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Characterizing Tumour Vessels using MRI and Histology - A novel dual injection MR protocol to study tumour.. Moosvi, Firas 2009

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Characterizing Tumour Vessels usingMRI and HistologyA novel dual injection MR protocol to study tumourblood vessel permeability.byFiras MoosviA THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFHON. BACHLEOR OF SCIENCEinThe Faculty of Graduate Studies(BioPhysics)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)April 2009c© Firas Moosvi 2009AbstractGalbumin, an MR contrast agent is characterized for use in a new class of animal MR experi-ments. It’s suitability as both a T1and T∗2agent was assessed and it was found that althoughGalbumin’s relaxivity (4.33 to 5.77 (mM ·sec)−1was comparable to Gd-DTPA, the solution wasnot available at a high enough concentration to achieve similar T1weighted effects. Further, itwas deemed an unworthy candidate for T∗2-weighted imaging as it’s magnetic susceptibility wasmuch too low (2.95 ppm/mM). Finally, we established a theoretical basis for a novel dual contrastagent MR protocol to investigate blood vessel permeability, extracted from previously publishedwork [1] on modelling MR contrast agents. The over-arching goal of this study is to use the liveimaging capabilities of MR combined with traditional immunohistochemical techniques to moreaccurately characterize tumour vessel permeability.Firas Moosvifmoosvi@interchange.ubc.caiiTable of ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiTable of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iiiList of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viAcknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ixDedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Introduction, Motivation and Theory . . . . . . . . . . . . . . . . . . . . . . . . . 21.1 Introduction and Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Theory - Blood vessel and tumour biology . . . . . . . . . . . . . . . . . . . . . . 31.2.1 Blood Vessels, Extravasation and drug delivery . . . . . . . . . . . . . . . . 41.2.2 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.3 MR Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.3.1 Measuring T1Relaxation (using a Spin Echo Pulse Sequence) . . . . . . . 91.3.2 Measuring T∗2Relaxation using (CPMG Pulse Sequence) . . . . . . . . . . 111.4 MR Contrast Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.5 Modelling Contrast Agents - Kety Model . . . . . . . . . . . . . . . . . . . . . . . 152 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.1 Mouse Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.1.1 Preparing a mouse for an MR scan . . . . . . . . . . . . . . . . . . . . . . 182.2 Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.3 MR Scans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.3.1 Diffusion-weighted EPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.3.2 T1weighted images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212.3.3 T∗2weighted images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222.4 Galbumin Relaxivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22iiiTable of Contents3 Results, Analysis and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.1 Galbumin Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.2 Galbumin as T1agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273.3 ADC (Apparent Diffusion Constant) Maps . . . . . . . . . . . . . . . . . . . . . . 304 T∗2Weighted Imaging: Galbumin & Feridex . . . . . . . . . . . . . . . . . . . . . 334.1 Galbumin as a T∗2agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344.2 Feridex as a T∗2agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354.3 Mapping Feridex Histologically . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374.3.1 Chemical Stain - Prussian Blue . . . . . . . . . . . . . . . . . . . . . . . . 375 Conclusions, Implications and Future Work . . . . . . . . . . . . . . . . . . . . . 385.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385.2 Implications and Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40AppendicesA Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43A.1 Susceptibility Equation Derivation . . . . . . . . . . . . . . . . . . . . . . . . . . . 43A.2 IDL Program to Calculate DSC Parameter Maps . . . . . . . . . . . . . . . . . . . 44A.3 Sample Staining Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46A.4 Feridex Staining Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48ivList of Tables1.1 A table of the typical divisions of MR contrast agents [2]. In this study, we useFeridex, Gd-DTPA and Galbumin (mainly a T1agent). Note, the table doesn’tquantify the difference between “significant” and “dramatic”, because there is arange of effects depending on concentration and relaxivities. . . . . . . . . . . . . . 151.2 EES refers to the space into which tracer can leak from a capillary and Ktransis thevolume transfer constant between blood plasma and the EES. Table parametersby: [1] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164.1 Table parameters from: [2] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35vList of Figures1.1 On the left, is a schematic of normal tissue vasculature. Notice in particular,approximate symmetry and order compared to the cancerous tissue, where thevasculature is non-uniform, inefficient and wasteful (in terms of nutritive delivery).Note also in the background, the growth of ordered layers of normal cell on theleft vs. the rapidly dividing cancer cells on the right. Image credit: [3] ....... 21.2 Histology analysis - image of a tumour section where individual cells can be re-solved. The colours on the tumour are explained in the legend on the right butthe main idea here is that around active blood vessels (stained dark blue), thereis cell growth. Cells close to the blood vessels, are more likely to receive nutrientsand are therefore actively dividing. The green rim, no less than 150 µm away fromblood vessels, represents cells that lack oxygen because it doesn’t diffuse that far.Above is an overlay of multiple images imaged at different fluorescent wavelengths.Data credit: Alastair Kyle (AIM lab), BCCRC . . . . . . . . . . . . . . . . . . . . . . 41.3 Image of a tumour frozen in an OCT medium being sectioned at a cryostat machine.10µm sections adhere to glass microscope slides due to a temperature gradient. . . 61.4 A schematic representation of the Galbumin molecule. 15-20 Gd-DTPA atomssurround a large 74 kDa Albumin protein and 3-5 fluorescent FITC tags allow forconvenient histological imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.5 On the left, a typical proton is shown with its magnetization vector pointing alongthe z-axis. On the right, the axes are rotated so the schematic is showing theproton’s spin vector, projected along the x-y-axis. . . . . . . . . . . . . . . . . . . 71.6 Classical diagram showing spins lining up to the external magnetic field B0re-sulting in a net magnetization M0. The spins align parallel and anti-parallel dueto a small difference in energy, as shown by the energy splitting diagram (due toZeeman splitting). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.7 Schematic representation of a Spin-Echo pulse sequence. Multiple iterations of thespin-echo sequence (180◦then 90◦) pulses are performed and the echo is recordedas a function of time. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10viList of Figures1.8 The signals (from figure 1.7) are Fourier transformed and here, the max signalintensity (of each echo) is plotted as a function of the variable delay. The timeconstant from the exponential recovery is T1. More precisely, T1is measured asthe time it takes for the signal intensity to return to 63 % of its’ maximum value. 101.9 Schematic representation of a CPMG pulse sequence. The initial 90◦pulse tipsthe magnetization pulse in the transverse direction, the magnetization then decaysrapidly due to field inhomogeneities and other spin-spin interactions. Applicationof a 180◦pulse flips the direction of the dephasing spins and causes them to rephase.As the spins rephase, a spin echo signal is generated. The T2decay curve in blueis represented in figure 1.10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.10 The curve above shows the T2relaxation process. The FID signal from figure 1.9is fourier transformed and the maximum intensity is plotted as a function of theEcho Time (TE). The decay constant is the spin-spin relaxation rate. . . . . . . . 121.11 On the left, the magnetization vector tipped and as it returns back to B0, itprecesses aroundB0. [4]. On the right, a schematic of slice-selection with a patientlying along the a magnetic field along their length. . . . . . . . . . . . . . . . . . . 131.12 Left: representation of the FID signal data acquired, notice concentration of dataaround the origin. Right: Typical MR image of a tumour after a Fourier Transform.Bright bulb at the bottom is the crosssection of a vial of water, used as a standardreference point. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141.13 Two compartment Kety Model with rate constants describing flow of contrast agentout into the EES (compartment A)and back into the vessels (compartment B). . . 161.14 Schematic of a dual contrast agent protocol - the first contrast agent is injected,the signal acquired and then the second agent is administered. Because both setsof data have been acquired, the signal intensities can be processed and the twoprocesses can be treated as independent. . . . . . . . . . . . . . . . . . . . . . . . 172.1 Description of Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . . . . 203.1 Typical tumour (HCT116) section stained for blood vessels (red, CD31) and Gal-bumin (green, FITC). Sections of the same tumour are stained and (fluorescently)imaged separately and overlaid on top of each other. . . . . . . . . . . . . . . . . 253.2 An area of the tumour above (figure 3.1) zoomed in. We note some vessels (red,CD31- bottom left) have positive Galbumin staining (green, FITC) while othervessels show no positive staining (top right). . . . . . . . . . . . . . . . . . . . . . 263.3 Plot of Galbumin extravasation as a function of distance from blood vessel in HCT-116 tumour xenografts. 4 time points from injection to sacrifice are shown and thegeneral trend is that over time, Galbumin equilibrates in the tumour. . . . . . . . 