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1 IMPLICATIONS OF ULTRAHIGH FIELD STRENGTH MRI FOR NON -INVASIVE IN VIVO CELL TRACKING By NICLAS EMANUEL BENGTSSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE R EQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009
2 2009 Niclas Emanuel Bengtsson
3 To my wonderful wife and family who have always supported and inspired me
4 ACKNOWLEDGMENT S Several people have supported me throughout my goal of receiving my doctorate degree. First, I would like to thank my mentor Dr Edward Scott for his friendship, support and guidance in matters relating to both science and the occasional trials of everyda y life Due to his mentorship, I have gai ned invaluable knowledge of how to be an effective scientist Secondly, I w ould like to thank Dr Glenn Walt er for mentoring me in the art of magnetic resonance imaging and my other committee members; Dr Bryon Peters en, Dr Maria Grant and Dr Jorg Bungert, for contributing with valuable advice during our discussions In addition, I thank Gary Brown for contributing with his expertise regarding the laboratory mouse. Many thanks a re also extended to the current and past members of the Scottlab, as well as people closely associated to the Scottlab, for their help and support. I would like to individually recognize, Dr. Koji Hosaka, SeungBum Kim Dustin Hart and Li Li n for providing me a helping hand when it was needed Fu rthermore, I would like to thank the staff of the Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) facility for their support and expertise. I would also like to take the opportunity to acknowledge the people that have been a source of great as sistance and support during the extent of my graduate career. I would like to thank Dr Wayne McCormack, Susan Gardener and Valerie Cloud Driver. Additionally, I would like to thank Joyce Conners for her ever present support, advice and patience. Her friend ship is one I will always value. Many thanks also go out to the staff at the University of Florida International Center for facilitating my life as a student in the United States of America. Finally, I would like to thank the most import ant people in my li fe Without the unconditional love and support from my wife Trina, my parents Gunder and Birgitta and my brother Joacim, nothing I have accomplished to date would have been possible.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 7 LIST OF FIGURES .............................................................................................................................. 8 ABSTRACT ........................................................................................................................................ 10 CHAPTER 1 BACKGROUND AND SIGNIFICANCE ................................................................................. 12 Stem Cells and Regenerative Medicine ..................................................................................... 12 Adult Stem Cells of the Bone Marrow (BM) ............................................................................ 13 The Hematopoietic Stem Cell (HSC) ................................................................................. 14 BM and HSC Niches ........................................................................................................... 15 The endosteal niche ...................................................................................................... 15 The vascular niche ........................................................................................................ 17 HSC Mobilization and Homing .......................................................................................... 19 Role of BM in Tumor Blood Vessel Formation ........................................................................ 20 Cellular In Vivo Imaging ............................................................................................................ 23 Fluorescent and Bioluminescent Imaging .......................................................................... 24 Computed Tomography/Positron Emission Tomography/Single Photon Emission Computed Tomography ................................................................................................... 26 Magnetic Resonance Imaging (MRI) ................................................................................. 27 Cellular MRI ........................................................................................................................ 28 2 MATERIALS AND METHODS ............................................................................................... 38 Animals ........................................................................................................................................ 38 Materials and Methods used in Chapter 3 ................................................................................. 39 Irradiation Induced I njury ................................................................................................... 39 BM Labeling with Feridex .................................................................................................. 39 Tibia Window Installment ................................................................................................... 39 Feridex Labeled BM Cell Tra nsplantation ......................................................................... 40 In Vivo Fluorescent Microscopy of DsRed BM Cells ...................................................... 40 MRI of BM Following Irradiation and Transplantation .................................................... 40 Histological Analysis of Feridex Labeled DsRed BM Cells ............................................ 41 Materials and Methods used in Chapter 4 ................................................................................. 42 S Gal Substrate Specificity Studies .................................................................................... 42 S Gal Staining of BM Cells ................................................................................................ 42 Intra C ellular Iron Accumu lation Determination .............................................................. 43 Cell Viability Assay ............................................................................................................. 43 Magnetic Resonance (MR) Measurements on S Gal Substrate and Cell Phantoms ...... 44
6 In Situ MRI of S Gal Labeled BM Cells ............................................................................ 45 In Vivo S Gal Labeling of Rosa26 Tissues ........................................................................ 45 Histological Analysis of S Gal Labeled Cells and Tissues ............................................... 46 Materials and Methods used in Chapter 5 ................................................................................. 47 Irradi ation Damage and BM Cell Transplantation ............................................................ 47 Galactosidase Expression in Peripheral Blood ............. 47 Tumor C ell Implantation ..................................................................................................... 47 In Viv o Fluorescent Imaging of Intra T umoral BM Derived Cells .................................. 47 In Vivo MRI of Tumor Recruited Myeloid Cell s. ............................................................. 48 Histological Analysis of Tumor Recruited BM Derived Myeloid Cells .......................... 49 3 IN VIVO VISUALIZATION OF BM CELL HOMING USING ULTRA HIGH FIELD MRI FOLLOWING IRRADIA TION AND CELL TRANSPLANTATION ......................... 50 Introduction ................................................................................................................................. 50 Irradia tion Induced Changes in BM Signal Inten sity at Ultra High Magnetic Field Strength .................................................................................................................................... 51 BM Labeling and In Vivo Fluorescence Tracking .................................................................... 52 In Vivo Tracking by MRI ........................................................................................................... 53 Histological Confirmation of In Vivo Findings ........................................................................ 53 4 LACZ AS AN IN VIVO GENETIC REPORTER FOR REAL TIME MRI .......................... 60 Introduction ................................................................................................................................. 60 S Gal Forms a MR Visible Precipitate with Ferric Ammonium Citrate ( FAC ) in the Gal .................................................................................................................... 60 Transverse Relaxation Rates ( R2) and Effective Transverse Relaxation Rates ( R2 *) i s Increase d in LacZ Expressing BM Cells Following S Gal/FAC Labeling .......................... 61 In Vivo Detection of LacZ Expressing BM Cells Following Intramuscular Transplantation ........................................................................................................................ 63 In Vivo Labeling of LacZ Expressing Tissues .......................................................................... 64 5 IN VIVO MRI OF BM DERIVED MYELOID CELL RECRUITMENT TO GROWING TUMORS USING A MR ACTIVE GENETIC REPORTER SYSTEM ........... 72 Introduction ................................................................................................................................. 72 In Vivo Fluorescen t Imaging Can Detect BM Derived Cell Recruitment to Growing Tumors ..................................................................................................................................... 73 Ultra High Field MRI Combined with a MR Active Genetic Repo rter Substrate Can Detect Intra Tu moral Recruitment of BM Derived Cells ..................................................... 74 Post Imaging Histol ogy of Intra Tumoral BM Cell Recruitment ............................................ 75 6 DISCUSSION .............................................................................................................................. 85 LIST OF REFERENCES ................................................................................................................... 89 BIOGRAPHICAL SKETCH ........................................................................................................... 106
7 L IST OF TABLES Table page 4 1 Comparison of cellular contrast enhancing effect of various iron based contrast agents ...................................................................................................................................... 71
8 LIST OF FIGURES Figure page 1 1 Schematic of anatomical locations of the bone and bone marrow (BM) and cell lineages produced by hematopoietic stem cells (HSCs) ...................................................... 32 1 2 HSC isolation characteristics ................................................................................................. 33 1 3 Illustr ation of proposed BM HSC niches .............................................................................. 34 1 4 Illustration of HSC mob ilization and homing fro m endosteal and vascular niches ........... 35 1 5 BM derived cell types implicated in tumor gr owth and blood vessel formation ............... 36 1 6 BM derived cells modulate tumor angiogenesis and growth .............................................. 37 3 1 Magnetic resonance imaging (MRI) clearly depicts changes in contrast between day 0 a nd day 14 following irradia tion ........................................................................................ 55 3 2 Fluorescent microscopy detection of initial BM cell h oming following transplantation ........................................................................................................................ 56 3 3 S uperparamagnetic iron oxide (S PIO ) labeled BM c ell homing visualized using MRI .... 57 3 4 Orthogonal validat ion of Feridex positive spheres .............................................................. 58 3 5 Histolog ical identification of iron la beled dsRed expressing BM cells .............................. 59 4 1 S Gal forms magnetic resonance ( MR ) active precipitates wi th ferric ammonium -galactosida se ( -gal) ..................................................... 66 4 2 S -gal expressing cells and results in increased intracellular iron accumulation .............................................................................................. 67 4 3 -gal expressing cells with S -Gal/FAC leads to increased effective transverse relaxation rates (R2*) that are enhanced with s tronger external magnetic field ......................................................................................................................................... 68 4 4 The in vivo detection capability of S Gal labeled Rosa26 BM cells i s increased with field strength ........................................................................................................................... 69 4 5 In vivo labeling of Rosa26 tibialis anterior (TA) muscles by intra -muscular delivery of S Ga l/FAC .......................................................................................................................... 70 5 1 BM engraftment check of peripheral blood in transplanted animals by fluor escein D -galactopyranoside) ( FDG ) and flow activat ed cell sorting (FACS) analysis ........ 77
9 5 2 Fluorescent imaging of donor derived LacZ expressing BM cell recruitment to the growing tumor ........................................................................................................................ 78 5 3 In vivo 17.6T three -dimensional ( 3D ) MRI of tumor infi ltrating donor derived BM cells ......................................................................................................................................... 79 5 4 Recruitment of BM derived cells detected at 11.1T magnetic field strength ..................... 80 5 5 Conditional Tie2 LacZ expression originating from tumor infiltrating donor derived cells, as visualized by in vivo 17.6T MRI ............................................................................ 81 5 6 High r esolution MRI of excised tumors ............................................................................... 82 5 7 Tumor histology confirms heavy infil tration of BM derived monocytes ........................... 83 5 8 Presence of BM derived Tie2 exp ressing monocytes within tumors ................................ 84
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Par tial Fulfillment of the Requirements for the Degree of Doctor of Philosophy IMPLICATIONS OF ULTRAHIGH FIELD STRENGTH MRI FOR NON -INVASIVE IN VIVO CELL TRACKING By Niclas Emanuel Bengtsson December 2009 Chair: Edward William Scott Major: Medical Sciences Molecular Cell Biology In vivo imaging has the potential to solve many unanswered qu estions regarding the spatial location s functions and fate s of transplanted cells in th eir normal physiological milieu Ultra high field M agnetic Resonance Imaging (MRI) comprise s one of the most exciting tools for the specific, non -invasive visualization of transplanted cells in vivo. Here we show that ultra high field MRI can achieve high resolut ion 3D images, of the bone marrow ( BM ) in live mice, sufficient to res olve anatomical changes in BM microstructures at tributed to lethal irradiation. Following transplantation with dsRed expressing BM cells labeled with superparamagnetic iron oxides, both fluorescent microscopy and MRI could be used to follow initial homing of cells within long bones in vivo. To date, MRI has been hampered by the lack of genetic reporters for molecular in vivo imaging. Therefore we propose d the novel use of a magnetic resonance ( MR ) active gene reporter system for the longitudinal tracking of cellular LacZ expression in vivo by MRI We determined that the commercially available substrate, S Gal, can be used to detect LacZ expressing cells by MRI. The effect and speci ficity of the reaction between LacZ and S Gal on MRI contrast was determined both in vitro and in vivo and was found to significantly increase effective transverse relaxation rates with increasing external magnetic field strengths
11 (4.7 17.6T) in phantom studies. Using both LacZ transgenic animals and LacZ tissue transplants, we we re able to detect labeled cells in live animals in real time. Similar to phantom studies, detection of the labeled cells/tissues in vivo was enhanced at higher magnetic fields. To test the applicability of S Gal for longitudinal cell transplantation studie s, systemic delivery of S Gal w as employed to track LacZ expression in a model of BM derived cell contribution to tumor angiogenesis. We found that ubiquitously or Tie 2 conditionally LacZ expressing BM derived cells could be detected within growing tumor s in vivo, using ultra -high field MRI and fluorescent imaging (FLI). T hrough histology, LacZ expressing myeloid cells were predominately found in avascular hypoxic areas while Tie2 expressing cells were mainly found in less obvious areas of hypoxia, base d on morphology and TUNEL staining, close to MECA32 expressing endothelium.
