenation and blood flow. These findings c ould be pivotal in time-scheduling the combinatorial cancer treatments such as chemotherapy and radiation therapy. Several of our findings were found to be consis tent with earlier work. Siemann et al have performed extensive investigations characterizi ng several vascular targeting agents, including VDAs such as CA4P and its analogue OXi4503[ 4, 30]. Results obtained from their tumor 54

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perfusion studies after OXi4503 treatment, us ing Hoechst-33342 staining, as well as using dynamic contrast-enhanced MRI (contrast agent: gadolinium-diethylenetriaminepentaacetic acid, Gd-DTPA) matched quite well wi th the results obtained thro ugh our technique. Their results indicated reduction in perfusi on of tumor microvasculature be ginning at about 30 minutes post treatment, with minimum perfusion at about 4 h ours post treatment. Recovery in tumor perfusion was observed from 24 hours to 48 hours post treatment. Tumor perfusion is an indirect measure signif ying the presence and functionality of the vasculature. It gives no info rmation regarding the oxygenation of the vessels in question. A structurally intact vessel may or may not be oxygenated. After the OXi4503 treatment, the blood flow is interrupted in the tumor vasculature. As we have demonstrated through our results, even though the function was lost in term of lack of oxygenation, sometimes the structure persisted throughout the treatment, and reappeared when th e blood flow was restored. Hence the perfusion studies may give false or incomplete informati on regarding the functionality of the vasculature. Inefficient staining of the perfusion dye could furt her contribute to the er ror. Using hyperspectral imaging direct graphic and quantitative eviden ce was obtained for structural and functional changes. Hemorrhaging vessels were clearly visible in the brightfield imag es obtained though our technique, whereas tumor vessel hemorrhages were assumed in their study[30] based on presence of red blood cells visible in the tissue hi stology sections within the tumor. It was also indicated that in the tumor as a whole, there was init ial decrease in the perfusion after 4 hours post-treatment and a significant recovery 48 hours later. The same changes were observed in the perfusion of the periphery, only that the amount of perfusion reduction was lesser with faster recovery. This fact is also very well corrobor ated in our oxygenation changes measurements, 55

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which show that the periphery was less de-oxygena ted and had faster recovery as compared to the tumor core. We added further piece of information regarding how the progression of structural and functional changes progresse d from tumor core towards periphery. Further, their study indicated vi a CD31 staining that there was in crease in vessel density in the tumor periphery as well as the whole tumor post-treatment. They showed that at 24 hours post treatment there was maximum vessel densit y at the periphery, whereas the whole tumor density was maximum at 48 hours post treatment. Th is information could be better interpreted now from our results showing ra pid recovery and neovasculariza tion starting at the tumor rim between 24 and 48 hours of treatment, and progre ssing towards the tumor core, thereby having higher density of vasculature at periphery at 24 hours and incr ease in overall tumor vascular density at 48 hours. 56

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CHAPTER 5 CONCLUSION Efficacy of vascular disrupting agent OXi4503 has been previously investigated via perfusion and necrosis based assays[4, 30]. In this study, we were able to demonstrate the efficacy of hyperspectral imaging technique for wi de-field regional assessment of structural alterations and functional change s in oxygen transport at microvesse l level after re peat treatment with vascular disrupting agent OXi4503. Through th e quantitative, direct and real time data obtained regarding treatment progr ession, we were able to confir m results indirectly shown by other investigators and c ontributed further to th e available information. 57

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BIOGRAPHICAL SKETCH Mamta Wankhede. Being one of the youngest kids in a traditional Indian family, of course she was over-protected. All her ed ucation was done in her home town Nagpur, and one of her strongest desires back then was to be able to get out and "see" the world on her own. So after she finished her Bachelors in El ectronics Engineering she wanted to study away from home. She had no idea what she was in for when she decide d to go for higher education in a foreign land on the opposite side of the globe. The family was sh ocked; probably because such an event was unprecedented in the family. Convincing them that she was not being crazy, and that she had thought this through, and that she would be able to manage everything on her own was the most challenging part. It has been an amazing experience so far. Mamt a just finished her Masters in Biomedical Engineering here at University of Florida, and is continuing as a PhD student. She considers it a great achievement on behalf of her entire family that she was able to come so far and pursued higher education in an intern ationally renowned university.