26viiList of Figures3.4 Plot of Hoechst extravasation as a function of distance from blood vessel in HCT-116 tumour xenografts. 3 time points from injection to sacrifice are shown. Com-pared to the Galbumin curve (figure 3.3) Hoechst tends to extravasate further awayfrom blood vessels (smaller molecule) . . . . . . . . . . . . . . . . . . . . . . . . . 273.5 Galbumin relaxivity data plotted and fitted according to equation 1.6 and theprocess laid out in that chapter. We determined the relaxivity of Galbumin to be86.62 ±5.26 (mM · sec)−1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.6 In this tumour, Gd-DTPA is the contrast agent being administered. T1-weightedgradient echo images before (A) and after (B) Gd-DTPA injection. Note the nearuniform enhancement throughout the tumour - because Gd-DTPA is not a bloodpool agent (diffuses everywhere), it provides MR signal enhancement everywhere. 293.7 A & B T1-weighted gradient echo before (A) and after (B) galbumin administra-tion. It is difficult to see an enhancement just with images (A) and (B) so (C) showsthe signal enhancement parameter map from DCEMRI acquired during Galbuminadministration (initial area under the curve IAUC60). Note areas of peripheralenhancement, potentially an area rich with larger vessels reside. . . . . . . . . . . 293.8 Apparent Diffusion Coefficient (ADC) map represents typical HCT-116 (left, [A])and HT29 (right, [B]) tumours. Greyscales are scaled from 0-255, 0 is black and255 is white, corresponding to an ADC ranging from 0 - 2 µmm2msrespectively.The HT29 tumour (right) has relatively low water difusion and equivalently, lessnecrosis. The tumour is outlined in red. . . . . . . . . . . . . . . . . . . . . . . . . 324.1 Inhomogeneities caused by the superparamagnetic contrast agent. . . . . . . . . . 334.2 Dual Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344.3 The data acquired for the experiment to determine Galbumin susceptibility. Thefinal value ∆χ was experimentally determined to be: 2.95 ppm/mM . . . . . . . . 354.4 This figure details the use of Feridex in the mouse, the parameter maps are at thetop left, the equations used to construct the maps on the right and a schematic ofthe intensity at the bottom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.5 The scale bar is 10 µm and the image has been taken under a light microscopeby Jennifer Flexman et. al [5]. The red and purple arrows indicate different aresof staining in a neuron. Jennifer Flexman assisted with developing a protocol forFeridex staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37viiiAcknowledgementsI would like to take this opportunity to thank my co-supervisors, Dr. Stefan Reinsberg fromthe UBC Physics and Astronomy Department and Jennifer Baker, Ph.D candidate from the BCCancer Research Centre. They have put in a tremendous amount of work into ensuring that thisundergraduate thesis project became and stayed a reality and also supported me in presentingmy research at six local, national and international scientific conferences. Without their supportand commitment to helping me with the experimental work and direction, this project wouldlikely not have happened. I would also like to thank Dr. Andrew Minchinton, a PI at theBCCRC and Jennifer Baker’s supervisor for his financial contribution in support of this projectas well as invaluable feedback on practice talks, posters and proposals. Dr. Piotr Kozlowski andAndrew Yung at the high field MRI group at UBC also guided the progress of this thesis and weacknowledge their contributions to the cause.ixDedicationThis thesis, the culmination of 5 years of undergraduate education, is dedicated to my parentsfor their perseverance in tolerating my abnormal schedules, unreasonable demands, expectationsand facilitating and funding my education.Thank you.1Chapter 1Introduction, Motivation and Theory1.1 Introduction and MotivationStopping the progress of cancerous tissue in the human body is one of the most actively researchedtopics in scientific research [6, 7, 8, 9, 10] . The spread of cancer has been attributed to manythings and as such, there are literally thousands of branches researchers take to reach the sameeventual goal - stop the inception and progress of cancer. Andrew Minchinton’s lab at theBCCRC is particularly interested in mapping tumours and studying the complex organization oftheir blood vessels [11].In studying cancer and tumours, traditional biochemical methods call for analyzing frozencross sections of tumour tissues [12, 13], staining and then fluorescently imaging them at highresolution. While this method certainly has its merits, situations and conditions are arising thatrequire dynamic imaging of live animals. Recent advances in the field of Biophysics have allowedresearchers the ability to correlate images taken with Magnetic Resonance Imaging (MRI) tothose using high-resolution fluorescent microscopy. While live imaging is possible using MRI,to reproduce much of the biologically relevant data acquired by fluorescent microscopy is nearimpossible. In this project, we begin first by setting up the biological problem, then proceedingthrough and developing a protocol using the principles of MR to attempt to solve it.Figure 1.1: On the left, is a schematic of normal tissue vasculature. Notice in particular, approxi-mate symmetry and order compared to the cancerous tissue, where the vasculature is non-uniform,inefficient and wasteful (in terms of nutritive delivery). Note also in the background, the growthof ordered layers of normal cell on the left vs. the rapidly dividing cancer cells on the right. Imagecredit: [3]21.2. Theory - Blood vessel and tumour biologyThe motivation behind this project is ultimately to better understand how blood vesselsbehave in tumours (referred to often as tumour vasculature, figure 1.1). The process where frozentissue cross sections are analyzed [13] is called immunohistochemistry as the tissue sections arestained (see section 1.2.2) to mark for a particular molecule (growth factor or protein) a cellexpresses [11, 14]. The marker is then imaged fluorescently at high resolution, and vast amountsof data can be acquired. However, this process doesn’t lend itself well for imaging dynamically tomeasure (for example) flow, study tumour evolution or even characterize response to combinationsof anti-cancer therapies [15, 16].With this study, our goal is to use magnetic resonance imaging to perform live scans of animalsusing a brand new contrast agent, Galbumin. This is an exciting development as for the first time,there is now a potential for direct pixel-pixel correlation between live MR image data and highresolution histology data. Combining these two analyses and imaging techniques is at the crux ofour solution [13]. The implications for such a protocol are staggering, mainly because MR allowsrepeated scans for longitudinal studies and histology allows for high resolution data acquisition forcellular level resolution. Figure 1.2 shows a typical layered image from an immunohistochemicalanalysis.1.2 Theory - Blood vessel and tumour biologyIn contrast to normal tissue, tumours are highly variable but are generally characterized by avastly disordered organization of blood vessels and capillaries (figure 1.1) [17, 18]. Among otherthings, tumour vasculature is aberrant and is characterized by a deregulation of normal cell cycleprocesses. These abnormal processes allow us to target tumour cells specifically with radiationand chemotherapies. For example, a class of drugs could target blood vessels by inhibiting (orstimulating) angiogenesis, the creation of new blood vessels. Tumour vasculature is heteroge-nous and the uptake of compounds (glucose, oxygen, anti-cancer drugs etc.) is related, in someunknown way, to the permeability of these blood vessels.An added complication is the existence of many tumour types, each with several distinguish-ing characteristics. Here, for simplicity, we use two tumour models and exploit the intrinsicdifferences between them. The names of tumour types are not relevant, but tumour class A hashigh vasculature density (lots of blood vessels over a small area) while tumour class B has lowvasculature density. This differential allows us a convenient way to test, qualitatively the effectof permeability directly. Because we want to study blood vessels in tumours, we want to use aContrast Agent that stays inside blood vessels so we can extract information such as flow andpermeability using MRI.With this study, we hope to validate and characterize Galbumin as a valid high molecularweight contrast agent and apply its unique properties as a suitable contrast agent, as well as auseful tool in tumour mapping to the two cell types. In other words, we want to use the live31.2. Theory - Blood vessel and tumour biologyFigure 1.2: Histology analysis - image of a tumour section where individual cells can be resolved.The colours on the tumour are explained in the legend on the right but the main idea here isthat around active blood vessels (stained dark blue), there is cell growth. Cells close to theblood vessels, are more likely to receive nutrients and are therefore actively dividing. The greenrim, no less than 150 µm away from blood vessels, represents cells that lack oxygen because itdoesn’t diffuse that far. Above is an overlay of multiple images imaged at different fluorescentwavelengths. Data credit: Alastair Kyle (AIM lab), BCCRCimaging capabilities of MR, combined with traditional immunohistochemistry techniques to get amore accurate picture of flow in the tumour microenvironment using Galbumin, a high molecularweight contrast agent.1.2.1 Blood Vessels, Extravasation and drug deliveryIt has been claimed that vessel leakiness influences the tumour microenvironment, access totherapeutic antibodies and perhaps even angiogenesis [19]. Tumour vessels are at least an orderof magnitude leakier than normal vessels and there is general agreement [19, 20, 21] that thesevessels are highly abnormal, however, the mechanisms involved are not very well understood.The implications of these vessels, with irregular diameters and branching patterns to cancergrowth and metastasis rate has also largely been ignored, despite enormous advances in tumourangiogenesis.Water and other small water-soluble solutes see the largest variation in permeabilities amongvarious vascular networks. In particular, factors such as the number of tight junctions in