12 CHAPTER 1 BACKGROUND AND SIGNI FICANCE Stem Cells and Regenerative Medicine Regenerative medicine has become one of the most intriguing fields of research today. Many degenera tiv e diseases that suffer from lack of or ineffective treatments today, could potentially be successfully treated by stem cell therapy in the future. Stem cells are defined by their ability to self renew and give rise to progenitor cells that can differentia te into multiple types of specialized cells. The ability to self renew is crucial to prohibit a proliferative burnout of the stem cell population, which would ultimately lead to a loss of regenerative ability and ultimately death. The process of self renew al is believed to occur by two means. First, stem cells are believed t o be able to undergo asymmetrical cell division (1). This means that upon division, stem cells produce one exact copy of itself as well as one differentiated progenit or cell that can further divide and differentiate to give ris e to a multitude of terminally differentiated cells (1) Another possibility of self renewal is that following a symmetrical division, one of the identical daughter cells rema ins in the original microenvironment that promotes quiescence and maintenance of the stem cell phenotype while the other daughter cell migra tes to a microenvironment that is conducive to differentiation (1). The ability of a stem cell to give rise to multiple differentiated daughter cells is referred to as potency. Certain stem cells have the ability to differentiate into a larger variety of specialized progeny cells and therefore the term potency comes in the various degre es ; Totipotent pluripotent multi potent oligopotent and unipotent, with the latter two normally being reserved for progenitor cells an d not generally considered for true stem cells. Totipotent or omnipotent stem cells, by definition, have the ability to differentiate into every cell type of an orgainsm A fertilized egg, or zygote, is considered to be totipotent. Examples of pluri potent stem cells are
13 embryonic stem cells ( ESC ), which are derived from the inner cell mass of the blastocyst during embryonic develo pment. These cells have virtually unlimited ca pacity to differentiate into all cell type s of the three primary germ layers: ectoderm, endoderm and mesoderm. Adult stem cells such as; H ematopoietic stem cells ( HSC ) and mesenchymal stem cells ( MSC ), are generally considered to be multipotent. These cell types found in most adult tissues, have the ability to self renew but are limited in their differentiation capacity and can only give rise to more differentiated cells of the same cell lineage. Another important te rm in regenerative stem cell medicine is plasticity. The term plasticity or transdifferentiation, refer s to the ability of a n adult stem cell to under certain conditions, differentiate into a cell phenotype of a different lineage or even germ layer (2) The term plasticity has recently taken on a new meaning with the arrival of methods for indu cing pluripotent cells from more differentiated cell types (3). These cells have been named induced pluripotent stem cells (IPS). The ability to gene rate pluripotent stem cells by reprogramming differentiated cell types though still needing further investigation, brings immense potential to the field of regenerative stem cell biology. Stem cells are rare in normal tissues and apart for being expensive and time consuming to isolate some are also notoriously hard to expand in vitro (1). Therefore a method of reverting abundant differen tiated cells back to a stem cell phenotype could provide an almost inexhau stible source of cells for regenerative cell therapies. Adult Stem Cells of the Bone Marrow (BM) Bone marrow (BM) hosts at least two known types of adult stem cells. One of these is the MSC which upon differentiation has been shown, mainly in vitro to be able to generate various differentiated cell types, including: osteoblasts, adipocytes chondro cytes myocytes fibroblasts and endothelial cells (4). While it has been shown that MSCs can be reliably expanded and differentiated in vitro, there are still questions regarding how to sort these cells based on cell
14 sur face markers and few in vivo experiments have been successful in providing functional readout s to support its stem cell phenotype The most prominent adu lt stem cell in the BM is the H S C, which will be discussed in more detail here. The Hematopoietic Stem Cell (HSC) The HSC is derived from the mesoderm germ layer and is found in blood islands of the extra embryonic yolk sac, the aorta -gonod -mesonephros (AGM) region and placenta during mouse embryo development (5 7) Upo n further development the HSC migrates to the fetal liver before seeding to the spleen and ultimately the BM (8 10) The hallmark features of the HSC is its ability to self renew and to maintain all the different lineages of the blood (Figure 1 1) for the entire lifespan of the organism (11) T he hematopoietic stem cell (HSC ) was first discovered during the 1960s w hen Till et al. found that lethally irradiated mice could be rescued if given a BM transplant from a healthy donor (12) This provided researchers with functional evidence of the existence and importance of the HSC. Further support for the immense capabilities of the HSC was obtained through experi ments showing that single HSCs could upon transplantation reconstitute the hematopoietic system of irradiated hosts (13) More stringent evidence of the functionality and self renewal capability of the HSC were provided when it was shown that upon engraftment of these single HSCs, they could be serially transplanted into additional irra diated animals to restore hematopoiesis (14) To date, the HSC has been studi ed extensi vely and many different methods of isolating it exist based on certain characteristics and the expression of various surface markers (Figure 1 2 ). One of the most common ly accepted methods of HSC enrichment involves sorting lineage depleted (Lin-) BM cells for the expression of the cell surface mark ers stem cell antigen 1 (Sca 1) and the stem cell factor receptor (c kit) (SKL cells ) (15) F urther enrichment can be obtained by the DNA -binding Hoechst dye efflux ability of HSCs, which is referred to as the side population (16,17) and by employing a set of SLAM
15 family surface markers (18) The natural oc currence of HSC s within the BM is extremel y low with only about 1 in 10000 cells being a SKL -cell. Additionally, o nly a fraction of the SKL subset of HSCs are believed to be actual long -term repopulating HSC s (LT -HSC) that have the ability to maintain hema topoiesis for the rest of the animals life (1,18 20) The remaining cells are believed to be short term repopulating HSCs (ST -HSC) or multi potent progenitor cells which have limited self renewal activity and plasticity (1,19,21,22) The great versatility of the HSC have also been demonstrated by its ability to t ransdifferentiate into various cell typ es such as epithelium, myocardium, liver, and re -vascularize areas of ischemic injury (23 26) BM and HSC N iches The maintenance of HSC self -renewal and differentiation in vivo i s thought to depend on the specific micro -environment they reside in. In the late 19 70s, the concept of a specialized microenvironment or niche responsible for governing and directing HSC fates was introduced (27) Since then various such niches have been proposed and summarized (1,28 30) Th e first, and most stu died, niche to gain attention is the endosteal or osteoblastic niche, where stem cells are thought to reside in close proximity of cells that line the inside of bone within marrow cavities (30,31) A second vascular niche, where stem and progenitor cells have been identified to reside close to sinusoidal endothelium, has also garnered much attention lately (18) Next the evidence of these two proposed niche s will be presented and summarized. The endosteal n iche In the mid 1970s, the HSC was first shown to be in close contact to the bone surface wit hin the BM (32) Shortly ther e after a majority of HSC s identified as being CD45+ Lin-, were discovered to be in close contact with endosteal linings of BM cavities in the t rabecular regions of long bones (33) Meanwhile, the more differentiated hematopoietic progenitor cell s (HPCs) we re suggested to be located towards the centre of the BM (33) Th is interface between marrow
16 and bone was later termed the endosteal niche and place s the HSC in close contact w ith spindle shaped, N Cadherin expressing osteoblasts (SNO cells ) (34,35) The retention and maintenance of HSC quiescence within the endosteal niche is thought to be facilitated through a variety of mechanisms. One of these is adhesion through homotypic N -cadherin expressed by HSC s and osteoblasts, catenin/Wnt signalling (34 36) Additionally, SNOs express membrane bound stem cell factor (mSCF KitL, steel factor ), which has been shown to be crucial in both niche retention and in maintaining HSC quiescence (37,38) Its complementary binding partner c kit, expressed on HSCs is activated upon interaction (39,40) This causes HSCs to form strong adhesions with extracellular matrix and surrounding stromal cells mainly mediated by the upregulation of integrins, which ultimately plays a role in regulating ni che activity (37,38) Osteoblasts also express osteopontin (Opn), which upon binding integrins expressed by HSCs acts as an additional niche retention signal while also suppressing proliferation (41) The studies identifying these interactions also demonstrate d an intimate relationship bet ween the number of osteoblasts and functional LT -HSC s. When osteoblasts numbers are reduced the number of HSC s within the BM also decreases, resulting in extra -medullary hematopo i esis occuring in liv er, spleen and peripheral blood (42) Multiple additional signalling interactions have been shown to maintain H SC self -renewal and primitive phenotype in the endosteal niche. Notch is expressed by primitive HSCs while its receptor Jagged 1 is expressed on osteoblasts. Upon activation of this signalling cascade, HSC numbers have been shown to expand while still reta ining a primitive state (43,44) Interactions between Angi opoietin1 (Ang1) expressed on osteoblasts and the receptor Tie2 expressed by HSCs leads to adherence and induced HSC quiescence (45) Additionally, calcium sensing receptors (CaR)
17 have also been implicated in the retention of HSCs in the endosteal niche (46). In summary the endosteal niche appears to be p aramount for HSC functionality. The vascular n iche BM is made up of t wo main cellular compartments, the hematopoie tic and stroma l compartments The stromal compartment consists of nerve cells, fibroblasts, adipocytes and BM vasculature (47) The primary function of BM vasculature is to provide a barrier between the hematopoietic c ompartment and the circulatory s ystem It consists of arterial vessels entering the mar row space through the bone that later branch out in arterioles. Arterioles and capillar ies are spread throughout the marrow and supply BM sinusoids (47,48) BM sinusoids are highly permeable vessels made up by a single fenestrated endothelial layer without structurally supporting cells (49) In fact, the collapse of these vessels seem to be avoided by the pressure generated from large amounts of hematopoietic cells within the BM (48) Sinusoids finally drain into the central sinus, which is the largest vascular structure found in the BM, measuring approximately 0.1 mm in diameter (48) The i dea of a vascular HSC niche is by comparison relatively new. Initial support for the existence of this niche is t he fact that hematopoiesis and vascularization occur together during embryogenesis (50) Furthermore, both HSCs and endothelial cells have been suggested to stem from a common p recursor cell, the hemangioblast (50) The vascular niche has been propose d to contain more committed stem and progenitor cells than the relatively quiescent stem cells in the end osteal niche (31,51). However, recently a study identifying LT HSCs as CD150+CD48-CD41-Linshowed that they mainly are located more towards the centre of the bone marrow in close proximity to BM sinusoidal endothelial cells (BMECs) (18) This perivascular location would render HSCs ideally suited to monitor molecular signals carried through the blood in order to mount a r apid response to trauma. Yolk sac, AGM derived cell lines and pri mary endothelial
18 cells have been shown to promote HSC maintenance and clonal expansion in vitro (52,53) However, vascular endothelial cells from non-hematopoiet ic tissues have been proven incapable of maintaining HSCs in culture (54) This may suggest that BMEC s are functionally and phenotypically different possessing special properties for HSC maintenance. Some of these properties involve the constitutive expression of the chemo k ine SDF 1 (or CXCL12) and the adhesio n molecules E -selectin and VCAM. These signalling molecules are all involved in HSC homing, engraftment and mobilization and will be discussed in more detail later (55 57) Hematopoietic recovery following myelosuppression by means of cytotoxic drugs, such as 5 Fluorouracil (5 FU) or irradiation is dependent on BM vascular s tructures. These treatments do not only affect HSC/HPCs, but also BMECs through the collapse of BM vascular structures (47,48) The BM vascular system has to be regenerated before hematopoiesis can be restored (47,48) Findings of Tie 2 upregulation on BMECs following myelosuppression supports this notion, since blocking of Tie 2 lead to delayed hematopoietic recovery (58) An interpretation of the existence of the two distinct niches co uld be that the HSC/HPCs residing close to the sinusoids continuously monitor the hematopoietic system by sensing hematopoietic factor s transported in the bl ood. When the hematopoietic system comes under stress, they could potentially mount a rapid res ponse by inducing division and differentiation and restore homeostasis. If the perivascular HSC/HPCs are insufficient to meet the demands of the injury, they cou ld signal HSCs lodged in the endosteal niche to exit quiescence and start producing additional cells until homeostasis is restored. The outline and interactions between these two niches are illustrated in ( Figure 1 3 and Figure 1 4). Taken together, the e ndosteal and vasc ular nich es seem to cooperate extensively, and both seem to be indispensable in control ling HSC quiesc ence and self renewing activity.
19 HSC Mobilization and Homing Mobilization and homing are two processes closely r elated. Mobilization invo lves the exodus of HSC/HPCs from the BM into circulation and homing is the opposite of this event. HSC mobilization and homing can be accomplis hed by several means. These include the use of mobilizing chemokines cytotoxic drugs or whole body irradiation (59) HSC mobilization from the endosteal niche to the vascular niche and ultimately into circulation normally occur s when stress induces changes of mainly SDF 1 levels in th e BM (60) One commonly used method in the clinic, is the administration of granulocyte colony stimulating factor (GCSF). The mechanism of stress or GCSF induced mobilization is not fully known but is in part acco mplished by the release of certain protea ses from neutrophils such as MMP 2, MMP9 cathepsin -G and elastase (61 63) These proteases cleave niche retention signals like mSCF SDF 1 and VCAM 1 (61 63) W hen cleaved, m SCF turns into its soluble form, soluble SCF ( sKitL) which have been shown to induce HSC/HPC proliferation, differentiation and mobilization (42,63) SDF 1 is a powerful chemoattractant that is responsible for the migration of many types of ce lls. SDF 1 has been found to be secreted, as well as bound and presented by proteoglycans on stromal cells such as osteoblasts and BMECs in the BM (64 67) The bound and presented form of SDF 1 have been shown to increase HSC extravasation through sinusoidal endothe lium during homing which was especially evident when presented by selectins under sheer flow (66) Additionally, this bound form of SDF 1 when presented by osteoblasts or BMECs is partly responsible for retaining HSC s /HPCs within the niche through the binding of its cognate rece ptor CXCR 4, expressed on HSC s/ HPCs. MMP 9 released from neutrophils in response to stress, have been reported to cleave the N terminal end of SDF 1 and thereby releasing HSC/HPCs from the niche (61) Ultimately, these events allow the HSC/HPCs to be released into circul ation in order to migrate
20 to distant sites of ongoing injury or to initiate restoration of homeostasis following myelosuppression. HSC migration from the BM does not only occur during induced mobilization but also to a lesser extent during normal homeosta sis (68) .The reasons for this are still largely unknown, but the small numbers of circulating HSCs might serve as a reservoir t o repopulate injured BM after trauma or be a result from the constant bone remodelling tha t goes on within the BM Following tra nsplantation, HSCs in circulation home back to their distinct niches within the BM This process requires the HSC s to follow chemokine gradients consisting of for instance SDF 1, that are carried by the blood from the BM (59,6467) Upon reaching the BM vasculature, SDF 1 have stimulated circulating HSCs to e xpress integrins such as: very late antigen 4 (VLA 4), leukocyte function antigen 1 (LFA 1) and h yalu ronan binding -cellular adhesion molecule (CD44) (69) These in turn, interact with VCAM, ICAM, E and P selectins expressed on BMECs which slows down the circulating HSC/HPCs in a process known as rolling (69) Following rolling, firm adhesion and subsequent endothelial trans -migration into the hematopoietic compartment is mainly accomplished by VLA 4 VLA 5 and LFA 1 interactions (69 71) O nce extravasated, the cells migrate along extravascular hematopoietic cords towards their specific niches through a SDF 1, FGF 4 or receding oxygen gra dient originating from the supporting osteoblastic or endothelial niche cells (60,64,67) A summary of the complex interactions between the endosteal and vascular niches during mobilization and homing is provi ded in Figure 1 4 Role of BM in Tumor Blood Vessel Formation Adult blood vessel formation is believed to occur by two means, vasculogenesis and angiogenesis. Both result in the formation of new blood vessels which in general consist of a single layer of endothelial cells lining the interior of the v essel wall. Structural support, to
21 prevent leaking, is provided in part by pericytes and in the case of larger vessels smooth muscle fibre lining the outside of the vessel. Vasculogenesis can best be described as de novo generation of blood vessels by recr uiting undifferentiated progenitor cells to the site of vessel formation where they differentiate into vascular endothelium. The circulatory system is formed this way during embryonic development. Postnatal neovascularization has been attributed to angioge nesis, a process characterized by sprouting of new capillaries from pre -existing blood vessels (72) This usually occurs as a result of injury when reduced blood flow leads to local hypoxia in the damaged area (72) The formation of new blood vessels are thought to be a process involving the division of existing endothelial cells, recruitment of circulating endothelial cells (CECs), endo thelial progenitor cells (EPC s) and perhaps even HSC/HPCs (73) Tumor blood vessel formation differs in many ways from normal angiogenesis. The usually rapid growth of tumors often create regions of acute hypoxia and necrosis when it outgrows its blood supply (74) In order for the tumor to continue to grow and metastasize, blood flow must be restored in these areas. Growing evidence sugge sts a major role for BM derived cells in m odulating tu mor angiogenesis. Apart from various stem and progenitor cell involvement in producing tumor neovessels, recruited myeloid cells seem to be crucial for angiogenesis to occur (75 78) The development of hypoxic regions inside a tumor ini tiates a cascade of events that lead to the recru itment of various cell types, which upon arrival in the ischemic area contribute to blood vessel formation (75,76,79) Tumor associated macrophages (TAMs) have been observed to accumulate heavily within hypoxic tumor areas (80) This accumulation is though t to depend on the release of CCL 2 (MCP 1) from malignant or stromal tumor cells (80) Two phenotypes of TAMs are thought to exist, the M1 and M2 phenotypes. M1 TAMs are believed to possess the ability to kil l tumor cells. However, most TAMs in tumors are
22 believed to be of M2 phenotype, which appears to enhance tumor progression (81,82) During hypoxic conditions, TAMs respond by upregulating hypo xia inducible transcription factor s (HIF) (83,84) Among several things, HIF 1 enhances the production of vascular endothelial growth factors (VEGF) and SDF 1 which have been shown to greatly influence angiogenesis by recruiting and inducing angiogenic phenotype in ECs, HPC s and BM derived myeloid cells (85) Upon hypoxic stimulation, tumor infiltrating macrophages (TAMs) have also been shown to secrete proteoly tic enzymes, such as MMP 9, which could help break down basement membranes and cell to matrix i nteractions to create a road for newly formed vessels as w ell as to facilitate metastasis (86,87) Tie2 expressing monocytes (TEMs) are a different type of myeloid cells which are also recruited by tumors. These cells have main ly been observed to localize perivascular to newly formed tumor blood vessels but also in hypoxic, avascular areas (88 90) The intra -tumoral recruitment of TEM s seem to differ from TAMs in that instead of CCL 2, angiopoietin 2 is the main factor responsible (91) TEM involvement appears important, since ablation of TEMs reduce tumor angiogenesis and growth, without affecting intra tumoral TAM numbers (89) The specific role of TEMs remains to be discovered, but initial experiments have suggested roles in increasing microvessel density through FGF 2 production and inhibiting anti angiogenic cytokines (89,91) Apart from TAMs and T EMs, other cell types have been implicated in tumor blood vessel formation. CEC s are believed to be adult endothelial cell s that have detached from the basement membrane due to some form of injury before incorporating into newly formed tumor blood vessels (92) EPCs, are BM derived cells characterized by, in addition of primitive hematopoietic cell markers, their express ion of CD133 and VEGFR2 (93,94) These cells have in response to
23 VEGF been observed to be mobilized during tumor neovascularization and incorporate in large amounts into formed endothelium during the early stages of tumor growth (95,96) Tumor cells also secrete placental growth factor (PlGF ) which recruit VEGFR 1 expressing HPCs to perivascular sites of tumor angiogenesis (97) These HPCs in addition to secreting more pro angiogenic factors, also provide help in stabilizing newly formed vessels (98,99) Additionally, myeloid -derived suppressor cells (MDSC s), dendritic cells, neutrophils and m ast cells also have been shown to have roles in: evading immune responses, MMP 9/VEGF secretion, and secretion of a number of angiogenic factors upon de -granulation (100104) A summary of BM derived cell types proposed to play important roles in tumor progression is provided in Figure 1 5 Additionally, an illustration of tumor growth and angiogenesis is provid ed in Figure 1 6 Cellular In Vivo I maging To date, many significant discoveries have been made in regards to cell function, post transplant localizations and pathology due to the use of histological analysis. However, great emphasis has recently been put on being able to follow cells carrying out their function in vivo. Being able to study living cells and tissues in vivo offers new opportunities for investigating pathological or cell migration details that previously were inaccessible. Various forms of n o n inv asive imaging have been shown promising in monitoring cell fate in vivo Where histological sections of tissues of interest can only provide snapshot images of the events that are taking place, in vivo imaging has the potential to provide unique advantages in tracking cell migration, engraftment, fate and tissue regeneration at multiple time points in the same study subject (105) Vario us successful techniques for in vivo imaging have been presented, each having their own distinct advantages and disa dvantages. The most common of these techniques will be described and compared in this section.
24 Fluor es cent and Bioluminescent I maging Fluorescen t imaging for this section will be divided up in two subcategories, in vivo fluorescent microscopy (FLM) and n o n invasive fluorescent imaging ( FLI). In vivo FL M has the best spatial resolution capability of all various imaging modalities and has been used extensively to track rare cell populations. The main advantages of FLM apart from providing s ingle cell resolu tion are: the availability of many different fluorophores for labeling multiple cell types of interest and short image acquisition times. Short acquisition times provide the ability to record video in real time of migrating cells. The main disadvantage wit h FLM is depth penetration. The inability for using FLM for deep tissue imaging mainly stem from light absorption, tissue associated light scattering and autofluorescence. Light scattering in s kin, during imaging of subcutaneous xenotransplants is usually dealt with by the temporary removal of the overlaying skin tissue or by permanent replacement with a transparent window (106108) In order to image deeper structures, microscope objectives have been designed that following surgical procedures can be inserted through the skin and allow for ima ging of organs situated at deeper locations (57,109) Recent discoveries regarding the location of stem cell niches have been due to t he use of FLM (110,111) I n vivo imaging of stem cell homing to bone marr ow indicated that both the endosteal and vascular niches were in close contact with each other and may very well be the one and the same (110) These exp eriments were performed in the mouse calvarium, where the shallow depth of the marrow cavity allows for direct optical visualization of stem cell homing However, because of the narrow marrow space in this location, distinguishing these two possible niches is difficult. While these approaches have been proven extremely effective, they are by no means non-invasive and the effects of the surgical procedures on the normal milieu being investigated are hard to predict.
25 FLI and bioluminescent imaging (BLI) are t wo imaging modalities producing similar imaging results by differing methods. Both produce non invasive planar projection images that are pseudo colored to match the signal intensity and overlaid on a greyscale picture of the animal. Use of sensitive camer as and exclusion of outside light sources are crucial parameters in order to detect the low amount of light that escapes absorption and scattering within the animal tissues being imaged Cell tracking using FLI involves the use of either transgenic cells, expressing fluorescent proteins, or cells labelled with fluorescent dyes or particles prior to transplantation. The best results using FLI are usually obtained by using near infra red (NIR) emitting dyes since these, due to the longer wavelength of emitted light, generate the least amount of tissue light scattering and autofluorescence (112) BLI exhibits less autofluorescence and greater sensitivity due to the fact that light is produced at the site of the cells of interest and therefore no excitation light source, contributing to autofluorescence is necessary (113) BLI typically depends on the introduction of a gene encoding for luciferase being inserted into cells of interest (114) After the cells have been transplanted into the animal, a substrate, luciferin, is administered which is oxidized by luciferase to provide a bioluminescent reaction that can be registered by a camera (115) Both FLI and BLI makes it possible to detect signal from deep tissue structures, as well as being able to perform multiple measurements over sever al time points (116) This has been used to show bone precursor cells within bone marrow of living mice as well as longitudinally during the HSC engraftment process following irradiation and transplant (117119) In general, b oth suffer from the same limitations of absorption, tissue associated light scattering and autofluorescence whi ch limit spatial resolution capability. Additionally, FLI and BLI lack the ability to produce three dimensional tomographic images, and signal originating from deep within the tissue will appear
26 less bright than the signal originating clos e to the surface without a reliable way of distinguishing the two from each other. The detection sensitivity is also a concern for tracking rare cell populations since at least 2500 cel ls expressing luciferase were required for optimal detection in an earlier report (120) Attempts are be ing made to improve on these shortcomings by developing mathematical models of light scattering and addition of multiple light detectors at different angles for improved image reconstruction (121,122) C omputed Tomography /Positron Emission Tomography /S ingle P hoton Emission C omputed Tomography Positron emission tomography (PET) and single photon emission tomography (SPECT) have been used extensively in the clinic especially in comb ination with X -ray computed tomography (CT), and proven especially useful in scanning for tumour metastases in human patients (12 3) PET /SPECT, in short, relies on the detection of positronor photon emi ssion decay of injected radiotracers to create 3D data sets of molecular events (124) The radioisotopes are usually incorporated into a biologically active molecule that accumulates in target tissues, where the rate of its decay provides spatial metabolic information (124) SPECT, which uses radiotracers with longer half -lives, exhibit lower sensitivity and resolution capability than PET, has been used for detecting macrophage tissue migration and nanoformulated drug delivery (124,125) The resolution capability of PET is one of its limiting factors. Therfore, PET is often used together with CT or magnetic resonance imaging ( MRI ) in order to combine the great metabolic or probe specificity of PET with the great anatomical resolutions of CT and MRI. Apart for low resolution capability, additional concerns regarding PET include the difficulties regarding handling and availability of radiotracers as well as radiation exposure associated with th e use of the administered radioisotopes (124)
27 Magnetic Resonance I maging (MRI) One of the most promising technique s, due to relatively recent advance s in hardware and contrast agent design is magnetic resonance imaging (MRI) MRI uses external magnetic fields to align a majority of nuclear spins of hydrogen in water molecules along the direction of the applied field. Following an applied radiofrequency pulse that alters the spin -state of these hydrogen nuclei, the time it takes for these to return to their normal relaxed states is measured. Two (or three) different relaxation times can be measured, the longitudinal relaxation time (T1), the transverse relaxation time (T2) and (effective transverse relaxation time (T2*)). These relaxation times vary from tissue to tissue, providing a basis f or detecting injury and ongoing pathologies. MRI is widely used today in both research and clinical settings due to its ability to provide, excellent soft tissue contrast, great three dimensional anatomical detail s and relatively high resolutions MRI cannot achieve the same spatial resolution as fluorescent mi croscopy However, spatial resolutions of approximately 5 m and routi nely below 100 m have been reported, with a theoretical lower limit of 1 m (126129) This is far better than any of the other no n invasive imaging modalities and sufficien t to visualize cell clusters, small tissue structures and even single cells (130,131) Additionally MRI does not suffer from tissue penetration issues or autofluoresce nce. Instead the major limiting factors normally associated with MRI are a lack of sensitivi ty in detecting cells of interest, due to signal to -noise (SNR) limitations and methods of generating specific molecular contrast The field of MRI is aimed at improving MR sensitivity by increasing signal to noise and contrast levels through the use of h igher magnetic field strengths, high performance gradients and better designed RF coils (132) The driving force behind producing stronger exte rnal magnetic field magnets is the increased SNR achieved at ultra high magnetic fields which can be traded for enhanced resolution and shorter scan acquisition times.
28 Cellular M RI In order to track cells of interest by MRI in vivo they have to be label ed with specific contrast agents (CAs) to make them different from surrounding cells and tissues. In general, MR CAs highlight regions of interest by altering the relaxation times of target cells or tissues. This usually occurs by either shortening T1or T2 relaxation. Depending on which relaxation time is affected the most by the CA, they are named T1 or T2 CAs. The inverse of T1,2 is called relaxation rate (R1,2). By correlating the relaxation rate to the millimolar concentration of the metal responsible for the generated contrast, the term relaxivity (r1,2)is obtained, which allows different CAs to be compared to each other. The defining relaxation feature of a contrast agent is governed by its metal compo sition. P aramagnetic lanthanides such as gadolini um (Gd) and dysprosium (Dy) mainly affect T1 relaxation at lower magnetic fields (<4.7T) by interacting with water protons through dipole -dipole interactions (133) However, they also affect T2 and T2* relaxation times and reports suggest that this beco mes predominant at higher field strengths (134136) Apart from creating contrast through affecting relaxation times, contrast can also be created through chemical exchange saturation tran sfer (CEST). Paramagnetic lanthanides such as Dy3+ and europium (Eu3+) possess certain exchangeable protons that resonate at a different frequency than that of the bulk water of the tissue. By applying a radiofrequency pulse that is offset to this frequenc y, these protons can be saturated and transferred to the bulk water, resulting in decreased signal intensity (137) This enables contrast to be switched on and off simply by applying this offset radiofrequency pulse. Iron based CAs, s uch as the superparamagnetic iron oxide (SPIO) and FDA approved Feridex mainly reduce T2 and T2* relaxation times. SPIOs work especially well in reducing T2* relaxation because of its ability to dephase neighbouring water protons (138) This is called ma gnetic susc eptibility effect and is used during susceptibility weighted imaging. The generation
29 of magnetic susceptibility, observed as strong hypointense signal s on T2* weighted gradient echo (GE ) scans, are not j ust encompassing the Feridex particles themselves, but also the dephased water protons surrounding them leading to a blooming effect (139,140) The refore SPIOs are many times more sensitive than non -superparamagnetic CAs and provides the best option for detecting single or small a mounts of labelled cells by MRI (137,140) Iron based CAs have been used extensively in experiments aimed at tracking cells non invasively by MRI (117,138,141146) Most of these experiments have relied on pre -labeling of cells of interest in vitro with the contrast agent prior to in vivo administration (117,143149) S ome of these methods have involved the use of electroporation (145) cationic/anionic transfection agents (146,149151) liposomal agents (152,153) dendrimers (144) viral tran sfection methods (143) and conjugated receptor targeting antibodies (154) The method of pre labeling cells i n vitro and then tracking them in vivo has been shown to be very promising, but they also introduce unique problems for studying rapidly dividing and hard to transfect cell populations (155,156) The i nitial homing, migration and engraftment of hematopoietic stem and progenitor cells (HSC/HPCs) derived from the bone marrow has been studied using lower field strength MRI and pre -labeled cells (152,153) However, events such as; long term engraftment, vascular remodeling and organ regeneration that involve large scale proliferation and differentiation of the transplanted stem cells lead to a rap id and unavoidable dilution of contrast agent to undetectable levels, which limits the possible use of these methods to very short term tracking of HSC contribution during events that could take weeks or months (138,153,155,156) Concerns have also been raised about potentially negative effects on cell viability (147,157) and how extended in vitro labeling times could negatively effect stem or progenitor cell function (145,158) Furthermore there are also questions related to the fate of
30 contrast agent released from dead or dying cells Free contrast agent could easily be taken up by resident phagocytic cells, such as; monocytes and macrophages potentially resulting in false positives (138,152,159) Noninvasive gene reporter systems are attractive alternatives for l ong -term cellular imaging. When cells divide, the gene reporter will theoretically still be expressed in both cells at equal levels and still provide a similar contrast change upon activation. Previously proposed methods of genetic cell tracking systems by MRI includes the over -expression of cell surface proteins, such as, the transferrin receptor (TfR) and Her 2/neu receptor (160,161) More successful methods of creating MR contrast by gene manipul ation have been obtained by the over -expression of the ferritin complex subunits (H and L) or the transferrin receptor together with the ferritin H -subunit (162164) Up regulating ferritin expression lead to an increa se in intracellular iron accumulation when cells were grown in an iron rich medium that could be detected on T2 or T2 weighted MR scans (162164) The resulting contrast difference was further enhanced by increasing the magnetic field strength from 1.5 to 7T (164) The cell ular expression of the bacterial enzyme -galactosidase Gal) has also been considered for MR imaging. Reports using this form of reporter system have relied on: coating contrast agents with gal and thus activates the contrast agent, or by using 19F chemical s gal (165,166) Wh ile these approaches are promising, many still require in vitro manipulation of target cells prior to labeling. In order to effectively evaluate potential effects of stem cell therapies, it is highly desirable to be able to accurately follow transplanted c ells and their fates in vivo. The goal of d eveloping highly sensitive, non invasive imaging techniques that are capable of detecting cell migration and fates in vivo are actively being pursued. Ultra -high field strength
31 MRI comprises one of the most promis ing methods for achieving this goal. T herefore, its capabilities for improving in vivo cell tracking using established methods and a novel molecular MR gene reporter in models of homing to BM niches and growing tumors are investigated here.