theendothelium lead to different permeabilities with the lowest values found in the brain (blood brainbarrier and much higher in the intestines. It is also important to understand why size matterswhen studying permeability. Small solutes move through mainly by Diffusionwhereas largermolecules move through by convection and extravasation is coupled to flow and permeability.41.2. Theory - Blood vessel and tumour biologyResearchers[22, 23] are highlighting the importance of distinguishing between the permeabilityof a vessel wall vs. the “accumulation of extravasated fluid and solutes outside vessels” and [19].Ostensibly, these accumulated fluids may be present due to a variety of reasons including butnot limited to forces that drive solutes across vessel walls, and the permeability surface areaproduct [1].In the tumour vasculature community, there exists a school of thought that is of the opinionthat all efforts should be made to “normalize the tumour vasculature” [19] before attempting totreat the tumour using traditional chemotherapy drugs. This seems slightly counter intuitive atfirst, however a normalization of vessels may in theory, increase blood flow by eliminating vesselabnormalities such as irregular branching and outlier diameters. One problem with this theoryis that it may also stimulate tumour growth as nutrient flow is made more efficient as well. Inorder to test similar theories on the implications of vasculature in tumour growth, it is clear thattumour blood vessels will be at the forefront of the tumour microenvironment community.Another interesting feature of tumour vessels is that the extravasation of proteins from vesselsis slower than what it would be in a normal blood vessel with pores the size of typical tumourvessels [19]. This suggests that there is an additional physical process occurring at the cellularlevel. It turns out that due to the high interstitial hydrostatic pressure within tumours, thereis an unusually small hydrostatic gradient from the inside of vessels to the outside. This gra-dient reducing the forces that drive convection of the molecules. Ultimately, it causes proteins(macromolecules) to move out of vessels mainly by Diffusion, a slower process for larger molecules.Using the rationale above, it now is clearer why macromolecule size is a factor in studying vesselpermeability. In this study, we will use a traditional MR contrast agent (Gd-DTPA) attached toan albumin protein to increase its effective size and take advantage of its low permeability acrosstumour vessels.1.2.2 ImmunohistochemistryImmunohistochemistry is the process that refers to localizing biomolecules in cells or frozentissue sections. Often, specific antibodies that bind to particular antigens are used in histologyprocedures. The antibodies have attached fluorophores that fluoresce at specific wavelengths. Inthis project, histology is used to localize the MR contrast agent as well as blood vessels (usingCD31 and carbocyanine / hoechst). Tumours are first excised from a Mouse Type, then frozenand sectioned using a cryostat (figure 1.3) with a thickness of 10 µm and imaged using a digitalmicroscope. The tumours were typically 25 - 50 mm2in area and were captured at a resolutionof 1 pixel/mm2.Once the images were acquired the layers were overlaid, the CD31 and carbocyanine (orhoechst) layers were thresholded. The images show CD31 vessels that are perfused (dark blue)surrounded by regions of perfusion (light blue), unperfused vessels (red), and BrdUrd labeling(greyscale). An unperfused vessel is defined as any CD31 positive area that is negative on the51.2. Theory - Blood vessel and tumour biologyFigure 1.3: Image of a tumour frozen in an OCT medium being sectioned at a cryostat machine.10µm sections adhere to glass microscope slides due to a temperature gradient.carbocyanine layer (has no light blue staining). Glowing Galbumin is a commercially available(BioPal, USA) contrast agent suitable for MR imaging. It is the low molecular weight contrastagent Gd-DTPA (Gadolinium-DiethyleneTriaminePentaacetic Acid) labelled with a high molec-ular weight bovine albumin protein (≈ 74 kDa). A schematic representation (i.e. not to scale) ofGalbumin is given in figure 1.4. Glowing Galbumin, has the added advantage of 3-5 fluorescentFITC tags conjugated to Galbumin that allow for a convenient immunohistochemical analysis tolocalize the molecule following the study.Figure 1.4: A schematic representation of the Galbumin molecule. 15-20 Gd-DTPA atoms sur-round a large 74 kDa Albumin protein and 3-5 fluorescent FITC tags allow for convenient histo-logical imaging.61.3. MR Theory1.3 MR TheoryThe basic principles of nuclear magnetic resonance (NMR) involve the intrinsic spins of nucleiand their behaviour in the presence of strong external magnetic fields.Microscopically, atoms consist of both a nucleus (composed of protons and neutrons) andorbiting electrons. A net angular momentum arises in atoms that have an unmatched composi-tion of protons and neutrons (referred to as “MR-active” nuclei, e.g.1H19F,23Na,31P etc...).Semi-classically, each MR-active nucleus also has a distribution of non-zero charge (protons) andbecause the nucleus has an intrinsic angular momentum, a magnetic moment is induced. Considera sample of water with an abundance of protons: at rest and unperturbed, the sample containsprotons whose nuclei (figure 1.5) are oriented in all possible directions and the net angular mo-mentum of all nuclei is zero. This is because on average, for each spin orientation there existsanother spin oriented in exactly the opposite direction. Similarly, the net magnetization is alsozero.Figure 1.5: On the left, a typical proton is shown with its magnetization vector pointing alongthe z-axis. On the right, the axes are rotated so the schematic is showing the proton’s spin vector,projected along the x-y-axis.71.3. MR TheoryFigure 1.6: Classical diagram showing spins lining up to the external magnetic field B0resultingin a net magnetization M0. The spins align parallel and anti-parallel due to a small difference inenergy, as shown by the energy splitting diagram (due to Zeeman splitting).However, after some time in the presence of a strong magnetic field (B0), the nuclei tend toalign their axis of rotation to the applied magnetic field (figure 1.6). Quantum mechanically thepicture is slightly different: the1H is a spin-12particle and exist simultaneously in two possiblespin states, spin-up and spin-down. The spin-up state (s=−12in figure 1.6) is more energeticallyfavourable, since it has a slightly lower ground state energy. However, the energy differencebetween the two eigenstates is less than random thermal fluctuations at room temperature (E =KBT) so slightly less than half of the nuclei are in the spin-down state, aligned anti-parallelto the external magnetic field. The small excess of nuclei in the spin-up state, are exploitedin NMR. The ratio of these populations can be approximated by differences in the Boltzmannenergy distributions,nupndown= e∆EkBT(1.1)Even at a relatively strong magnetic field of 7 Tesla, the difference in the number of nucleiis about 1 in 20,000. MR measurements are made by measuring the time it takes for perturbedmagnetizations to decay back to equilibrium. However, after the magnetic field is exerted onthe sample, the longitudinal component of the net magnetization M eventually points along thedirection of external magnetic field (see figure 1.5), making it almost impossible to measure. This81.3. MR Theoryis why MR measurements are made in the the transverse direction (x and y if the B is directedalong +z). Since all of the atoms are spinning out of phase with each other, the net transversemagnetization is initially zero.Longitudinally, the initial magnetization vector (M0can be tipped away from its thermalequilibrium value by applying an orthogonal oscillating RF pulse tuned at the Larmour frequency.The time it takes for the longitudinal component of the magnetization vector to decay back toequilibrium (aligned with B0) is referred to as the spin-lattice relaxation time or in short, T1relaxation.Initially when the sample is first placed inside the magnetic field (before the spins aligneither parallel or anti-parallel to B0oriented in the z-direction), the spins precess around B0.The spins eventually reach equilibrium and the parallel and anti-parallel orientations yield ina net magnetization vector M0. If an oscillating magnetic field is now applied to the samplein the x or y-axis, the magnetization vector is simply rotated. The duration of this oscillatingmagnetic field, usually in the radio-frequency range (RF - tens of Mhz) controls the extent ofthe magnetization vector rotation. For instance, a 90◦pulse tips the pulse from the z-axis to thex-y-plane and a 180◦or pi pulse tips it from +z to -z. Once rotated, due to the persistent presenceof the external magnetic field, the nuclei spins immediately begin dephasing (T2relaxation) andthe magnetization in the z-direction is re-established (T1relaxation). To measure T1and T2relaxation, various pulse sequences are used to measure the free induction decay (FID) in thetransverse direction. Typical sequences are shown in figures 1.7 and 1.9 to measure longitudinaland transverse relaxation.1.3.1 Measuring T1Relaxation (using a Spin Echo Pulse Sequence)One way to measure T1is to use a Spin Echo pulse, shown in figure 1.7. The induced voltagein the coil is measured as a function of time and then fourier transformed to given intensity.These intensities are then plotted against the “variable delay times”, and a decaying exponentialis fitted to the data to produce the longitudinal relaxation time, T1.91.3. MR TheoryFigure 1.7: Schematic representation of a Spin-Echo pulse sequence. Multiple iterations of thespin-echo sequence (180◦then 90◦) pulses are performed and the echo is recorded as a functionof time.Figure 1.8: The signals (from figure 1.7) are Fourier transformed and here, the max signal intensity(ofeachecho) is plotted as afunctionof thevariabledelay. Thetime constantfrom theexponentialrecovery is T1. More precisely, T1is measured as the time it takes for the signal intensity toreturn to 63 % of its’ maximum value.101.3. MR Theory1.3.2 Measuring T∗2Relaxation using (CPMG Pulse Sequence)T∗2is commonly measured using a pulse sequence called a CPMG sequence. Figures 1.9 and 1.10detail the sequence and the method for determining T∗2and T2using the CPMG sequence. Noticethat