32 Figure 1 1. Schematic of anatomical locations of the bone and bone marrow (BM) and cell lineages produced by hematopoietic stem cells (HSCs) BM is a major source of HSCs in adult mammals. It consists of a wide variety of hematopoietic and mesenchymal derived cells e ncapsulated by bone, as well as an intricate and highly specialized vascular network that extends throughout the marrow space HSCs can be found at various locations throughout the BM in close contact with bone lining cells, s tromal cells and specialized endothelial cells. These supportive cell types govern t he main responsibility of the HSC to maintain all the different cell lineages of the blood for the entire lifespan of the organism. (This figure was adapted from the NIH website, 2001 Terese Winslow ).
33 Figure 1 2 : HSC isolation characteristics. Various properties and cell surface markers have been proposed for the characterization of various stages of HSC maturity L ong -term repopulating HSCs (LT HSC) are considered to have an unlimited ability to s u stain the production of all cell lineages of the blood. They differ in surface marker profile from more specialized short term repopulating HSCs (ST -HSC) and multi potent progenitor cells (MPP) ST HSCs and MPPs are more committed HSCs that possess limited self renewal capability therefore can only sustain blood cell production for a limited amount of time. ( Adapted from Wilson and Trumpp, 2006 Nat Rev ).
34 Figure 1 3. Illustration of proposed BM HSC niches HSC fate is believed to be controlled by spe cific BM microenvironments or niches. Two such ni ches have been proposed to date. The endosteal or osteoblastic niche is thought to harbor mainly LT HSCs, maintaining cell quiescence and self renewal through a variety of cell -to -cell in teractions with b one lining osteoblasts (Top inset) Additionally, asymmetric cell division arising from uneven distribution of intra -cellular fate determination cues or slight differences in the external microenvironment are also thought to occur inor in the vicinity of endosteal niches. The vascular niche (bottom inset) has been suggested mainly to contain more committed HSC subsets through interactions with specialized bone marrow vascular endothelial cells. While positioned in close proximity to BM sinusoids, these HS Cs could closely monitor molecular cues carried in the blood and mount a rapid response to injury. ( Adapted from Wilson and Trumpp, 2006 Nat. Rev.)
35 Figure 1 4 Illustration of HSC mobilization and homing from endosteal and vascular niches In response to stress, HSCs have the ability to exit the BM and migrate to distant sites within the body. Stress caused by peripheral injury, irradiation, myeloablation or mobilization agents lead to increased chemotactic signals carried in the blood. Upon reaching th e BM compartment, these molecules stimulate neutrophils to release a variety of proteases. This causes the cleavage of niche retention signals and migration of HSCs into peripheral circulation. HSCs in circulation also have the ability to home back to thei r supportive niches. Following irradiation, SDF 1 released from surviving osteoblasts and megakaryocytes induce the migration of HSCs to the BM. Within BM sinusoids, circulating HSCs are slowed down by interactions with selectins on endothelial cells in a process known as rolling. S trong integrin mediated adhesions are then formed and induce HSC extravasation into the BM stromal compartment. Once inside, HSCs migrate along hematopoietic chords towards chemotactic gradients until they reach and lodge withi n preferred niches. ( Adapted from Wilson and Trumpp, 2006 Nat. Rev and Yin et al 2006 J. Clin. Invest )
36 Figure 1 5 BM derived cell types implicated in tumor growth and blood vessel formation BM derived cells have been shown to greatly influence t umor angiogenesis, growth and metastasis. HSCs/HPCs (hemangiocytes) and more differentiated cells of myeloid lineage, play distinct roles in promoting tumor growth. Certain cell types accomplish this through the secretion of a wide variety of chemotaxis a nd angiogenesis stimulating molecules, as well as matrix remodeling proteases. Other cell types, such as myeloid derived suppressor cells (MDSCs), serve mainly to suppress an immune response to the growing tumor. BM derived cell types shown to incorporate into growing tumors and the identified mouse cell surface markers for ea ch cell type is provided in the t op portion of the figure ( Adapted from Murdoch et al. 2008 Nat.Rev).
37 Figure 1 6. BM derived cells modulate tumor angiogenesis and growth. Rapid t umor growth often leads to the development of hypoxic regions in the center due to an inadequate blood supply (bottom portion) Through up regulation of hypoxia induced transcription factors chemokines such as SDF 1, VEGF and CCL 2 are secreted which init iates recruit ment of HSCs, HPCs, EPCs and a wide variety of myeloid cells Monocytic cells, such as T AMs and TEMs, incorporate within these hypoxic hot spots and have been shown to modulat e angiogenesis induce basement membrane remodeling and stabilize n ewly formed vessels Additionall y, TAMs have been proposed to be able to transdifferentiate into an endothelial phenotype as well as to further enhance stem and progenitor cell recruitment through the release of additional chemotactic signals. Ultimately the complex interactions between tumor cells and recrui ted BM derived cells lead to re vascularization, progression to malignancy and metastasis. ( Adapted from Wels et al ., 2008 Genes Dev. ).
38 CHAPTER 2 MATERIALS AND METHOD S The optimized materials and me thods described here have been developed and adapted over a number of years from multiple previous reports and own observations. This chapter will discuss the various procedures used during these experiments including, parameters for optimized magnetic res onance imaging (MRI) scans, magnetic resonance ( MR ) relaxivity measurements flow cytometry, cell labeling and tissue preparations for histology. Additionally, all experimental procedures performed were in accordance with the University of Floridas Instit utional Animal Care and Use Committee. Animals Control C57BL6J (BL6) and FVB mice were purchased from Charles River Laboratories. Homozygous dsRed transgenic animals (Tg(CAG DsRed*MST)1Nagy/J, express ing the red fluorescent protein variant DsRed in all tis sues under the control of the chicken beta actin promoter coupled with the cytomegalovirus (CMV) immediate early enhancer were obtained from The Jackson Laboratory. B6 Rosa26 (B6.129S7Gt(ROSA)26Sor/J) mice that ubiquitously express the L acZ gene in most a dult tissues were obtained from The Jackson Laboratory (Bar Harbor, Maine). Tie2LacZ (FVB/N Tg(Tie2 -LacZ)182Sato/J) mice, conditionally expressing LacZ under the control of the Tie2 promoter, were obtained from The Jackson Laboratory (Bar Harbor, Maine). T he L acZ gene encodes galactosidase Gal). Bacterial -gal has differing substrate specificity from its mammalian counterpart. LacZ is a widely used generic reporter system used both in vitro and in vivo, particularly in the field of developmental biology where countless L acZ transgenic mouse strains have been generated.
39 Materials and Methods u sed in Chapter 3 Irradiation Induced I njury Two cohorts of 6 irradiation. The first cohort of animals was s ubsequently transplanted with approximately 2106 BL6 whole bone marrow (BM) cells by retro orbital sinus (ROS) injection one day following irradiation (n=15). This cohort was used for investigating irradiation induced morphological BM changes as well as c hanges in MR characteristics over two weeks following irradiation. The second cohort was used for subsequent in vivo imaging of BM cell homing (n=5). Antibiotics (Enrofloxacin) were added to the drinking water during the first 2 weeks of engraftment to pre vent infection. BM Labeling w ith Feridex BM cells for homing studies were harvested from homozygous male dsRed transgenic animals. These cells were incubated with a mixture of Feridex (Berlex laboratories) and Protamine sulfate (Sigma) as previously descri bed (167) Following overnight labeling cells were purified using 10 U/ml Heparin (Sigma) to remove excess Feridex that had not yet been taken up by the cells. The cells were then washed, counted and re -suspended in PBS before injection (168) Cytospins of labeled BM cells were stain e d with Prussian blue, AccustainTM Iron stain (Sigma Diagnostics inc, MO, USA) for 3 minutes to visualize cellular iron uptake. Cell labeling efficiency was estimated by manual counting of Prussian blue positive cells. Tibia Window Installment 24 hours post irradiation, animals from the second cohort were sedat ed by intra peritoneal Avertin injections ( 600 mg/kg, Aldrich). Tibia windows were installed by first carefully exposing and subsequent thinning of the bone in the diaphysis region of the tibia. Great care was observed to not injure the marrow itself while thinning the bone. A custom fitted coverslip was then mounted in place using Vectashield ( Vector laboratories inc.) over the exposed BM and
40 attached using superglue This procedure provided a window for optical microscopy of BM cell homing that could be correlated with MRI. Feridex Labeled BM Cell T ransplantation For studying BM cell homing, y oung animals were of great importance due to loss of BM signal intensity (SI) on T2 weighted MRI scan s as the animals grew older, in accordance with previous reports (169) Therefore, 6 week old BL6 female mice (n = 15) were used for opti mal initial MR SI properties. 24 hours after irradiation and directly following the installatio n of the tibia window, approximately 10 000 F eridex labeled dsRed BM cells were transplanted via the left femoral artery (FA) while the animal was still anesthetized. FA injection was performed by placing a ligature around the FA proximally to the injection site, making a small incision distally to the ligature and injecting approximately 10 l of the cell suspension. This was followed by, incision cauterization, ligature removal, suturing the skin and covering it with antibiotic ointment. Following FA injec tion, a booster dose of 2106 BM cells were delivered via ROS injection to ensure that a sufficient irradiation rescue dose of BM cells were given In Vivo Fluorescent Microscopy of DsRed BM Cells In vivo fluorescent microscopy of dsRed BM cell homing was performed directly after cell delivery via the FA while the animals were still sedated I mages and videos were acquired through a Texas Red filter at 10X magnification using a LEICA 5500B microscope (LEICA, Wetzlar, Germany), a Hamamatsu 3CCD camera and Volocity 4.2.1 software (Improvision, MA, USA). MR I of BM Following Irradiation and Transplantation MRI was performed at 17.6T (750MHz) magnetic field strength following transplantation (Bruker Biospin,) using Paravision 4.0 software ( Bruker ). Animals were s edated during the imaging process by breathing a mixture of I sofluorane and oxygen. Respiration and body
41 temperature was monitored during the imaging process ( S A Instruments). A custom built single tuned 811 mm surface coil ( Doty Scientific ) was placed ov er the knee and tibia, allowing imag ing of the BM space. Three -dimensional (3D) gradient echo ( GE ) scan sequences were obtained with imaging parameters of; repetition time ( TR ) = 80 ms, echo time (TE ) = 2.5 ms, field of view ( FOV ) = 22.214.171.124 cm3, m atrix size = 39321483, spectral width = 75 kHz and 4 signal averages, resulting in a resolution of 282860 m3. For ex vivo imaging of excised legs, a 10 mm birdcage coil and 3D GE scan sequence was used with imaging parameters; TR = 70 ms, TE = 3 ms, FOV = 126.96.36.199 cm3, m atrix size = 566300200, spectral width = 75 kHz and 8 signal averages. These imaging parameters resulted in a resolution of 303030 m3. BM SI was measured by n ormalizing the SI of BM to the SI of an adjacent portion of the tibialis an terior (TA ) muscle for each time point. MR images and 3D renderings were processed using OsiriX v.3.5 ( http://www.osirix -viewer.com ). Histological Analysis of F eridex Labeled DsRed BM Cells Freshly harvested bones were fixed overnight in 4% paraformaldehyde (PFA) decalcified overnight in 5% formic acid and embedded in paraffin. Hematoxylin and eosin (HE) staining was used to determine BM cellularity. For the an imals receiving Feridex labeled d sRed BM cell s an tibody retrieval was performed on 5 m tissue sections using DACO heat retrieval and stained with a primary dsRed antibody (1: 200 dilution, Pharmingen). This was followed by a peroxidase secondary antibody with 3, 3 Diaminobenzidine ( DAB ) development and Prussian blue stain for iron detection. Prussian blue iron development was only allowed for 2 minutes, instead of the manuf acturers recommended 10 minutes, in order to limit the development of endogenous iron.