T∗2is always shorter than T2because the T∗2relaxation time includes spins dephasing dueto both spin-spin interactions as well as field inhomogeneities.Figure 1.9: Schematic representation of a CPMG pulse sequence. The initial 90◦pulse tips themagnetization pulse in the transverse direction, the magnetization then decays rapidly due to fieldinhomogeneities and other spin-spin interactions. Application of a 180◦pulse flips the directionof the dephasing spins and causes them to rephase. As the spins rephase, a spin echo signal isgenerated. The T2decay curve in blue is represented in figure 1.10.111.3. MR TheoryFigure 1.10: The curve above shows the T2relaxation process. The FID signal from figure 1.9 isfourier transformed and the maximum intensity is plotted as a function of the Echo Time (TE).The decay constant is the spin-spin relaxation rate.As shown in figure 1.11, when a magnetic moment is tipped so the angle between the mag-netization vector and the original magnetic field is α, the field exerts a torque on the vector Mcausing it to precess. This precession frequency is referred to as the Larmor frequency and isgiven by,ω0= γB (1.2)Here, γ is the gyro-magnetic ratio, an intrinsic property of the nucleus andB is the magneticfield, dominated by the static field B0with a small contribution from a magnetic field gradient.This magnetic field gradient is the key to NMR imaging as it changes the total magnetic fieldstrength at a particular spatial location and thus, by equation 1.2, the Larmour frequencies of thenuclei vary by location and the spatial coordinates can be encoded into the measured resonantfrequencies.The following general description show some basic NMR procedures to image a sample.Basic Steps for acquiring MR data1. Place sample in constant magnetic field B0, length aligned parallel to B0and the z-axis(figure 1.11).2. Apply a continuous magnetic field gradient across the sample3. Simultaneously, apply a radio frequency (RF) pulse close to the larmour frequency ω0.121.3. MR TheoryFigure 1.11: On the left, the magnetization vector tipped and as it returns back toB0, it precessesaround B0. [4]. On the right, a schematic of slice-selection with a patient lying along the amagnetic field along their length.4. The magnetic field gradient causes each slice along the z-axis to have a slightly different netB and by equation 1.2, a different Larmour frequency.5. Recall that the RF pulse tips the magnetization vector so with the RF pulse at ω0, only aparticular slice has its’ vector M tipped and all the other nuclei are still aligned with theB field they encounter (function of distance along z).6. By tuning ω to match ω0, we effectively “select” the slice we’re interested in imaging.7. The magnetization vector precesses around B0and eventually decays back to its steadystate (aligned with B0). The longitudinal component of this relaxation is referred to as T1and the transverse component as T2.8. Receiver coils in the MR scanner pick up the voltages induced by the precessing magneti-zation9. The FID’s (or echoes with T1) are fourier transformed and the maximum intensities areextracted and plotted as in figure 1.9.10. Similar gradient and pulse sequences in x,y and z can be used to encode frequency andphase so the final image looks like figure 1.12.The human body is composed of about 60% water, so hydrogen nuclei are a natural choicefor MR imaging experiments in both humans and animals. Contrast agents allow a furtherenhancement and with T1or T2weighted imaging, can increase the amount of detail present inimages taken in-vivo. In this study, we will be considering two types of weighted imaging, andour novel protocol will require the use of both T1and T∗2weighted imaging simultaneously.131.4. MR Contrast AgentsFigure 1.12: Left: representation of the FID signal data acquired, notice concentration of dataaround the origin. Right: Typical MR image of a tumour after a Fourier Transform. Bright bulbat the bottom is the crosssection of a vial of water, used as a standard reference point.1.4 MR Contrast AgentsMR images are created based on the signal from MR-active nuclei relaxing under the presence ofan external magnetic field. Image contrast is primarily based on the distribution of these nucleiinherent in different tissues (e.g. water or lipid content). To increase contrast, MR contrastagents are used to selectively alter the relaxation times of hydrogen nuclei in tissues where thecontrast agent is present. The driving force behind using MR contrast agents (and other imagingmodalities) is to improve image contrast and visualize areas that would otherwise not appear onthe scan. This can often occur when the tissue properties of an area of interest is too similar tothat of its surroundings and the effect averaged out. In a T1weighted image, areas with shortT1relaxation times such as fat, appear bright and water with significantly higher T1relaxationtimes, appear dark [24].In other imaging modalities, such as X-ray or CT scans, iodine or barium contrast agents areused that result in a direct effect in image contrast (tissues containing barium or iodine results ingreater attenuation of x-rays. MR contrast agents work indirectly as no signal is derived from thecontrast agent itself - the relaxation times in hydrogen nuclei is still measured but the times havebeen altered due to the proximity of the nuclei to the contrast agent. The best MR contrast agentstherefore are those that produce the largest gradient in relaxation times. Table 1.4 categorizescontrast agents into three major groups:Recall that the spin-lattice (T1) and the spin-spin (T2) relaxation times are related to theirrelaxivities (relaxation rates) reciprocally:141.5. Modelling Contrast Agents - Kety ModelCA Type Effect on T1Effect on T2or T∗2ExamplesParamagnetic Agents decreases decreases Gd-DTPASuperparamagnetic particles None Significant Decrease Feridex, CombidexFerromagnetic particles None Dramatic decrease fluidMAGTable 1.1: A table of the typical divisions of MR contrast agents [2]. In this study, we useFeridex, Gd-DTPA and Galbumin (mainly a T1agent). Note, the table doesn’t quantify thedifference between “significant” and “dramatic”, because there is a range of effects depending onconcentration and relaxivities.r1=1T1and r2=1T2(1.3)The relationship between relaxation rates and contrast agent concentration can then be pre-dicted by the Solomon-Bloembergen equations [22, 25]:1T1=1T10+r1[Gd] (1.4)1T2=1T20+r2[Gd] (1.5)where [Gd] is the contrast agent concentration, usually a Gd-based agent.Using equation 1.5, we can now find a relationship for CGd(t) empirically:CGd(t)=R10−R1(t)r1(1.6)Once the contrast agent relaxivity has been determined, assumed concentrations of CA inthe blood will result in predicted T1values. Each T1value will yield a signal intensity (from ashort-TRgradient echo sequence), given by the following well established equation [25]:S.I. = g · ρparenleftbigg1−eTRT1parenrightbigg1−cos(α)e−TRT∗1· e−TET∗2sin(α) (1.7)1.5 Modelling Contrast Agents - Kety ModelThe 2-compartment pharmacokinetic model called the Kety-model later expanded by Tofts et.al. [1] is the industry standard in modelling contrast agent uptake in humans and animals. Themodel was initially intended for use in the brain, where the effect blood brain barrier was a keyassumption. Figure 1.13 shows a schematic of the model - the Kety model relates the movement151.5. Modelling Contrast Agents - Kety Modelof contrast agent between two compartments, the blood vessel (containing plasma) and the inter-stitial space. The red arrow in figure 1.13 termed Ktransis the forward rate, and it encompassesboth blood flow and (see [26] for details) the vessel permeability surface area product (PS - see[1]). The reverse rate, Kepis the rate constant between the EES and blood plasma (see Table 1.2for details on these quantities).This model can be applied in two limiting circumstances: flow-limited and permeability lim-ited. Low molecular weight agents such as Gd-DTPA, considered freely diffusing agents transferbetween the blood and the EES of the tumour with a ktransthat is proportional to the the flowrate. Larger, minimally diffusing agents result in a Ktransthat is proportional to the permeabilitysurface area product PS.Parameter Units Physical MeaningEES none Extravascular extracellular spacevenone Volume of EES per unit volume of tissueKtransmin−1Volume transfer constantKepmin−1Rate constant between EES and blood plasmaCpmM Tracer concentration in arterial blood plasmaCtmM Tracer concentration in tissueTable 1.2: EES refers to the space into which tracer can leak from a capillary and Ktransis thevolume transfer constant between blood plasma and the EES. Table parameters by: [1]Figure 1.13: Two compartment Kety Model with rate constants describing flow of contrast agentout into the EES (compartment A)and back into the vessels (compartment B).161.5. Modelling Contrast Agents - Kety ModelThe kinetic tracer model can best be described in the following form [1]:dCtdt= Ktrans(Cp−kepCt) (1.8)When Ct=Cp= 0 at time t=0, equation 1.8 solves to the following relation:Ct(t)=Ktransintegraldisplayt0Cp(τ)e−kep(t−τ)dτ (1.9)The dual contrast agent protocol we hope to implement has this idea at its’ core - in orderto determine the permeability of tumour vessels, two agents may be used in succession. Thefirst, a small molecular weight agent would yield a Ktransrelated to both permeability and bloodflow. Then, in the same mouse, a second high molecular weight agent would yield in a Ktrans-ostensibly related only to the blood flow as the agent is too large to extravasate out of vessels.Figure 1.14 shows a schematic of this protocol.Figure 1.14: Schematic of a dual contrast agent protocol - the first contrast agent is injected,the signal acquired and then the second agent is administered. Because both sets of data havebeen acquired, the signal intensities can be processed and the two processes can be treated asindependent.17Chapter 2Materials and MethodsIn summary, 5 NOD/SCID mice with subcutaneous HCT-116 (3 mice with human colorectalcancer) and HT29 (2 mice with human Caucasian colon adenocarcinoma) xenografts received aninitial scan (under anesthesia) for water-diffusion. The mice then received a baseline T1weightedMRI scan prior to injection of the first contrast agent. Then a dynamic scan was taken for 30minutes with the low molecular weight agent (Gd-DTPA). A follow up T1weighted scan wastaken to re-establish