42 Ma terials and Methods used in Chapter 4 S Ga l Substrate Specificity Studies S Gal (3,4 cyclohexeneoesculetin-B D -galactopyranoside salt) is one of the commercially available histological stains for L acZ Gal, S -Gal chelates ferric iron, derived from ferric ammonium citrate (FAC), to create a dark, colored reaction product (170) Traditionally, this dark stain is used to rapidly identify cells expressing L acZ in cultures his tological sections, or in whole mounted,fixed embryos (171,172) S Gal labelin g solution was prepared by mixing 1mg/ml S Gal Sodium salt (Sigma, St Louis, MO, USA) and 0.5 mg/ ml FAC (Sigma Aldrich, St Louis, MO, USA) in Dulbeccos Phosphate Buffered Saline (DPBS) (GIBCO, Grand Island, NY, USA ). Following mixing, the S Gal labeling solution was filtered to remove precipitants. -gal enz yme (US Biological, Swampscott, MA, USA) was added to yield a final concentration of 0.06 U/ml in one of the S G al/FAC samples and allowed to react for 1 2 minutes at room temperature. Cont rol samples consisted of just PBS, 0.5 mg/ml FAC and 0.5 mg/ml FAC with 1 mg/ml FAC without enzyme addition. Substrate phantoms for subsequent MRI were then manufactured by diluting the sample solutions with an equal amount of 1% Ultra -PureTM Agarose (Invi trogen, Carlsbad, CA, USA). The resulting mixtures were then injected into glass capillary tubes (Curtin -Matheson Scientific, Broomall, PA, USA) and allowed to solidify on ice to eliminate substrate sedimentation during imaging. S Gal Staining of B M Cells BM cells were harvested from BL6 and Rosa26 mice by flushing BM from femurs and tibias with 1X DPBS. Cells were then passed through a 26 gauge needle until single cell suspensions were obtained. Cells were re -suspended with 1 mg/ml S Gal and 0.5 mg/ml FAC in DPBS, 0.5 mg/ml FAC in DPBS or only DPBS in ClicksealTM micro centrifuge tubes (National
43 Scientific, Claremont, CA, USA) and incubated at 37C for 2 hours. Following incubation, cells were washed twice with 1X DPBS to remove free S -Gal and FAC. Cells we re subsequently filtered through FACS tubes with cell strainer caps (BD Falcon, Franklin Lakes, NJ, USA) to remove large aggregations of S -Gal and cells. We found that this step was essential since unfiltered cell and substrate aggregations seemed to gene rate strong non-specific magnetic susceptibility artifacts on T2* weighted scans. Finally cells were counted and re -suspended at a concentration of 8107 cells/ml in 100 l DPBS. Intra -C ellular Iron Accumulation Determination Intra -cellular iron accumulation was determined by a ferrozine iron assay (173) 3106 BM cells labeled as described above were incuba ted at 60C with 50 mM NaOH, 10 mM HCl and iron releasing reagent (equal volumes of 1.4M HCl and 4.5 % KMnO4) for 2 hours (n=5). The samples were allowed to cool down to room temperature and an iron detecting reagent was added consisting of; 6.5 mM ferrozi ne, 6.5 mM neocupoine, 2.5 M Ammonium acetate and 1 M Ascorbic acid. The mixture was allowed to react for 30 minutes at room temperature before measuring absorbance at 562 nm in a plate reader. Intra -cellular iron amounts was determined by extrapolating ab sorbance values on a standard curve of known iron concentrations. Measurements were repeated on five sets for each sample. Statistical significances between sample groups were determined by one -way analysis of variance ( ANOVA ) tests using GraphPad Prism 4. 0 software Data are presented as average standard deviation ( SD ) of measurements. Cell Viability Assay Cell viability was determined by Trypan blue (Invitrogen, GIBCO, Grand Island, NY, USA) exclusion at 40 minutes and 2 hours of labeling. 4x104 BM cell s selected from the labeling solutions were re -suspended in a 1:10 dilution with equal amounts of DPBS and Trypan blue.
44 Cell viability was then estimated by counting the dye excluding and dye retaining fractions to obtain a percentage of viable cells. Mea surements were repeated five times for each sample and data are presented as average SD of the measurements. Magnetic Resonance (MR) Measurements on S Gal Substrate and Cell Phantoms Cell phantoms was made by adding 100 l 1% Agarose to the cell samples and injecting the resulting solution into glass capillary tubes to keep cells or precipitates from sedimenting during the imaging steps. Final cell concentration for the imaging steps was 4x107cells/ml. The capillary tubes were placed in FC 43 fluorinertT M (3M) to minimize susceptibility artifacts during imaging. Cell and (substrate) phantoms were kept on ice until imaging at 4.7, 11.1, 14.1 and 17.6 T magnetic field strength magnets with Paravision software (PV3.02;Bruker). For transverse relaxation (R2) measurements a spin echo (SE), multiple slice multiple echo (MSME), scan sequence was used with param eter settings: TR = 15 s, TE = 7 ms (100 echoes), matrix si ze = 12864, FOV = 2.51.25 cm2, slice thickness 1mm. Effective transverse relaxation (R2*) me asurements use d a series of GE fast low angle shot (FLASH), scan sequences with the parameters: TR = 6 (or 18) s, TE = 4.0 140 ms, FOV = 2.82.8 cm2, matrix size = 256256, spectral width = 60 kHz and 1 mm slice thickness. Regions of interest (ROIs) fo r each sample were then drawn to contain the entire cross section of each of the samples and Paravision software was used to calculate R2 values. R2* values were determined by plotting the mean SI w ithin the ROIs against TE using nonlinear least squares curve fitting. These measurements were repeated on five sets for each sample. Statistical significances between sample groups at each magnetic field strength as well -gal containing samples at varying magnetic field strengths were determined by one -way ANOVA tests using GraphPad Prism 4.0 software Data are presented as average SD of measurements
45 In Situ MRI of S Gal Labeled BM Cells Rosa26 and BL6 animal s were used as BM cell donors. Cells were labeled as des cribed above with S Gal/FAC. BL6 recipient mice were anesthetized by intra peritoneal Avertin injections and the hair on their lower hind limbs were removed to facilitate a consistent intra muscular d elivery of cells. Subsequently, 0.5106 S Gal/FAC labeled L acZ expressingor control BM cells suspended in 40 l DPBS were injected into the TA muscles of the left and right legs respectively (n=3). The animals were kept stationary during the imaging step s by breathing a mix of Isofluorane and oxygen. The hind limbs were placed inside a custom made single tuned (4.7T) proton solenoid coil or a custom made proton loop gap coil (11.1T) and imaged with 3D GE scan sequences at 4.7 and 11.1 T magnetic field str engths with; TR = 100 ms, TE = 5 ms, spectral width = 100 kHz, 30 pulse angle, FOV = 1.451.202.40 cm3 and matrix size = 38419264. Images were acquired with Paravision Software and analyzed with OsiriX so ftware In Vivo S Gal Labeling of Rosa26 Tissue s First, 2 mg/ml S Gal with 1 mg/ml FAC or just 1 mg/ml FAC, was suspended in 200 l PBS containing 25% dimethyl sulfoxide (DMSO, Sigma, St Louis, MO, USA) and 30 40 l were injected into the leftand right TAs respectively of Rosa26 animals (n=5). Animal s were anesthetized and positioned in the coil as mentioned previously and imaged at 11.1T magnetic field strength at 1, 5 and 24 hours post injection using; a 3D GE scan sequence with: TR = 100 ms, TE = 3.8 ms, spectral width = 100 kHz, 30 pulse angle, F OV = 1.501.202.40 cm3 and matrix size = 38419264 or a MSME scan sequence with: TR = 2 s, TE = 5.7 ms (6 echoes), spectral width = 100 kHz, FOV = 1.5 1.4 cm2, matrix size = 256 128 and 1 mm slice thickness. MR data was subsequently analyzed using Os iriX software.
46 Secondly, a group of BL6 animals (n = 3) underwent a procedure where an approximate 2 mm3 piece of Rosa26 muscle was surgically implanted in their left gastrocnemius muscles. These animals underwent a pre injection scan followed by an intra -muscular injection of 50 l S Gal labeling solution (1 mg/ml S -Gal 0.5 mg/ml FAC in PBS with 10% DMSO). The animals left hind limbs were then imaged 1 and 24 hours post injection with 3D GE scan sequences with: TR = 100 ms, TE = 3.8 ms, spectral width = 1 00 kHz, 30 pulse angle, FOV = 188.8.131.52 cm3 and matrix size = 38419264. MR data was subsequently analyzed using OsiriX software. Histological Analysis of S Gal Label ed Cells and Tissues Samples of labeled and non -labeled BM cells were spun onto positi vely charged microscope slides (Fisher scientific) at 100 rpm/min for 1min using a Shandon Cytospin 3 and stained with Prussian blue to identify intracellular iron accumulation according to the manufacturers specifications. The slides were fixed for 2 minu tes in 100% methanol, mounted with CytosealTM XYL (Rich ard -Allan Scientific) and cover sliped. Brightfield Images were acquired at 40X with a LEICA 5500B microscope and Volocity 4.2.1 software. Following MRI, mice were sedated and euthanized by cervical dis location. TA muscles were dissected out and frozen immediately with liquid nitrogen for optimal preservation of morphology. Following storage at 80C overnight, 10 micrometer tissue sections were cut and fixed for 5 mi nutes with 4% PFA Staining of tissue sections were then performed with either Prussian blue iro n stain, HE or Nuclear Fast Red (NFR). Slides were mounted with CytosealTM XYL and cover sliped. Brightfield Images were acquired with a LEICA 5500B microscope and Volocit y software
47 Materials and Methods used in Chapter 5 Ir radiation Damage and BM Cell Transplantation 6 8 week old BL6 and FVB mice were given 950 Rads of whole body irradiation and subsequently trans planted by ROS injection one day following irradiation with approximately 2106 BL6, Rosa26 or Tie2LacZ whole BM cells respectively. Antibiotics (Enrofloxacin) were added to the drinking water during the first 2 weeks of engraftment to prevent infection. BM Galactosidase Expression in Peripheral Blood Three months following irradiation and transplantation, BM cell engraftment was Gal expression using flow activated cell sorting (FACS) analysis of peripheral blood (PB) (174) Prior to FACS analysis, PB cells were loaded with the fluorescent substrate Fluor D -galactopyranoside) Gal, emits fluorescence in the FIT Gal expression Rosa26 derived PB was simultaneously analyzed using CD11bPE conjugated antibodies. Tumor Cell Implantation Lewis lung carcinoma (LLC) (ATCC) and Lung AB carcinoma (LAP0297) (a generous gift from Dr Peigen Huang at Harvard medical school) were used for BL6 and FVB respectively. After confirmation of successful BM engraftment, 1106 to 2 06 t umor cells were inj ected into the TA muscles of the engrafted animals. T he tumor cells w ere allowed to grow for 14 days during which in vivo MRI of the grow ing tumor was performed. Approximately 14 days after initial tumor cell implantation, the animals were sacrificed due to the size of the formed tumor. In Vivo Fluorescent Imag ing of Intra -Tumoral BM Derived Cells gal substrate FDG was used over mo re recently developed and improved substrates due to its shorter wavelength and therefore decreased ability for emitted light to penetrate tissue (175) This was desirable since light emitted from engraf ted cells within the BM
48 would interfere with the light emitted by BM derived cells within the tumor. Animals were given 25 mg/kg of FDG solution by ROS injection approximately 30 minutes prior to imaging. An excitation of 500 nm and a recorded emission of 540 nm were used to collect the image data using an IVISSpectrum biophotonic imager (Xenogen). In Vivo M RI of Tumor Recruited Myeloid Cells. MR im aging was performed at 4.7, 11.1 and 17.6T magnetic field strength using Paravision 3.01 and 4.0 acquisition softw are (Bruker). 3D SE and 3D GE scans were obtained of the tumor containing lower hind limb prior to and f ollowing S Gal administration. SE scan s were acquired in order to visualize the extent of the growing tumor based on the increased T2 relaxation time that is characteristic for tumors. GE scans were used to visualize iron containing cells within the tumor based on iron induced T2* shortening. In vivo MR imaging at 17.6T was performed using a custom built surface coil with dimensions 811 mm (Doty Scientifi c) placed over the animals TA muscle s 3D GE scan sequences were acquire d using; TR = 70 ms, TE = 3 ms, s pectral width = 50 kHz, 4 signal ave rages, matrix size = 31425788, FOV = 184.108.40.206 cm3, resulting in a resolution of 353580 m3. In vivo MR imaging at 11.1T was performed on tumor containing lower hind limbs using a custom built solenoid coil. 3D SE rapid acquisition with relaxation e nhancement ( RARE) scan sequences were acquired with imaging parameters: TR = 1 s, effective TE = 41.3 ms, spectral width = 75 kHz, rare factor = 10, 1 signal average, matrix size = 204120120, FOV = 220.127.116.11 cm3. 3D GE, gradient echo fast imaging (GEF I ), scan sequences were then acquired with imaging parameters: TR = 100 ms, TE = 5 ms, spectral width = 61 kHz, 1 signal average, matrix size = 440200150, FOV = 18.104.22.168 cm3, resulting in a resolution of 108108108 m3 for the SE scan sequence and 50 65 87 m3 for the GE scan sequence
49 Following basel ine scans, animals were given ROS injections with 100 l of a mixture of 2mg/ml S Gal and 1 mg/ml FAC in PBS. Animals were allowed to rest for approximately 3 hours following imaging and injection before the start of post contrast imaging. In vivo imaging at 4.7T was performed 24 hours post S Gal injection s to visualize the extent of the growing tumors in both LacZ transplanted and control animals. Images were acquired using a custom built loop gap coil an d a 3D SE (RARE) scan sequence with imaging parameters: TR = 1.5 s, effective TE = 42.83 ms, spectral width = 75 kHz, 1 signal average, rare factor = 4, matrix size = 25612880, FOV = 22.214.171.124 cm3, resolution = 9898156 m3. Excised fixed tumors wer e submerged in FC 43 fluorinert (3M) to improve signal to noise ratio (SNR) and imaged ex vivo at 17.6T using a 3D GE scan with parameter settings: TR = 80 ms, TE = 3.2 ms, spectral width = 50 kHz, 8 signal averages, matrix size = 433300300, FOV = 1.30. 90.9cm3 and resolution = 303030m3. Post -processing of images was performed using OsiriX v.3.5 software. Histological Analysis of Tumor Recruited BM Derived Myeloid Cells Following imaging, animals were sacrificed by cervical dislocation. The tumors wer e exc ised and freshly frozen in optimum cutting temperature embedding compound (OCT) Ten micrometer thick sections were cut and placed on positively charged glass slides. Sections were stained for LacZ expression using X -gal (Sigma), iron accumulation usi ng Prussian blue i ron stain, HE CD11b monocytic cell marker, F4/80 macrophage cell marker, MECA32 endothelial marker and terminal deoxynucleotidyl transferase nick end labeling ( TUNEL ) for cells undergoing apoptosis.