a new baseline T1and were then injected with the second contrast agentand another dynamic scan was taken. After the scans, the mouse was removed from the scannerand hoechst was injected i.v. 5-20 minutes before excision.2.1 Mouse ProtocolThe following is a reasonably accurate procedure for implanting the mice with tumours (for moredetails see [8, 11, 27]:1. Seed 2 x T-175 flasks with 1.5 x 106cells (1:10) days before implant2. Split to 4 x T-175 flasks in 3-4 days or whenever 70% confluency is achieved3. Collect cells to 1.6 x 108cells / mL (for 8 x 106cells per 50µl implant)4. Anaesthetize mice5. Sterilize sacral region using isopropyl alcohol6. Implant 50 µl cells per mouse7. Return mice to cage8. Measure tumours once or twice a week9. Tumours are ready in 3-4 weeks2.1.1 Preparing a mouse for an MR scanAnimal handling was performed according to the ethics guidelines approved by the Animal CareCommittee valid until December 31, 2008. The animals were stored at the Animal Research182.1. Mouse ProtocolCentre at the BC Cancer Research Centre and transported to the 7T MRI facility at the UBCLife Sciences Centre the day of the experiment and were sacrificed at the end of the experimentthat day. Refer to figure 2.1 and the following outline for the animal preparation procedure.1. Begin Mouse Prep Protocol2. Turn on water blanket to thermoregulate anaesthetized mice while in the MR scanner (45◦Cset point, pump = fast).3. Turn on ParaVision (imaging software), electronics, unlock magnet room, check anestheticlevels, refill anaesthetic if necessary, turn on medical air.4. Record information on mouse: Type (HCT116 or HT29), number of tumours (1 or 2), age,gender, identification code, tail markings.5. Weigh mouse (and record weight) prior to scan and injections6. Insert catheter into tail vein with appropriate connector.7. Use heat lamp to warm up mouse and increase blood flow (helps identify tail vein forinjection)8. Prepare solution of heparin (anti-coagulant) diluted in saline 1:109. Inject 200-250 µL of saline into mouse to keep it hydrated10. Place mouse into anaesthesia box, wait about 30 seconds11. Squeeze toes to check if suitably anaesthetized12. Check breathing13. Remove mouse from restraint, make sure it is still anaesthetized. Leave on heat lamp.14. Apply protective lubricant on eyes to prevent drying/scratching.15. Move mouse from the anaesthesia box to the scanning coil16. Place mouse’s mouth and nose over the anaesthetic hood on the coil (make sure mouthpieceis in the proper orientation.17. Adjust mouse position to have the tumour(s) in place directly inside the coil18. Insert rectal temperature probe, use Vaseline on the end of the probe. Tape down.19. Transport mouse from the animal room to the scanning room, attach anaesthetizing tubein the scanning room to the connector on the coil192.2. HistologyFigure 2.1: Description of Experimental setup20. Attach respiratory monitor onto mouse with tape. and tape the catheter, syringes any otherwires/tubes to the bed.21. Place water blanket over mouse, check temperature and breathing rate22. Monitor respiratory rate and temperature. Monitor anesthetic levels (≈ 1 unit of air, 0.5to 1 unit of isofluorane).23. End Mouse Prep Protocol2.2 HistologyAn automated microscope with x-y-z slide stage was used to acquire entire tumour-section images.Hoechst (perfusion), CD31 (vasculature), Haematoxylin (nuclei) and Galbumin (contrast agent)were fluorescently imaged independently from tissue sections corresponding to MR scan slices(see [11, 27] for details). Galbumin and Hoechst distribution was mapped as a function ofdistance away from blood vessels in the two tumour types. Extravascular extracellular space,marked with Coll IV was measured and correlated with ADC from diffusion-weighted EPI.202.3. MR Scans2.3 MR ScansMR scans were taken at the high field MR research centre at the Life Sciences Institute underthe direction of Dr. Stefan Reinsberg, Dr. Piotr Kozlowski and Andrew Yung. Imaging was per-formed on a 7T Bruker Biospec 70/30 using a custom-built 4 turn distributed-capacitor solenoid.The final dual contrast agent MR protocol is summarized below:1. Tripilot (scout) (2 minutes)2. Diffusion weighted EPI (3-4 minutes)3. High-Res image for baseline T1(5 minutes)4. Begin acquisition: T1DCE-MRI (30 minutes)5. Administer contrast agent 1 after at least 30 seconds of acquisition6. High-Res image for T1(5 minutes)7. Begin acquisition: T1weighted DCE-MRI (30 minutes)ORT∗2weighted DSC-MRI (30 minutes)8. Administer contrast agent 2 after at least 30 seconds of acquisition9. High-Res image post-contrast T1(5 minutes)2.3.1 Diffusion-weighted EPIDiffusion-weighted EPI with TR/TE= 3000/26.9, b-values 0,500 was performed and apparentdiffusion coefficient (ADC) maps were calculated. See figure 3.8 for a representative set.2.3.2 T1weighted images30 minutes of the T1-weighted FLASH (TR/TE=113/2.145) FLASH were acquired following a100-150µL/mouse bolus of Galbumin (25 mg/mL). A two-TR FLASH protocol (TR=226 msand 113 ms, TE= 2.145 ms) was used for calculating contrast agent concentration with timeresolution of 14.5s. Scanning was conducted for 30 minutes because unlike with T∗2-weightedimaging, enhancement lingers as the agent equilibrates over the tumour. Because the the effect ofthe T∗2agent relies upon introducing magnetic field inhomogeneities, the effect rapidly decreases(signal intensity recovers quickly) as it circulates through the blood stream.212.4. Galbumin Relaxivity2.3.3 T∗2weighted images150s of the T∗2-weighted FLASH (TR/TE=46.875/10.0) FLASH were acquired following a 100-150µL/mouse bolus of Galbumin (25 mg/mL). Flip angle was set to 30◦ and two slices of thetumour were acquired with a time resolution of 150s for complete acquisition.2.4 Galbumin RelaxivityReferring to equations 1.5 and 1.6, we can construct a protocol to experimentally determine therelaxivity of Galbumin if we take several samples with a known concentrations of Galbumin,record measured T1values and plot the signal intensity (equation 1.7). The protocol used isdescribed below:• Prepare vials with at least 5 precisely known concentrations of contrast agent• Perform a T1weighted MR scan on each of the vials independently• Record T1values for each of the 5 known concentrations• Plot R1vs. CGd(t) and fit to a linear function (f(x)=mx+b)• Slope of this graph (figure 3.5) is the relaxivity22Chapter 3Results, Analysis and Discussion3.1 Galbumin HistologyGalbumin (commercially available from BioPal, U.S.A) is a contrast agent that lends itself wellto histological analysis immediately after the tumour has been frozen. In other words, once theMR scan is complete (≈ 45 minutes) and the mouse has been sacrificed with the tumour excisedand frozen (≈10 minutes), the tumour can be sectioned and directly imaged (fluorescently) forGalbumin. This is the most direct method of imaging as it avoids several washing and fixing stepsthat may potentially result in washout artefacts for the target molecule (Galbumin in this case).To localize Galbumin with respect to other histological features such as blood vessels however, thewashing and fixing steps are required. Due to the versatility of the FITC tags, the slides can beimaged again to account for intensity differences, bleaching and washout effects. Figure 3.1 showsa typical tumour section stained for blood vessels (red, CD31) and Galbumin (green, FITC). Infigure 3.3, we mapped the presence of Galbumin (detected by positive fluorescence intensity) as afunction of distance from the nearest perfused vessels(determined by the presence of both CD31and hoechst). Contrast figure 3.3 - mapping a large molecule to perfused vessels, to figure 3.4,where the presence of hoechst is mapped as a function of distance from perfused vessel in thesame tumour. Qualitatively, most of the Galbumin is localized to within 50µm away from vesselswhereas Hoechst, the smaller molecule, perfuses/diffuses as far as 100 µm away from vessels.Quantitatively, there exist several problems with comparing extravasation curves. Primarily,there exist intrinsic differences in the molecules’ ability to fluoresce at particular wavelengths. Itmay be that the tissue exposed to 488nm light, may have a higher background compared to 546nmor 350 nm. Further, the exposure time of tissue to the fluorescent lamp greatly influences thefluorescence signal Intensity. Finally, to accurately compare two extravsation curves and extractmeaningful quantitative information, we require there to be several controls imaged at variousexposure times, with and without the target molecule to predict the contribution of backgroundfluorescence.The tumour microenvironment is heterogeneous and the vasculature disordered and in fig-ures 3.1and 3.2 there is a clear differential in vasculature behaviour among vessels (in the sametumour). Since the differential appears to be uncorrelated with any known effect, this suggeststhat chemotherapeutic drugs administered would likely reach their target at different times, ifat all. This idea is of crucial importance because in order for a tumour to be fully eradicated,233.1. Galbumin Histologyall cancer cells must be killed. One can imagine that if drugs take effect in certain areas earlier,surrounding cells may compensate for this and the effect of the drug would effectively, be reduceddrastically. A possible solution, and one that is used often, is the drug is administered in dosesmuch larger than strictly required to ensure maximal effect. Of course, this has the disadvantageof increased toxicity and other unintended physiological consequences.Future studies would look at the difference in vessels in various areas and attempt to charac-terize their behaviour more accurately. The ideal method for accomplishing this is a long term(longitudinal) study on a group of mice that tracks and characterizes the behaviour of the tumourand vessels over time. Pike et. al.