50 CHAPTER 3 IN VIVO VISUALIZATIO N OF BM CELL HOMI NG USING ULTRA HIGH FIELD MRI FOLLOWING IRRADIATIO N AND CELL TRANSPLAN TATION Introduction Since the discovery of the hematopoietic stem cell (HSC), much has been learned about its phenotype and biological function (12) Even though much is known about the function of the HSC, there is an ongoing debate regarding where the HSC res ides within the bone marrow (BM ) and what are the underlying molecular cues which determine its fate (1). As discussed in chapter 1, tw o distinct niches, have been proposed to be crucial for HSC maintenance and function, the endosteal and vascular niche. BM remodeling and subsequent restoration of homeostasis following irradiation relies heavily on the ability of transplanted cells to home back to these niches (17 6) Therefore, the function of vascular structures w ithin the BM is of great importance. Irradiation induced changes of vascular structures within the BM have been elegantly visualized ex vivo using vascular casting and electron microscopy (48) This study indicated that 5 days post 5 FU treatment or irradiation, marked swelling and fusion o f BM sinusoids and dilation of the central sinus vein occurred together wi th a decrease in BM cellular ity (48) Changes in BM signal intensity (SI) on magnetic resonance imaging ( MRI ) scans have previously been described and attributed to the red to yellow marrow conversion that occurs with age in was described to be crucial for the conversion to occur (169) This report in dicated that this conversion occured between 2 and 5 months of age. Another study described the c hanges in t he blood to BM barrier using MRI following irradiation (177) By administering a blood -pool contrast agent, disruption of the BM vasculature could be visualized by a gr eater change in SI of irradiate d animals versus nonirradiated (177) These studies are valuable in elucidating physiologica l changes on the macro imaging level within the
51 BM, but have not been able to provide sufficient resolution to determine specific spatial location s of transplanted BM cells. In vivo magnetic resonance ( MR ) ch aracteristics of the BM and cell homing at Ultra -high magnetic fields of 17.6T have not been described to date Here the ability of ultra high field strength MRI for achieving high resolutions images of the initial homing of transplanted superparamagnetic iron oxide (SPIO ) labeled BM cells to specific l ocations within the BM was investigated. The hypothesis was that the increased MR sensitivity at higher magnetic field stre ngths would enable the noninvasive visualization of sin gle or small clusters of homing iron labeled cells that could be further confirmed using in vivo fluorescent microscopy and histology. Irradiation Induced Changes in B M Signal Intensity at Ultra High Magnetic Field S tren g th In order to establish BM MR characteristics at ultra -high magnetic field strength, young animals were lethal ly irradiated and transplanted with normal unlabeled BM cells. These animals were imaged throughout the first two weeks of engraftment in order to determine the optimal tim e frame for tracking SPIO labeled BM cell s. This was neces sary because BM SI has pre viously been reported to change drastically following lethal irradiation, with a marke d drop in SI occurring from day 3 f ollowing irradiation, due to hemorrhage associated shortening of T2 (178) Since we planned to use FeridexTM, due to the strong negative contrast generating effect associated with SPIOs we needed to ensure that labeled cel ls would be optimally distinguished from non-specific, irradiation induced T2 shortening. Following lethal irradiation and BM transplan tation, host BM SI wa s observed to decrease steadily during the two weeks the animals were followed (Fig ure 3 1a, top and Fig 1b). This drop in SI coincided with a prevalent decrease in BM cellularity (Fig ure 3 1 a, bottom) and increased iron deposition within the mar row space, especially towards the central portions of the diaphysis, observed towards later time points (> 1 w eek post irradiation) Decreasing BM cellularity
52 coupled with increased iron deposition are likely reasons f or the observed drop in SI on three dimensional (3D) renderings of the BM space (Fig ure 3 1c, d). A previous report has indicated that a loss in BM cellularity leads to the swelling of BM sinusoids through a lack of structural support (48) This effect led to the ability to visualize sinusoidal vasculature by MRI at 7 days post irradiation (Figure 3 1c) Eventually, between week 1 and 2, the s ignal originating from BM cells and smaller vascul ar structures was markedly decreased leaving mostly signal originating from the larger, centrally located vascular structures, that by this time were becoming enlarged (Fig ure 3 1c, d ). Rupturing of BM sinusoids as a result of irradiation has previously been described to occur (48) This could be a likely explanation for the disappeara nce of previously observed sinusoidal vas culature on MRI scans by day 14 following irradiation (Figure 3 1d). Based on these observations, we found that 6 8 week old mice were ideal candidates for SPIO labeled BM transplantation due to the fact that their BM still exhibited r elatively high SI and that it was maintained at a reasonable level for up to 5 days follow ing irradiation and transplantation Additionally, increased amounts of non -specific iron within the BM, as a result of hemorrhage from ruptured BM sinusoids, could potentially lead to the detection of false contrast on susceptibility based MR scan sequences. However, no such iron accumulation was observed prior to one week following irradiation and therefore should not contribute significantly to observed MR contrast from SPIO labeled BM cells during early time points. BM Labeling and In Vivo Fluorescence T racking Prior to BM transplant ation c ytospins of dsRed expressing BM cell s labeled overnight with the SPIO contrast agent FeridexTM, were perf ormed in order to ensure a r easonable labeling efficiency. C ellular e xpression of dsRed fluore s cent protein was confirmed by direct fluorescent visualization (Fig ure 3 2a) and the iron cell la beling efficiency was estimated by manual counting to be approx imately 40 percent following Prussian blue iron staining (Fig ure 3 2b).
53 Using in vivo fluorescent microscopy (FLM) dsRed fluorescent cells could be observed to e nter the BM space through a bone window installed on a thinned portion of the animal tibia w it hin minutes following systemic delivery via the femoral artery (FA) Arriving cells were observed to join groups of previously lodged cells (Figure 3 2c) The resulting clusters seemed to form at favorable niches located close to the bone surfa ce undern eath the tibia window (Fig ure 3 2d ). In Vivo T racking by MRI Following confirmation of transplanted BM cell arrival by FLM, h igh resolution in vivo MRI was performed for u p to 48 hours post transplant. SPIO labeled cells were detected as small regions of s ignal void s, located throughout the limb within muscle as well as within the BM cavity. These areas of signal void s measured approximately 80 110 m in diameter which indicated the presence of small clusters of labeled cells While l abeled c ells were main ly observed close to the bone surface, they were also observed more centrally located away from the tibia window (Fig ure 3 3a) The use of ex vivo MRI of excised and fixed hind limbs enabled the acquisition of additionally enhanced resolution data sets tha t further confirmed the presence and spatial localization of transplanted BM cells wi thin the marrow space (Fig ure 3 3b, c) Orthogonal slices in all three directions confirmed the presence of single or small clusters of labeled cells as negative contrast ing spheres (Fig ure 3 4) Histological Confirmation of In Vivo F inding s Post -imaging histology of the imaged tibia identified cells with enhanced iron content at the very sites indicated by FLM and MRI. Using a shorter iron development period on the tissue sections ensured that main ly Feridex containing cells, responsible for the hypointense SI areas observed by MRI, were staining positive inside the marrow space (Fig ure 3 -5a) Subsequent antibody staining for dsRed protein coupled with Pr ussian blue iron stain further proved that
54 s mall clusters of iron containing cells at the locations indicated by MRI, were indeed of donor origin (Fig ure 3 5 b ). In summation, dual tagged cells obtained by harvesting BM cells from dsRed expressing transgenic mice and labeling them in vitro using SPIOs, could be used to visualize homing of transplanted cells in vivo by both FLM and MRI Once delivered, these tagged BM cell s could be observed from the first moment of arrival t o the BM by FLM and subsequently by MRI. This validates the use of high r esolution MRI with FLM confirmation for the visualization of BM cell homing to spatially resolved locations within the marrow space in vivo Furthermore, u ltra -high field strength MRI (17.6 T) possesses the sensitivity to be capable of high resolution, non invasive cell tracking during the complex events occurring inside the normally inaccessible BM cavity follo wing irradiation and BM cell transplantation. I n order to validate the presence of transplanted cells within the marrow space, in vivo F L M or BLI co upled with hist ology provides a great advantage and is vital in confirming the MRI findings. However, issues concerning label dilution due to cell division as well as MR contrast originating from dead cells cannot satisfactorily be addressed using SPIO lab eled cells. In the next chapter, a MR active gene reporter system for the longitudinal tracking of viable cells will be proposed and characterized.
55 Figure 3 1. M agnetic resonance ima ging (MRI) clearly depicts changes in contrast between day 0 an d day 1 4 following irradiation. a : As SI within the BM space decreased, BM cellularity was also observed to dec rease (bottom panel, hematoxylin and eosin (HE) staining). Normalized BM SI of irradiated animals was observed to drop steadily during two weeks follow ing irradiation (b) (n = 5). 3D MR renderings of the BM space following irradiation enabled the visualization of swollen sinusoids at day 7 (c) BM s inusoids were no longer visible at day 14 and i nstead signal was generated predominately from the larger mo re central ly located vascular structures (d)
56 Figure 3 2. Fluorescent microscopy detection of initial BM cell homing following transplantation. DsRed fluorescence (a) and presence of Feridex (b) within labeled cells were detected prior to systemic del ivery into the host animals (n = 5) In vivo fluorescent microscopy was used to detect the arrival of dsRed fluorescent BM cells inside the marrow cavity within minutes of femoral artery injection (c) The arriving BM cells seemed to be seeding into favora ble niches (d) (n = 5)
57 Figure 3 3. S uperparamagnetic iron oxide (S PIO ) labeled BM cell homing visualized using MRI. The bone window (* ) and SPIO labeled cells are visible on an inverted signal overview of an excised hind limb (a ). S mall clusters of tr ansplanted Feridex labeled BM cells (arrowheads) could be visualized by in vivo MRI as small hypointense areas in close proximity of the bone window (b) (n = 5) Ex vivo MRI provided further enhanced resolution which led to even easier distinction of these hypointense areas (c) (n = 1) The majority of the Feridex labeled BM cells seem ed to be in close proximity of the endosteum of the central portion of the tibi a. Animals receiving dsRed BM cells without Feridex label lacked the presence of hypointense regions within the same area underneath the tibia window (d) (n = 1)
58 Figure 3 4. Orthogonal validation of Feridex positive spheres The thinned tibia bone and window were visible both in vivo (a) and ex vivo (b) on orthogonal MR slices. Negatively contra sting spheres (arrows), indicative of iron loaded cells could be distinguished in all three orthogonal directions.
59 Figure 3 5. Histological identification of iron labeled dsRed expressing BM cells. a: HE staining combined with Prussian blue iron staining shows iron positive cells (black arrows) located at sites close to the bone (*) as indicated by MRI. Enhanced iron uptake was also observed in one endothelial cell close to iron la beled BM cells (white arrow). b: Dual staining using dsRed antibody and Prussian blue iron stain verified that clusters of transplanted dsRed cells also contained enhanced intracellular iron levels (arrow).
60 CHAPTER 4 LACZ AS AN IN VIVO GENETIC REPORTER FOR REAL TIME M RI Introduction Ultra high field magnetic resonance imagin g ( MRI ) comprises a very attractive tool for cell tracking As seen in Chapter 3, the e xcellent resolution capability and use of highly sensitive iron based contrast agents ( CAs ), enables the non invasive visualization of small amounts of transplanted cell s I ron as a contrast agent, is well known for its magnetic properties and fairly high tolerance by living organisms (138,142) However, t he vast ma jority of iron based CAs require external manipula tion of nonphagocytic cells (i.e. most stem cells) to induce label up take (147,148) Additionally, once these labeled cells are transplanted into the host it can be difficult to distinguish whether contra st is generated by via ble or dead cells (138,147) The us efulness of MRI for real time longitudinal in vivo studies, would be greatly enhanced by an effective genetic reporter system that is ca pable of creating magnetic resonance ( MR ) contrast on demand As mentioned in Chapter 2, S Gal was specifically designe d to form large, poorly diffusible, non -toxic, intracellular accumulations of iron -containing reaction products in the presence of -galactosidase ( -gal) and ferric salts (170) While S Gal has most commonly been used as a colorimetric stain on histological sections, we reason ed that accumulation of such large iron -containing deposits within lacZ -expressing cells could be visualized using ultra high fi e ld MRI. S Gal Forms a MR Visible Precipitate w ith Ferric Ammonium Citrate ( FAC ) in the G al T he specificity as wel l as the contrast enhancing effe ct of the reaction between S Gal and -gal -gal to a solution of S Gal and ferric ammonium citrate ( FAC ). The following reaction resulted in a change from a clear to a dark colored solution within 1 minute of labeling at room temperature ( Fig ure 4 -1a ). Subsequent MR
61 -gal enzyme containing phantoms exhibited a significant increase in transverse relaxation rate ( R2) and effective transverse relaxation rate (R2* ) when compared to controls at all magnetic field strengths ( N = 3; P < 0.05). Control R2 and R2* values were not significantly different from each other ( P >0.05 ), except at 4.7T where the R2 of the SG al/FAC compared to Blank and FAC were significant ly different ( P <0.05 ). R2 (Figure 4 1b) and especially R2* (Figure 4 1c, d) were enhanced with higher magnetic field strength for enzyme treated samples ( P < 0.05). However, only between 14.1 and 17.6T was there no significant differences in R2 or R2* ( P > 0.05). The observed increase in relaxivity with field is consistent with previous reports where T2 and susceptibility (T2*) based contrast agents have been shown to increase their ability to generate contrast with increasing external magnetic fields (164,179181) For instance, there is a 10 fold reduction in the minimal concentration of ferritin necessary, when used as a contrast agent at 11.7 T compared to 1.5T (180) Th erefore, we decided to investigate whether LacZ -expressing bone marrow (BM ) cells labeled in vitro with S Gal, would exhibit enhanced relaxivity with increasing magnetic fields. Transverse Re laxation Rates ( R2) and Effective Transverse Relaxation Rates ( R2 *) i s I ncreased in LacZ Expressing B one M arrow Cells F ollowing S Gal /FAC L abeling To evaluate the use of S Gal/FAC for cellular detection by MRI, we harvested lacZ expressi ng Rosa26 and contr ol C57BL6 BM cell s. Three different samples containing Rosa26 cell s labeled with S Gal/FAC, FAC, or left untreated were compared to control C57BL6 cells incubated with S Gal /FAC. Following labeling Rosa26 and control cells could be easily distinguished based on color. Rosa26 cell pellets turned dark while control cells maintained their original color ( Fig ure 4 2a ). Intra -cellular iron accumulation was determined by Prussian blue staining and ferrozine assays. Cytospins of S Gal/ FAC labeled Rosa26 BM cells revealed an increased intracellular
62 iron accumulation when compared to controls ( Fig ure 4 2b ). Iron accumulation determination by ferrozine assay showed that all samples accumulated significantly different amounts of iron ( N =5; P < 0.05). However, Rosa26 cells incubated with S -GalTM/FAC accumulated significantly more iron (0.3 pg/cell ) than any of the control samples (Fig ure 4 2d) ( N =5; P < 0.05). This intracellular iron accumulation is approximately an order of magnitude higher than that following the c o -expression of transferrin and ferritin (164) but lowe r than that reported by inducing the bacterial reporter gene MagA (0.6 pg/cell) (182) As anticipated this also represents less than the 0.7 30.1 pg/cell reported using direct labeling with iron oxides (183,184) As increased iron levels have been proposed to be metabolically cleared in vivo, we assumed that the accumulated intercellular free iron should be transported out from nonlacZ expressing cells (185,186) However, the cleaved S Gal product was specifically designed to chelate ferric iron into large, non -dif fusible intercellular complexes (170) Once assembled inside the cell these large complexes would most likely be ret ained In fact, only LacZ expres sing cells, incubated with S Gal / FAC, still possessed elevated intracel lular iron levels 24 hours following incubation compared with control cells (16%), as determined by the ferrozine assay. Additionally, S Gal /FAC labeling did not appear to have a negative effect on cellular viability as revealed by Trypan blue exclusion te st following 40 minutes and 2 hours of labeling. This test showed a slight but not a significant drop in viability for Rosa26 cells treated with S Gal/FAC compared to control cells after 40 minutes and 2 hours of labeling (Fig ure 4 2c) ( N = 5; P > 0.05). S ubsequent MR relaxometry was performed on cell phantoms at 4.7, 11.1 and 17.6T magnetic field strengths. R2 was observ ed to be increased for the S -Gal /FAC treated Rosa26 cells compared to controls at the differen t magnetic field strengths (Figure 4 3a). R2 values for all
63 samples were observed to be significantly different from each other ( N = 5; P < 0.05). R2* values were obt ained by acquiring gradient echo ( GE ) images at varying echo times (TEs ) (Fig ure 4 3b, lower) (Images acquired with TE = 60ms) and plotting the mean signal intensities of the samples with respe ct to TE (Figure 4 3b, upper) (representative measurements with normalized SI plotted against TE). R2* was observed to be drastically increased for Rosa26 cells labeled with S Gal /FAC when compared to controls at varyin g magnetic field strengths (Figure 4 3d) ( N = 5; P < 0.05). Control R2* values were all significantly different from each other, except for Rosa26/FAC compared t o Rosa26/Unlabeled and C57BL6/S G al/FAC at 17.6T ( P > 0.05). Both R2 and R2* values for S Gal /FAC labeled Rosa26 cells were significantly enhanced with higher magnetic field strengths ( P < 0.05) resulting in a cellular relaxivity of r2 = 16.7 (mmol Fe)1s1 and an apparent r2* of 198 (mmol Fe)1s1 at 17.6T. As previously prop osed, this large increase in relaxivity is likely an effect of intracellular iron accumulation and subsequent clustering (162,164,181,183) The change in R2* with increasing magnetic field strengths is also demonstrated by representative curves of normalized signal intensity ( SI ) plotted against TE for S Gal / FAC treated Rosa26 cells in Figure 4 3c. In V ivo D etection of LacZ Expressing BM Cells Following Intramuscular T ransplantation Next we determined if transplanted BM cell s labeled with S Gal/FAC could be detected in vivo Furthermore we determined if th e observed increase in relaxivity with magnetic field observed in vitro would translate into enhanced detection of cells at the higher magnetic fields in vivo. Control or Rosa26 cells were labeled as described above with S Gal/FAC for 2 h ours in PBS. Follo wing labeling, and 24 hours prior to imaging, C57BL6 mice were injected with 5105 of the labeled Rosa26 and control cells into the tibialis anterior ( TA ) muscles of the left and right hind limbs respec tively (n=3). The animals were anesthetized and the hi nd limbs of these mice were then imaged at 4.7T and 11.1T magnetic field strengths with three -dimensional ( 3D ) GE
64 scan sequences. At 4.7T magnetic field strength, signal loss was observed at the injection site of the labeled Rosa26 cells while minimal effe ct was observed in the leg receiving labeled control cells ( Figure 4 4d, e ). At 11.1T, the region of signal loss at the injection site of the labeled Rosa26 cells was markedly increased while the effect from the labeled control cells was relatively unchang ed ( Figure 4 4a, b ). Subsequent histology, 24 hours after injection, verified the presence of iron containing cells at the Rosa26 cell injection site (Figure 4 4c, f) while iron levels were too low to be detected in the TA receiving control cells (data not shown). In V ivo L abeling of L acZ Expressing T issues The potential use of S -Gal, for in vivo detection of L acZ expression by MRI, was investigated in two steps. First, by direct delivery of S Gal/FAC into TA muscles of Rosa26 animals and secondly, by establishing L acZ+ (Rosa26) muscle transplant into C57BL6 mice gastrocnemius muscles followed by S Gal/FAC injection. S Gal/FAC was dissolved in a PBS/DMSO solution to f acilitate the transport of the substrate across the cell membranes of L acZ expressing muscle fibers. Following injection, h ypointense signal areas (green) were observed on T2* weighted GE scan sequences in the left TA, which received S Gal /FAC (Figure 4 5a) T he right control leg, receiving only FAC, showed significantly less effect (Figure 4 5a) The hypointense areas in the left leg were detectable for approximately 4872 hours follow ing initial labeling. Additionally, i ncreased cellular permeability was evidenced in both legs by an increase in extra -cellular water accumulation (elevated T2 cont rast) (blue areas), as seen on T2 weighted spin echo ( SE ) images (Figure 4 5b). The a nimals were later sacrificed for visual confirmation and dark a reas indicative of S -gal specific reaction were observed in the left TA (Figure 4 5c, d (Top)), as well as increased iron accumulation determined by Prussian blue staining (Figure 4 5d (Bottom)). The surgically implanted muscle tissues were not visible o n T2* weighted scan sequences before injecti ng the labeling solution (Figure 4 5e). Administration of
65 labeling solution, led to cleavage of the S Gal substrate by the implanted L acZ -expressing muscles, creating strong hypointense signal areas (green) surrounding the muscle within 1 hour post injection (Figure 4 5f), which is also depicted in 3D (inset). In summary, the results obtained in this chapter indicate that S Gal provides the ability to detect cellular L acZ expression by MRI both in vitro and in vi vo A comparison between using S Gal, previously reported genetic reporter systems and SPIO labeled cells to generate MR contrast is provided in Table 4 1. H igh magnetic field strengths are important for optimal contrast generation using this iron based system However, the sensitivity of creating cell specific contrast following systemic delivery of S Gal still needs to be addressed The effectiveness of the MR active gene reporter system presented here for longitudinal in vivo studies will be examined and presented in the next chapter.
66 Figure 4 1 S Gal forms magnetic resonance (MR) active precipitates with ferric ammonium -gal) a) -gal to S Gal /FAC led to a rapid reaction, resulting in a color s -gal added to S Gal / gal. S Gal -gal samples showed significantly (P<0.05) increased R2 (b) and R2* (d) when compared to controls at 4.7 17.6T (n = 3) Error bars represent the SD in each case. R2 gal treated samples are further enhanced when compared to R2 and increases significantly with magnetic field strength. c) Normalize d signal intensities of S Gal gal plotted with respect to echo time for R2* determination at varying magnetic field strengt hs.
67 Figure 4 2. S Gal -gal expressing cells and results in increased intracellular iron accumulation. a) 8106 BM cell s after incubation with S Gal /FAC. Left: Rosa26 BM cell -gal, right: C57BL6 BM cell s (control). Cytospins of labeled Rosa26 BM cell s stained with Prussian blue iron stain for intracellular iron accumulation (b) (Top left): unlabeled, (Top right): FAC, (Lower left): Control C57BL6 BM cell s incubated with S Gal /FAC, (Lower right): Rosa26 labeled with S Gal /FAC showed increased amounts of intracellular iron compared to controls. c) Viability assessment by Trypan blue exclusion showed ne gligible effect of S Gal /FAC incubation on cellular viability following 40 minutes and 2 hours (P>0.05, n = 5) d) Ferr ozine iron assay indicated a large increase in intracellular iro n in Rosa26 BM cell s following S Gal /FAC incubation compared to controls (P<0.05, n = 5) Error bars represent SD.
68 Figure 4 3. gal expressing cells with S Gal/FAC leads to increased effective transverse relaxation rates (R2*) that are enhanced with stronger external magnetic field Rosa26 BM cell s incubated with S Gal /FAC showed a slight increase in R2 (a ) but large increase in R2* ( d ) when compared to controls at varying fi eld strengths (P<0.05, n = 5) Furthermore, this increase in R2* was obser ved to be significantly increased with magnetic field strength (P<0.05, n = 5) Error bars represent SD. b) Images of cell phantoms were acquired at different magnetic field strength s with varying TEs (bottom) and the signal intensities were plotted with respect to TE to obtain R2* (top). R2* curves corresponding to Rosa26 BM cell s incubated with S Gal /FAC for the different field strengths are shown in (c ).
69 Figure 4 4. The in vivo detection capability of S Gal labeled Rosa26 BM cell s is increased with field s trength. (a, d) 3D GE data rendering from 11.1T (a) showed a large region of signal void (green) at th e site of S Gal labeled Rosa26 BM cell injection that was less pronounced a t 4.7T (d) (n = 3) A much smaller effect was observed in the right leg receiving labeled control cells (purple). (b, e) Representative MR cross -section of mouse hind limbs corresponding to the area of histology sectioning (same hind limbs imaged at (a) 11 .1T and (d) 4.7T). Histology cross -sectio ns of the TA injected with S Gal labeled Rosa26 BM cell s are shown with hematoxylin and eosin (H E ) staining in (c) (40X) and with Prussian blue iron stain in (f) (40X).
70 Figure 4 5. In vivo labeling of Rosa26 t ibialis anterior (TA) muscles by intra -muscular delivery of S Gal /FAC. a: 3D GE images showed a strong hypo intense region (gr een) in the left TA due to S Gal -gal specific reaction ( n = 5 ). b: Increased permeability within the TA as evidenced by extracell ular wat er accumulation (blue). c: S Gal precipitant was easily observed within the left TA (bottom), Control (Top). d: Top: Nuclear fast red ( NFR ) stain at 5X magnification showed p resence of S Gal in left TA (left image), control TA without S -Gal stainin g (right image). (Bottom): Prussian blue staining of left TA at 5X and 40X magnification confirmed increased iron a ccumulation at the location of S Gal staining e: Implanted Rosa26 muscle tissu es were not visible before S Gal substrate administration. f: Following i ntra -muscular injection of S Gal /FAC, strong hypointense signal areas surrounded the site of muscle implantation (green) which is depicted in 3D (inset)) (n =3)
71 Table 4 1. Comparison of cellular contrast enhancing effect of various iron based contrast agent s MR contrast method I ron content/ labeled cell R 2 (Magnetic field strength) R 2 (Magnetic field strength) Reference # S Gal/F AC labeling of LacZ expressin g mouse BM cells 0.3 pg 11.5 s 1 (17.6T) (s) 46 s 1 (17.6T) (s) Transferrin/transf errin receptor up regulation in C17 (mouse neural progenitor cells) 0.03 pg 26 s 1 (7T) (p) 58 s 1 (7T) (p) (164) Induced bacterial MagA expression in 293FT cells 0.6 pg 17.5 s 1 (3T) (p) N/A (182) SPIO labeled HeLa cells 11.8 pg 87.2 s 1 (1T ) (s) N /A (183) SPIO labeled human cervical carcinoma (CG 4 ) cells 14.7 23.25 s 1 (1 T (s ) N/A (183) SPIO labeled mouse lymphocytes 1.82 pg 10.2 s 1 (1 T) (s) N/A (183) SPIO labeled human mesenchymal stem cells (MSCs) 30.1 pg 80.6 s 1 (1 T) (s) N/A (183) Methods of creating iron based MR contrast generally rely on increased intracellular iron accumulation H ere, the use of S Gal as a MR active genetic reporter system is c ompared to previously described methods of generating MR contrast Listed above are the methods used to label various cell populations, resulting intracellular iron accumulation and their effect on R2 or R2* relaxation rates. Relaxation measurements were made on cell suspensions (s) or cell pellets (p). Relaxation rate values tended to be slightly higher for measurements ma de on cell pellets due to increased cell densities within the imaged volume (N/A = not available).