[? ] conducted a similar study and looked at tumour growthover time. We intend to extend that study to investigate possible explanations to vasculaturedifferential including the age of vessels, proximity to other established/unestablished vessels aswell as a fundamental characteristic of the tumour cell line. We used mainly HCT116 (humancolon carcinoma) and HT29 (human colon adenocarcinoma grade II). Comparing vasculature be-haviour more rigourously among these two cell lines using histology and MRI is a potential goalfor this study.243.1. Galbumin HistologyFigure 3.1: Typical tumour (HCT116) section stained for blood vessels (red, CD31) and Galbumin(green, FITC). Sections of the same tumour are stained and (fluorescently) imaged separatelyand overlaid on top of each other.253.1. Galbumin HistologyFigure 3.2: An area of the tumour above (figure 3.1) zoomed in. We note some vessels (red,CD31- bottom left) have positive Galbumin staining (green, FITC) while other vessels show nopositive staining (top right).Figure 3.3: Plot of Galbumin extravasation as a function of distance from blood vessel in HCT-116 tumour xenografts. 4 time points from injection to sacrifice are shown and the general trendis that over time, Galbumin equilibrates in the tumour.263.2. Galbumin as T1agentFigure 3.4: Plot of Hoechst extravasation as a function of distance from blood vessel in HCT-116 tumour xenografts. 3 time points from injection to sacrifice are shown. Compared to theGalbumin curve (figure 3.3) Hoechst tends to extravasate further away from blood vessels (smallermolecule)3.2 Galbumin as T1agentThe initial intent of the dual contrast agent injection protocol was to use Galbumin as the highmolecular weight agent and Gd-DTPA as the low molecular weight agent. Because Galbumin isa new product on the market, it had to be characterized in terms of its effect on T1relaxation.In figure 3.5, we determined the relaxivity of Galbumin to be 86.62 ±5.26 (mM · sec)−1, muchhigher than published values for each Gd-DTPA molecule at 8.5T (we used a 7T magnet) 3.870.06 (mM · sec)−1[28]. However, this differential is easily accounted for when considering thecomposition of the Galbumin molecule. BioPal claims that anywhere from 15-20 Gd-DTPA atomsare attached to an Albumin protein. The adjusted value for Galbumin per Gd-DTPA moleculeresults in a value ranging from 4.33 to 5.77 (mM · sec)−1, closer in line with published values.273.2. Galbumin as T1agentFigure 3.5: Galbumin relaxivity data plotted and fitted according to equation 1.6 and the processlaid out in that chapter. We determined the relaxivity of Galbumin to be 86.62 ±5.26 (mM ·sec)−1.283.2. Galbumin as T1agentFigure3.6:Inthistumour,Gd-DTPAisthecontrastagentbeingadministered.T1-weightedgradientechoimagesbefore(A)andafter(B)Gd-DTPAinjection.Notethenearuniformenhancementthroughoutthetumour-becauseGd-DTPAisnotabloodpoolagent(diffuseseverywhere),itprovidesMRsignalenhancementeverywhere.Figure3.7:A&BT1-weightedgradientechobefore(A)andafter(B)galbuminadministration.Itisdifficulttoseeanenhancementjustwithimages(A)and(B)so(C)showsthesignalenhancementparametermapfromDCEMRIacquiredduringGalbuminadministration(initialareaunderthecurveIAUC60).Noteareasofperipheralenhancement,potentiallyanarearichwithlargervesselsreside.293.3. ADC (Apparent Diffusion Constant) MapsFigures 3.6 and 3.7 show images from typical DCE (dynamic contrast enhanced) scans withGd-DTPA and Galbumin. Despite the fact that Galbumin had a similar relaxivity to Gd-DTPA,Galbumin did not provide a large enough enhancement. Using the signal intensity equationdiscussed in chapter 3 (equation 1.7) we can calculate the net change in intensity from a measuredchange in T1before and after Galbumin administration. With a 100 µL dose of Galbumin injectedI.V., the calculated T1value is about 418 ms. The T1of blood plasma is around 1200 ms andwith the other parameters set as in chapter 2, applying the signal intensity equation twice yieldsa net signal intensity increase of 151%. However, signal enhancement only occurs in vessels andareas with blood. We can estimate that about 5% of the total volume of a typical mouse at 20 gis blood volume and extending that to each voxel, only a total signal intensity increase of 7.5%is achieved.Several attempts were made to continue using Galbumin as the high molecular weight agentin the dual contrast agent protocol because it is an ideal compound to use histologically. Thefirst strategy was to increase the dosage from 100 µL to 200 µL. This had several side-effectsas the tolerance of the mouse (to the contrast agent) seemed to decrease with the added volumeinjected I.V. through the tail vein. More importantly, as expected the increase in intensity wasstatistically insignificant. Attempts to concentrate the mixture were quickly derailed because themanufacturer suggested that it would become too viscous past 4x concentrated. The alreadycostly agent would increase in price by a factor of at least 4. Consider all this, we concluded thatGalbumin was not a suitable contrast agent for T1weighted imaging at the concentration themanufacturer provides in our dual-contrast agent protocol. One insight we’ve gained from thispilot study is that having a larger contrast agent results in requiring more of the agent to achievethe same effect as a smaller agent if the active molecules (Gd-DTPA) are the same. In searchingfor more suitable contrast agents, one restriction we can now place is that the relaxivity of theagent has to be several times higher.3.3 ADC (Apparent Diffusion Constant) MapsWhile the mouse is in the scanner, before contrast agent administration, it is often convenient todo a simple diffusion weighted MR scan. This serves several purposes and in a longitudinal study,diffusion-weighted MR scans can be time coursed and the changes studied over time. The changein the apparent diffusion coefficient (ADC) of water is hypothesized to be an indicator of tumournecrosis. DW scans were acquired using echo-planar imaging (EPI - TR/TE=3000/26.9) andapparent diffusion coefficient (ADC) maps were calculated. Figure 3.8 shows the ADC maps forthe two tumour models used in this study. HCT116 (left) is characterized by higher propensityto necrosis and leakier vessels. DCEMRI parameter maps (figure 3.7) show this higher leakinesseven for macromolecular contrast agent Galbumin. HT29 (right) is characterized by moderatenecrosis (corresponding lower ADC values) and reduced leakiness. In future studies, we hope to303.3. ADC (Apparent Diffusion Constant) Mapsmake use of ADC maps in studying tumour growth and as such, included them in the protocoldevelopment stage.313.3. ADC (Apparent Diffusion Constant) MapsFigure3.8:ApparentDiffusionCoefficient(ADC)maprepresentstypicalHCT-116(left,[A])andHT29(right,[B])tumours.Greyscalesarescaledfrom0-255,0isblackand255iswhite,correspondingtoanADCrangingfrom0-2µmm2msrespectively.TheHT29tumour(right)hasrelativelylowwaterdifusionandequivalently,lessnecrosis.Thetumourisoutlinedinred.32Chapter 4T∗2Weighted Imaging: Galbumin &FeridexMagnetic susceptibility is a fundamental property of matter and is defined as the ability of theexternal magnetic field to affect the nucleus of an atom and magnetize it. The role of contrastagents in general, is to introduce changes in the local magnetic field. Using molecules with highsusceptibility is one way to induce small local inhomogeneities in B and from equation 1.2, achange in the larmour (precession) frequency is caused to the nuclei(figure 4.1). The signals fromthe sum of these precessions collectively decay quicker than if there was noB field inhomogeneity.This is the underlying principle of T∗2weighted imaging and referring to figure 1.2, we notice thatFeridex is commonly used as an agent in T∗2weighted imaging (it is a superparamagnetic ironoxide particle - SPIO). However, Galbumin has not yet been eliminated as an agent affecting T∗2.Figure 4.1: Inhomogeneities caused by the superparamagnetic contrast agent.Our dual-contrast agent protocol can be readily adapted to T∗2weighted imaging (as opposedto T1weighted, figure 1.14). Figure 4.2 shows the modification, following the T1agent injection,we simply change the scan type and proceed as usual. The parameters measured in T∗2weightedimaging are slightly different so we won’t get a ktrans, but rather the rBF, rBV and MTT (seesection 4.2).334.1. Galbumin as a T∗2agentFigure 4.2: Dual Protocol4.1 Galbumin as a T∗2agentThe magnetic susceptibility of Galbumin was measured according to previously published meth-ods [29] and it was found that Galbumin susceptibility was not high enough to allow effective T∗2weighted imaging. Reference susceptibility values are shown in Table 4.1. Equation 4.4 was usedto find the susceptibility ∆χ,∆χ = −2∆φB0TEγproton1parenleftbigcos2(θ)−13parenrightbig (4.1)(4.2)∆χgalbumin=2.9465x10−6(4.3)(4.4)The parameters used in determining the susceptibility curve (figure 4.3) are below:• θ = 10.1 degs• φ = -0.60 rads• B0= 7 T• TE= 2.145 ms = 0.002145 secs• Gyromagnetic ratio of protons γproton=42.576MHz/T,344.2. Feridex as a T∗2agentFigure 4.3: The data acquired for the experiment to determine Galbumin susceptibility. The finalvalue ∆χ was experimentally determined to be: 2.95 ppm/mMSubstance Susceptibility ∆χ (ppm)Blood -0.3 to 1.5Gd-DTPA 2.7 ± 0.1AMI-25 40 ± 2Feridex TBATable 4.1: Table parameters from: [2]4.2 Feridex as a T∗2agentIn T∗2weighted imaging, the contrast agent (Feridex here) acts as a tracer (that stays in vessels)during at least the first pass through the circulatory system [30]. Applying the appropriatemodels, we can obtain the relative blood flow (rBF) and relative blood volume (rBV) within atumour. There is also a third useful parameter, the mean transit time (MTT) which is the ratioof the rBV and the rBF. In this study, we have only acquired preliminary data using Feridex,and analysed it sparingly.354.2. Feridex as a T∗2agentRecovery and intensity parameters are shown from two slices of an MR scan are shown infigure 4.4. Since T∗2weighted imaging results in an intensity dip due to magnetic field inhomo-geneities caused by the contrast agent (Feridex), we would expect to see a recovery in signalintensity once the agent has equilibrated in the blood. The recovery maps show the relationshipbetween two intensities: middle of the scan (lowest intensity) and at the end (return to intensity)and both slices (slice 1 and 2) show no recovery in the intensity 140 seconds after injection.Theintensity maps show us which region of the tumour had the largest decrease in signal intensity.The areas that are black in the parameter maps show no decrease in intensity, and consequently,we can expect that those areas saw no contrast agent flow (necrosis or interstitial space).Figure 4.4: This figure details the use of Feridex in the mouse, the parameter maps are at thetop left, the equations used to construct the maps on the right and a schematic of the intensityat the bottom.364.3. Mapping Feridex Histologically4.3 Mapping Feridex HistologicallyFeridex (generically ferrumoxide), is a sterile aqueous colloid of superparamagnetic iron oxide(SPIO) associated with dextran. Administered intravenously, it is an MR contrast agent usuallyused for the detection of liver lesions. Feridex is taken up by macrophages, found only in healthyliver cells but not in most tumors. We want to use Feridex in the DSC weighted portion ofour protocol but the major disadvantage of Feridex is that it does not lend itself well to beingcharacterized histologically. As a result, the tracking and/or mapping the movement of Feridexas it extravasates out of tumour vessels. The absence of the FITC tags requires using morerigourous, less versatile staining methods (such as chemical staining). We have recently discoveredthat the Feridex contains a dextran coat, which could potentially be useful in manually attachingfluorescent tags or even using antibodies specific to that dextran for immunohistology.4.3.1 Chemical Stain - Prussian BluePrussian Blue is a synthetic pigment commonly used in histology to detect the presence of non-hemoglobin iron. More specifically, ferric ions (Fe+3) ions in the tissue combine with ferrocyanide(reagent) and results in the formation of a bright blue pigment called Prussian Blue. See figure 4.5for sample staining [6]. The general protocol is described in the appendix A.4,Figure 4.5: The scale bar is 10 µm and the image has been taken under a light microscope byJennifer Flexman et. al [5]. The red and purple arrows indicate different ares of staining in aneuron. Jennifer Flexman assisted with developing a protocol for Feridex staining.37Chapter 5Conclusions, Implications and FutureWork5.1 ConclusionsIn this study, we have managed to establish, at least theoretically, a dual contrast agent pro-tocol that will serve to accurately extract tumour vasculature permeability information that isgenerally coupled to vasculature flow in Ktrans. Following this, we considered Galbumin, a novelcontrast agent (commercially available from BioPal USA) as a candidate for our dual CA proto-col. Through both experimental and theoretical means, we established that while the relaxivityof Galbumin (4.33 to 5.77 (mM · sec)−1per Gd-DTPA chelate) was close to the relaxivity ofGd-DTPA (3.87 ± 0.06 (mM ·sec)−1[28]), the required level of signal enhancement was not seenwhen imaging with the scans T1weighted. Next, we adapted our protocol to include T∗2weightedimaging and thus considered Galbumin again as a T∗2contrast agent. Again, through experimen-tation and as expected, we found that Galbumin susceptibility (2.95 ppm/mM) was very closeto Gd-DTPA susceptibility (2.7 ppm/mM ±0.1), effectively eliminating it from consideration asa candidate for a T∗2agent. Ultimately, Feridex, a well-studied SPIO (superparamagnetic ironoxide particle), emerged as the leading candidate for a high molecular weight contrast agent inour protocol. Preliminary experiments were conducted with Feridex in mice and it was foundthat the particle did not extravasate out for at least the first 150 seconds (figure 4.4). Further,it’s susceptibility was quite high and thus made it ideal for a T∗2contrast agent.In addition to the MR work above, we also developed, refined and optimized several histologyprotocols for use in the future. For example, we now have a set protocol that dictates precisely thesteps to take from when the MR scan is complete up until tumour sections have been obtained.Several challenges were encountered in maintaining the tumours in the orientation they wereexcised to match with MR images as closely as possible. Once we have established the right highmolecular weight contrast agent to use in the MR protocol and determined the most effectiveway of visualizing it histologically (whether it be a chemical stain/dye or a fluorescent marker),we are confident the histology portion of the study has been reasonably optimized.385.2. Implications and Future Work5.2 Implications and Future WorkThe work presented in this thesis is likely going to be the basis of a Masters project. A lot of theideas presented here seemed deceptively simple at first, several challenges and roadblocks wereencountered in the process of arriving at some of the conclusions. The following is a list of itemsthat are at the top of our list to attempt over the next few months:• Study (rigourously) Galbumin extravasation from vessels• Look into using dextran-DTPA contrast agents (as high as 500 kDa)• Consider other high molecular weight T1agents suitable for dual-injection protocol• Experimentally determine Feridex Susceptibility• Investigate labelling Feridex with FITC tags• Ensure Feridex is an intravascular agent• Investigate mapping Feridex using antibodies specific to the dextran coating• Devise and test a method to extract vessel permeability from the dual-injectionprotocol• Implement the use of fiducial markers in more accurately determining the orientations oftumour from MR scan to histological section• Investigate vascular permeability over time in a longitudinal study• Study qualitatively and quantitatively the effect of anti-angiogenesis drugs on vessel per-meability39Bibliography[1] P Tofts, G Brix, D Buckley, and J Evelhoch. 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Dcemri-oncology-chptr4. page 15, Nov 2008.42Appendix AAppendixA.1 Susceptibility Equation DerivationHere we derive the susceptibility equation ( [2]) used to experimentally determine the suscepti-bility of contrast agent (Galbumin in our case),∆B =∆χ2parenleftbiggcos2(θ)−13parenrightbiggB0(A.1)i.e. for θ=0,∆B =χB03(A.2)ω = γ∆B (A.3)∆φ = −γ∆BTE(A.4)∆φ = γχB03TE(A.5)(A.6)and finally, for θ negationslash=0∆B =parenleftbiggcos2(θ)−13parenrightbigg∆χB0TE(A.7)∆χ = −2∆φB0TEγproton1parenleftbigcos2(θ)−13parenrightbig (A.8)43A.2. IDL Program to Calculate DSC Parameter MapsA.2 IDL Program to Calculate DSC Parameter Maps;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;; Creating DSC Image Maps ;; DSC_MAP_Firas.pro ;;;; April 1st, 2009 ;; Programmed by: Firas Moosvi ;; Assisted by: Stefan Reinsberg ;; Applied on Data set: 30Dec08 + 31Dec08 ;; Feridex Susceptibility ;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;;/// Turn the program into a routinepro dsc_map_firas,scanid=scanid;/// Load scan data into a matrix ’a’;/// Create 2 float arrays called map and map2 of size 64;/// These arrays hold the final map dataa=mm_load_scan(scanid=scanid)map=fltarr(64,64,4)map2=fltarr(64,64,4);/// Begin 3 for loops spanning x,y,z;/// z is the number of slices, in this case 2;/// Position of z as the last loop is importantfor x=0,63 do beginfor y=0,63 do beginfor z=0,1 do begin;/// take three averages of each pixel, given by (x,y) for each z;/// the first average is prior to the injection (beginning);/// the second average is some time after the injection (middle);/// the third average is the intensity average at the end44A.2. IDL Program to Calculate DSC Parameter Mapsavg1 = mean(a[x,y,z,1:5])avg2 = mean(a[x,y,z,15:25])avg3 = mean(a[x,y,z,35:*]);/// This portion of the program allows you to have an image with 4 sections;/// Section 1 and 2 are for z=1 and z=2 for avg2/avg1;/// Section 3 and 4 are the same as above except avg2/avg3;/// 1 - Avg2/Avg1 = 1 - Middle / Beginning -----> Large Dip = More white (closer to 1);/// 1 - Avg3/Avg1 = 1 - End / Beginning / -----> Large Recovery = More white (closer to 1);/// map[x,y,z]=((1-avg2/avg1) > 0)<1;/// map[x,y,z+2]=((1-avg3/avg1) > 0)<1;/// Here we try a map test to see if the Feridex is leaking out;/// 1 - (Middle / End) -------> No difference between middle and end = more white (closer to 1)map2[x,y,z] = ( (1-avg2/avg3) >0)<1;/// print, map[x,y,z]print, map2[x,y,z]endfor endfor endfor;/// create colour barscale=rebin(transpose(findgen(32)/32),5,32)help,scalemap2[5:9,16:47,0]=scale;/// set window sizewindow,0,xs=512,ys=512;plot 4 images, two from each zfor z=0,1 do begintvscl, rebin(map2[*,*,z],256,256),zendforfor z=2,3 do begintvscl, rebin(map2[*,*,z],256,256),zend45A.3. Sample Staining WorksheetA.3SampleStainingWorksheetDate:12-Apr-09Purpose: CD31 & Coll IV stainingFor use with capillary gap staining system5pairs of slidesRoom temperature with shaker (400 rpm, 1.5 cm)160µl/paircut 10 µm cryosections+Primariesdry overnight+778.4µl PBS+0.1% tween1:2004µl rat anti CD31 (1:10)50%MeOH/50%acetone101:5001.6µl rabbit anti Coll IVRinse in PBS 3x10 sec+1:5016µl Goat SerumWash (PBS with 0.1% Tween 20) with stirring20SecondariesPut in capillary system778.4µl PBS+0.1% tween1:2004µl anti rat Alexa 647primaries 60min1:5001.6µl anti rabbit Alexa 546Rinse (PBS with 0.1% Tween 20) 3x10 sec+1:5016µl Goat Serumsecondaries 60minRinse (PBS with 0.1% Tween 20) 3x10 sec+Image+Notes:46A.4. Feridex Staining ReportA.4 Feridex Staining ReportSURGICAL PATHOLOGY - HISTOLOGY Date:STAINING MANUAL - MINERALS AND PIGMENTS Page: 1 of 2IRON -  PRUSS I AN BLUE RE A C T ION -  MA LLORY'S METHODPURPOSE: To demonstrate ferric iron in tissue sections. Small amountsof iron are found normally in spleen and bone marrow. Excessive amountsare present in hemochromatosis, with deposits found in the liver andpancreas, hemosiderosis, with deposits in the liver, spleen, and lymphnodes. PRINC I PLE: The reaction occurs with the treatment of sections in acidsolutions of ferrocyanides. Any ferric ion (+3) in the tissue combines withthe ferrocyanide and results in the formation of a bright blue pigmentcalled 'Prussian blue" or ferric ferrocyanide.CONTROL: A known positive control tissue.F IXA T I V E: 10% formalinTECHNIQUE: Cut paraffin sections 4µ.EQUI PMENT: Microwave oven, acid-cleaned glassware, non-metalicforceps.RE AGENT S:5% Po t a s s i um F e r r oc y an i de:Potassium ferrocyanide 25.0 gmDistilled water 500.0 mlMix well, pour into an acid-cleanedbrown bottle. Stable for 6 months.CAUT ION: Lo w  t ox i c i t y i f  no t  hea t ed.. Nuc l e a r - f a s t  Red:See Retic5% Hyd r och l o r i c A c i d:Hydrochloric acid, conc. 25.0 mlDistilled water 475.0 mlMix well, pour into brown bottle,stable for 6 months.C AUT ION: Co r r os i v e, a vo i d con t a c t  andi nh a l a t i on.Wo r k i ng So l u t i on:5% potassium ferrocyanide 25.0 ml5% hydrochloric acid 25.0 mlMake fresh, discard after use.CAUT ION: A vo i d con t ac t  and i nha l a t i on.48A.4. Feridex Staining ReportMINERALS AND PIGMENTSIRON Page: 2 of 2SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation. Potassium ferrocyanide; Low toxicity as long as it is not heated, it willrelease cyanide gas.Hydrochloric acid; target organ effects on reproductive system and fetaltissue. Irritant to skin eyes and respiratory sytem.PROCEDURE:1. Deparaffinize and hydrate to distilled water.2. *Working solution, * microwave, 30 seconds. Allow slides to stand insolution for 5 minutes, in the fume hood.3. Rinse in distilled water.4. Nuclear-fast red, 5 minutes.5. Wash in tap water.6. Dehydrate, clear, and coverslip.*Conventional method: room temperature for 30 minutes.RESULTS:Iron (hemosiderin) blueNuclei redBackground pinkREFERENCES:Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd Ed,1980, pp217-218, Battelle Press, OhioLuna L, Manual of Histologic Staining Methods of the AFIP, 3rd Ed, 1968, pp183, McGraw-Hill, NYCrookham,J, Dapson,R, Hazardous Chemicals in the HistopathologyLaboratory, 2nd ED, 1991, AnatechPrepared:                                        By:                           Approved:                                       By:                           Downloaded from WebPath: Internet Pathology Laboratoryhttp://www-medlib.med.utah.edu/WebPath/webpath.html49Physics 449 Report: Feridex Mapping April 14, 20091 Mapping Feridex histologicallyFeridex (generically ferrumoxide), is a sterile aqueous colloid of superparamagnetic iron oxide (SPIO) asso-ciated with dextran. Administered intravenously, it is an MR contrast agent usually used for the detectionof liver lesions. Feridex is taken up by macrophages, found only in healthy liver cells but not in most tu-mors. We want to use Feridex in the DSC weighted portion of our protocol, alongside Gd-DTPA as theDCE portion. The problem with using Feridex so far (instead of Galbumin) is tracking and/or mapping themovement of Feridex as it extravasates out of tumour vessels.1.1 Magneto-capsules - Dextran-specific FITC-conjugated antibody• LIM-Sun Method, described below in detail from the Barnett paper.Magnetoencapsulation. Magnetocapsule synthesis is based on a one-step modication (that is,Feridex addition) of the Lim-Sun method29. Our modication uses an electrostatic (van de Graaff)droplet generator, which produces smaller, stronger and more uniform capsules as compared with250Physics 449 Report: Feridex Mapping April 14, 2009Table 1: Description of FeridexDescription Valuer1,r2,Bo 40.0,160,0.47TConcentration 11.2 mg Fe/mLAvg. Formula FeO1.44Active Ing. 11.2 mg of FeInactive Ing. 1 61.3 mg of mannitolInactive Ing. 2 5.6-9.1 mg/mL dextranInactive Ing. 3 0.25-0.53 mg/mL citrateHydrodynamic Diameter 120-180 nm *(80-150 nm also cited)Size of Crystal Core 5.55 nmCoating Dextran T10 kDathe older air-jet technique. Before encapsulation, human cadaveric islets were passed througha 20-g needle. We suspended cells, adjusted to 400 islet equivalents per ml or 1.5 E7cells/ml(bTC-6), in 2% (wt/vol) ultrapuried sodium Protanal-HF alginate (FMC Biopolymers) and 20%(vol/vol) Feridex (Berlex Laboratories). We passed this solution through a needle at 200 ml/minusing a nanoinjector pump. We collected droplets, representing islet cells surrounded by the rstlayer of alginate, in a Petri dish containing 100 mM CaCl2 in 10 mM HEPES, and washed themthree times. We suspended gelled droplets in 0.05% poly-L-lysine (2224 kDa; Sigma) for 5 minto crosslink alginate and Feridex. We washed and resuspended droplets in 0.15% Keltone HVCRalginate (Monsanto) for 5 min, and then washed them again. For capsule rupture, we manuallyagitated magnetocapsules in a 50-ml conical tube lled with 1-mm glass beads.1.2 Alexa-647 conjugated Feridex• Refer to as the paper for the full methods of creating this• Preparing Alexa Fluor 647 conjugated Feridex• Feridex added to a solution containing KOH, (ddH2O), epichlorohydrin• Reacted for 12 hours with constant shaking• Concentrated ammonia added to the Feridex and reacted overnight at 37C• Feridex was reacted with 1 mg of Alexa Fluor 647 succinimidyl ester overnight at roomtemperature• Centrifuged and supernatant removed• Assuming an average particle diameter of 80 nm, we determined that there were approxi-mately 140 molecules per iron particleMore descriptive protocolPreparation of Fe[647] and Fe[750] Nanoparticles Synthesis of Alexa Fluor 647- andAlexa Fluor 750-conjugated Feridex (Fe[647] and Fe[750], respectively) was based on351Physics 449 Report: Feridex Mapping April 14, 2009previ- ously published methods [15]. Briefly, 1 ml of Feridex (11.2 mg/ml) was addedto a solution containing 1.6 ml of KOH, 0.7 ml of double-distilled H2O (ddH2O), and0.7 ml of epichlorohydrin [19]. The mixture was reacted for 12 hours with constantshaking. To produce reactive amines on the dextran coat, concentrated ammonia (0.5ml) was added to the Feridex and reacted overnight at 37C. Excess epichlorohydrinand ammonia were removed by extensive dialysis against ddH2O using 12,000 14,000molecular weight cut off tubing. Feridex was reacted with 1 mg of Alexa Fluor 647 or750 succinimidyl ester (Molecular Probes, Eugene, OR, http://probes. invitrogen.com)overnight at room temperature. Excess fluorophore was removed by centrifuging thesample at 160,000g for 30 min- utes. The supernatant was discarded, and the pellet wasresuspended in phosphate-buffered saline (PBS) buffer (pH 7.4). The centrifu- gationstep was repeated four times to ensure that unconjugated fluorophore was removedfrom the sample. Removal of excess fluoro- phore was confirmed using a fluorometer.To disperse and remove large nanoparticle aggregates, the sample was sonicated for 5minutes and filtered using a 0.2 M size-exclusion filter. The iron concentration wasmeasured using the method de- scribed by Stookey [20]. The number of fluorescentmolecules was calculated by using a standard curve of known Alexa Fluor 750 concen-tration. From these measurements, assuming an average par- ticle diameter of 80 nm,we determined that there were approximately 140 molecules per iron particle.1.3 Prussian Blue - Fe staining• To demonstrate ferric iron in tissue sections• The reaction occurs with the treatment of sections in acid solutions of ferrocyanides• Tissue stained and positive control• Paraffin sectioning 5µm thick• Ferric iron is blue and nuclei are red• See attached protocol for Prussian Blue stain.1.4 Radio-labeled Feridex• Wasn’t able to find situations of radiolabelled Feridex• I have a couple of papers that describe the structure of Feridex.• Could also use this to directly attach FITC tags to Feridex• I didn’t really understand them, but it might to someone that knows a bit morechemistry• Still have to read AK’s paper for the radio labelling protocol.452Glossaryblood brain barrier The blood brain barrier is a special feature (many tight junctions) of braintissue as it restricts the passage of molecules of a certain size between the blood stream andbrain tissue.. 4, 15carbocyanine Carbocyanine (DiOC7(3)) is a small molecular dye commonly used in histology tomeasure perfusion. Because of its small size, carbocyanine stains cells immediately adjacentto blood vessels outlines tumour vasculature. The dye commercially available (Invitrogen,SKU# D-378) fluoresces at 488nm and can be imaged using the digital microscope at theBC Cancer Center.. 5, 6CD31 An endothelial cell marker that when stained for, marks blood vessels.. 5, 20, 23Contrast Agent Radioactive, fluorescent or magnetic chemical compounds that aid in the vis-ibility of internal bodily structures in imaging. In this study, we will be using Galbumin(Gadolinium atoms attached to the protein Albumin) as the contrast agent.. 3dextran Complex sugar. In context, dextran is used to increase the effective size of the targetmolecule (Gd-DTPA in this case. 35, 37Diffusion This is the traditional, classical method of molecules moving according to brownianmotion.. 4, 5EES Extravascular extracellular space - referred to the space that isn’t occupied by vasculature(vessels) or cells. 16endothelium A layer of cells that line the interior of blood vessels.. 4extravasate To leak out of... In context, extravasation is a term used to denote the leakage offluid from a container.. 17, 36extravasation In general, this term refers to the leakage of a fluid out of its container. In thecontext of this study, it’s the leakage of molecules (contrast agents, drugs, markers etc.)out of the blood vessel and into the extra cellular matrix.. 4, 5, 37hoechst A small fluorescent biomarker that is commonly used to mark perfusion (from bloodvessels). 5, 2353Glossaryhydrostatic Hydrostatic - fluids at rest. In context, hydrostatic pressure is the force per unitarea delivered from water that is static (in equilibrium and not moving). 5immunohistochemical Immunohistochemistry is the process of localizing biological molecules(often proteins) at the cellular level using histology (study of thin sections, usually frozen)immunology (antibodies binding to antigens) and chemical principles.. 3, 6immunohistochemistry Immunohistochemistry is the process of localizing biological molecules(often proteins) at the cellular level using histology (study of thin sections, usually frozen)immunology (antibodies binding to antigens) and chemical principles.. 3, 4in-vivo Tumours will be xenografted in NODSCID mice of the cell type HCT116 and HT29. 8interstitial Area that surrounds cells and vessels filled with fluid.. 5, 16, 34intravascular Stays inside blood vessels. In contrast, large molecular weight contrast agentsstay confined to within blood vessels and do not extravasate.. 37metastasis Metastasis is referred to as the spread of disease (usually cancer) from one part ofthe body to another non-bordering part. One method of metastasis is cancer cells movingthrough the bloodstream from one part of the body to another.. 4Mouse Type NODSCID - Non-obese diabetic, severe combined immunodeficiency mice. 5necrosis Group (or area) of cells that is dying or dead. Some tumours show patches of necrosisin areas distant from blood vessels (nutrients) and these areas are essentially empty space..29, 34tight junctions Tight junctions are the closely associated areas of two cells whose membranesform an impermeable barrier to fluid. In context, generally the number of tight junctionsin tissue is related to the permeability of the tissue.. 4vascular networks A vascular network is the arrangement of blood vessels of a particular organor tissue.. 4vasculature The network of blood vessels of an organ or body part and includes distribution ofall vessels, including arteries, capillaries and veins.. 3, 5, 2054

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