72 CHAPTER 5 IN VIVO MRI OF BM DERIV ED MYELOID CELL RECRUITMENT TO GROWING TUMORS USING A MR AC TIVE GENE TIC REPORTER SYSTEM Introduction As discussed in Chapter 1, bone marrow ( BM ) derived myeloid cells have been implicated in several studies to influence tumor growth, angiogenesis and metas tasis (187189) These cells are recruited to the tumor where they contribute to the release of pro angiogenic factors that modulate the supply of nutrients to the tumor (187,188) Tumor associated macrophages (TAM) and Tie2 expressing monocytes (TEM) are two identified sub -populations of BM deri ved cells that have been shown to infiltrate certain tumors to induce the formation of blood vessels that sustain, facilitate growth and s pread of the tumor (187189) In order to assess and characterize the involvement and kinetics of BM cell contribution to t umor angiogenesis, growth and ultimately metastasis non invasive imaging methods could prove extremely useful for longitudinal tumor studies. Fluorescent imaging ( FLI ) and bioluminescent imaging (BLI ) have been proven to be extremely useful in vivo imagin g modalities (55,124) However, both methods, as discussed in chapter 1, are limited in providing high resolution three -dimensional ( 3D ) data sets Also, the use of fluorescent microscopy ( FLM ), is limited by shallow penetration depths (< 1 m m) and is inadequate for the non invasive imag ing of deeper structur es (124) While positron emission tomography ( PET ) and single photon emission com puted tomography ( SPECT ) demonstrate the ability to provide great molecular specificity and sensitivity, low resolution capability, rad iation exposure and handling of radiotracer s, are concerns that limit its use As presented in Chapter 3, ultra high fiel d magnetic resonance imaging ( MRI) provides both excellent resolution and high detection sensitivity of superparamagnetic iron oxide ( SPIO ) labeled cells in vivo. Indeed, previous studies, aimed at visualizing cells homing to tumors by MRI, have used cells th at were pre -
73 labeled with SPIOs prior to transplantation (190192) However, as discussed earlier, while pre labeling cells with SPIOs enables single cell detection in vivo it has the disadvantage s of providing potentially nonspecific contrast and suffers from label d ilu tion in dividing cells (138,159,193) In Chapter 4, a method of avoid ing these pitfalls was proposed by the novel use of a commercially available magnetic resonance (MR ) responsive L acZ stain. It was demonstrated that S G al could be used to read o ut LacZ expression in BM cells a nd that the sensitivity of this reporter was greatly enhanced at ultra high magnetic field strengths (17.6T) In order for this gene reporter system to be effective for longitudinal in vivo studies, the ability of S Gal to generate viable cell specific con trast upon systemic delivery needed to be addressed. Therefore the use of S Gal for non invasive track ing of LacZ expressing -, BM derived cell involvement in growing tumors will be examined next In V ivo Fluorescent Imaging C an D etect B M Derived Cell Recru itment to Growing Tumors In order to selectively visualize BM derived cell infiltration in tumors, chimeric mice with LacZ expressing myeloid cells were generated by lethal ly irradiating C57BL6 or FVB mice followed by Rosa26 or Tie2 -LacZ expressing BM ce ll transplant s respectively Three months following transplantation, flow activated cell sorting ( FACS ) analysis of peripheral blood ( PB ) cells in transplanted animals was performed to check for engraftment. U sing a fluorescent substrate f luo D galactopyranoside ) (FDG ), for detection of -gal) expression, t his analysis showed tha t approximately 39.6% of PB cells in Rosa26 transplanted animals expressed the LacZ gene product -gal. Of these, 10.5 % also expressed th e monocyte cell marker CD11b (Figure 5 1c) Animals transplanted with Tie2 -LacZ expressing BM cells showed only 5.6 % LacZ expre ssing cells in PB circulation (Figure 5 1d) gal expression has previously been reported using this system (194) and was observed as a small,
74 slight ly shifted population in wild type C57 BL6 animals (Figure 5 1b) Ten days following gal expression at the site of the growing tumor following retro orbital sinus ( ROS ) injection of FDG. Fluorescent signal originating from tumor infiltrating BM derived cells, was obs erved in Rosa26 transplanted animals (Figure 5 2 a, right), Tie2 LacZ transplanted animals (Figu re 5 2 b), but not in control C57 BL6 animals (Figure 5 2 a, left). LacZ expressing cells were on ly abundant enough to generate det ectable fluorescent signal s a t th e sites of growing tumors while contra -lateral tumor free hind limbs produced no detectable fluorescence Ultra High Field MRI Combined with a MR Active Gene tic R eporter Substrate C an Detect Intra -T umoral R ecruitment of B M Derived C ells Subsequent MRI an alysis of tumor bearing hind limbs showed a large change in signal inte nsity on pre versus post S Gal injection at specific locations w ithin the tumor mass (Figure 5 3 ). Rosa26 transplanted animal tumors exhibited numerous areas of specific negative contr ast (Figure 5 3 b), generated by the cleaved, MR active S Gal product, while control C57 BL6 animal tumors showed no visible change in contrast follow ing S Gal injection (Figure 5 3 c). T2weighted spin echo (SE) scans at 4.7T magnetic field strength clearly demonstrated the large extent of the growing tumors in both Rosa26 transplanted and control C57 BL6 animals (Figure 5 3 d, e). The extent of growing tumors (Figure 5 4a), as well as the involvement of BM derived LacZ expressing Rosa26 cells (Figure 5c, d, f) could easily be observed at 11.1T magnetic field strength (Figure 5 4) Tie2 -LacZ transplanted animal tumors showed much less, but still detectable, areas of negative contrast (Figure 5 5 b, d). The lesser contrast generation reflecting the much smaller pr evalence o f conditional Tie2 LacZ expressing cells versus Rosa26 cells that express LacZ ubiquitously This observed negative contrast appeared to be cleared within 48 hours post injection. Interestingly, MRI analysis of tumors prior to 1 week after tumor cell
75 implantation s howed no signs of cleaved S Gal product within the tumors. The lack of MR contrast generation prior to 1 week following tumor cell implantation may have be en caused by two reasons. Either BM derived cells had not been recruited in suffic ient numbers to the tumor, or the substrate was unable to reach the growing tumor due to an under developed blood supply to the tumor at e arlier time points. High resolution ex vivo imaging of excised tumors at a resolution of 303030 m3 provided addition al detail of the hypointense regions generated by recruited L acZ expressing cells (Figure 5 6 a c). Upon isolation and further magnification of the hypointense regions, small clusters and potential single cells could be resolved within these tumor hotspots (Figure 5 6 d). Post Imaging Histology of Intra -T umoral B M Cell R ecruitment Post imaging histology of Rosa26 transplanted animal tumors confirmed heavy involvement of LacZ expressing BM derived cells within avascular tumor hot spots which seemed to exhibit central areas of hypoxia and damage (Figure 5 7 a -d). These ar eas of LacZ positive BM derived cells responsible for negative MR contrast, also stained positive for the monocytic cell marker CD11b and macrophage marker F4/80 w hich suggests a possible TAM p henotype (Figure 5 7 g, h). This is in accordance with previous reports, where monocyte derived TAMs were observed to accumula te extensively in hypoxic areas due to a lack of functional blood vessels to sustain the growing tumor (74,80,195) Although f ew MECA32 positive tumor blood vessels were present in these avascul ar zones, there seemed to be a certain degree of co l ocalization between them and BM derived angiogenic monocyte s (Figure 5 7i). This may indicate the onset of an angiogenic switch within the tumor hot spot since it has been shown that upon arrival to thes e areas, TAMs respond to the hypoxic environment by upregu lating pro angiogenic factors that stimulate angiogenesis and induction of malignancy (85,86,196,197)
76 TEM s have relatively recent ly been suggested as a potential target for treating cancer (89,188) These cells are, in p art, characterized by its expression of the angiopoietin receptor Tie2. Except for Tie2, these cells also are believed to express the monocy te marker CD11b, F4/80 macrophage marker and a variety of other cell markers (187) Here TEMs were identified on tumor sections by conditional Tie2-LacZ (Figure 5 8 d), CD11b (Figure 5 8 b) and F4/80 (Figure 5 8 e) expression. These cells were observed perivascular to tumor b lood vessels in areas of ongoing angiogenesis as evidenced by MECA32 staining (Figure 5 8 c). This correlates to previous findings indicating that the perivascular location is ideal for stim ulating angiogenesis and metastasis (89) As opposed to the observations of ubiquito usly LacZ expressing BM cells, Tie2 -LacZ expressing cells mainly seemed to be recruited to sites less obviously affected by hypoxia, as seen by high tumor microvessel density, more homogenous tumor morphology and reduced amounts of TUNEL positive cells (Fig ure 5 8 a, f). The observed difference in locations of TAMs and TEMs, likely stems from a different mechanism of intra tumoral recruitment, since TEM recruitment has been su ggested to be modulated by angiopoietin 2, which is highly expressed by intra tumoral blood vessels (89,91,198) Apart from TAMs and TEMs, o ther cell types also known to infiltrate tumors include a number of cell types, such as: endothelial progenitor cells ( EPC ), circulating endothelial cells (CEC) and h ematopoietic stem and progenitor cells (HSC/HPC) (187) These cell types were not speci fically addressed in here though their contribution to the observed MR contrast generation, especially in the case of ubiquitous LacZ BM tran splants, cannot be excluded. However, these results demonstrate that S Gal as an MR active gene reporter exhibits sufficient sensitivity to detect viable LacZ -expressing BM derived cells, longitudinal ly in vivo.
7 7 Figure 5 1. BM engraftment check of peripheral blood in transplanted animals by f luor escein D -galactopyranoside) ( FDG ) and flow activated cell sorting ( FACS ) analysis. a: Wild type C57 BL6 PB cells without FDG labeling -gal expressing cells in circulation b: Wild type C57BL6 PB cells following FDG labeling showing weak endogenou g al expression as a slightly shifted population of cells c: Rosa26 transplanted animal PB cells labeled with -gal expressing -gal expressing cells also express monocytic CD11b (PE channel) d: Conditional T ie2 LacZ transplanted animal PB cells with FDG labeling -gal expressing cells, which correlates with the normally smaller prevalence of Tie2 expressing cells circulating in PB
78 Figure 5 2 Fluo rescent imaging of donor derived L acZ expressing BM cell recruitment to the growing tumor a: Strong fluorescent signal was generated following systemic FDG injection from the legs containing growing tumors (*) by infiltrating, Rosa26 derived LacZ express ing cells (n = 4 ). LacZ negative C57BL6 (BL6) control animals showed no fluorescent si gnal generation following FDG injection (n = 2) b: Animals transplanted with Tie2 LacZ expressing BM cells, also exhibited strong fluorescence emitted from tumor infiltr ating LacZ expressing cells (n = 4)
79 Figure 5 3 In vivo 17.6T three -dimensional ( 3D ) MRI of tumor infilt rating donor derived BM cells a: Rosa26 transplanted animal tumor (dotted line) prior to S Gal injection. Top: Sagit tal view, Bottom: Axial view. b: Following systemic S Gal injection, numerous negatively contrasting regions (arrowheads) are visible at sites of Rosa26 donor derived LacZ expressing cell infiltration (n = 4) c: Control BL6 animals showed no signs of specific negative contrast generat ion following S -Gal injection (n=2) The extent of the growing tumors in Rosa26 transplanted (d) and control BL6 (e) animals could be observed as regions of enhanced signal intensity at 4.7T using a T2 weighted SE scan sequence following S Gal injection.
80 Figure 5 4. Recruitment of BM derived cells detected at 11.1T magnetic field strength. a: Rosa26 transp lanted animal tumor (red ) growing in mouse hind limb prior to S Gal injection. Prior to S Gal injection, the extent of the growing tumor is observed a s an area of enhanced signal intensity (dotted lines) on SE scan sequences (b). Following systemic S -Gal delivery, n egatively contrasting regions ( arrows, arrowheads) are observed at sites of Rosa26 donor derived Lac Z expressing cell infiltration (n = 4) .( c : (SE), d : (3DGE), f : (GE)) that are absent in pre injection scans (b : (SE), e : (GE)).
81 Figure 5 5 Conditional Tie2 LacZ expression originating from tumor infiltrating donor derived cells, as visua lized by in vivo 17.6T MRI. a, c: Prior to S Gal injection few hypointense areas are visible within the large tumor (dotted line). Following systemic S Gal administration, regions of negative contrast were observed at sites of successful S Gal cleavage by recruited LacZ expressing cells (arrows) (n = 4) (b :(corona l view), d: (axial view)).
82 Figure 5 6 High resolution MRI of excis ed tumors. a: Strong hypointense signal area s on a T2* weighted GE scan indicates LacZ expressing cell presence (Top/left: Rosa26 transplanted animal tumo r, Bottom/right Control BL6) (n = 1) b: Rosa26 transplanted (Top left) and control BL6 (lower right) animal tumors imaged side by side with LacZ specific S Gal genera ted signal in white (3D maximum intensity projection (MIP ) ). c: Same 3D volume rendering but rotated and cropped to show the region of S Gal labeling. d: Magnified 3D surface rendering of the hypointense region visualized in (a), shows infiltrating LacZ expressing cells.
83 Figure 5 7 Tumor histology confirms heavy infiltration of BM derived monocytes a: Overview image of tumor and stroma clearly shows specific hot spots of LacZ expressing cells, morphologically similar to images obtained by MRI (X Gal, 5X). b: Hematoxylin and eosin ( H E ) staining of Rosa26 transplante d animal tumor hotspot (40X). c: Lac Z expressing (blue) BM derived cells are prominent withi n the same area (X -Gal, 40X). d: TUNEL staining (red) indicated that a level of hypoxia/necrosis is occurring in the area (40X). Control BL6 animal tumor sections show presence of a similar hotspot in (e) (HE, 40X), w ithout the presence of LacZ expression, as seen in (f), (X Gal, 40X). g: Prominent expression of LacZ (green) and CD11b (red) within hotspots (40X). h: Areas also stained for F4/ 80 (red), LacZ (green) (40X). i: LacZ expressing cells (green), occasionally l ocalized close to MECA32 positive endothelial cells (red) (40X).
84 Figure 5 8 Presence of BM derived T ie 2 expressing monocytes within tumor s a: H E of tumor region contain ing TEM. b: Tie2 Ab+ cells (green) within an area of CD11b expressing cells (Red ) (40X). c: Gal Ab+ cells (green) reside in close proximity to MECA32 expres sing endothelium (Red) (40X). d: Enzymatic X -Gal staining (arrowheads ) Gal (40X). e: Co Gal (green) and F4/80 (red) e xpress ion (40X). f: Few apoptotic cells are visible in the same region as evidenced by weak TUNEL stain (40X).
85 CHAPTER 7 DISCUSSION Earlier belief that magnetic resonance imaging ( MRI ) lacks great specificity and sensitivity has always be en regarded as the ma in limiting factor in using MRI for cellular in vivo imaging However, with the arrival of ultra high magnetic field strength magnets, especially for research purposes, these traditional weaknesses may not be as limiting as they once were thought to be. Th e advantage of using ultra high magnetic field strength MRI for detecting transplanted cells has been proposed earlier. For instance, u sing superparamagnetic iron oxide ( SPIO ) labeled embryonic stem cells that were imaged both in vitro and in vivo at 17.6T the authors determined that small amounts of labeled cells could easily be detected in the mouse brain at an isotropic resolution of 98 m following stereotactic injection (199) Though single cell detection at low magnetic field strength has been prev iousl y reported using SPIO labeled cells (141) th e use of higher magne tic field strengths (17.6T) enables the acquisition of much more detailed images of surrounding tissues as well as the detection of cells containing less or different forms of iron based contrast agents ( CAs ). The process of cell ho ming to the bone marrow ( BM ) has been described at lower magnetic field strengths In one case, human hematopoietic stem cells (HSCs) labeled with SPIOs were determined to be homing to t he liver, spleen and BM in mice based on the loss of signal of the ent ire target organ (153) In another case, SPIO labeled mesenchymal stem cells (MSCs ) were imaged within the BM of mice (117) In this case, t he authors circumvented the process of homing by injecting the labeled cells directly into the marrow cavity. While this study was elegantly performed using bioluminescent imaging (BLI) of transplanted cells to confirm the magnetic resonance ( MR ) findings, the resulting susceptibility artifacts from the injected cells affected the entire BM and eliminated single cell d etection as well as any spatial information of
86 the surrounding niche. Furthermore, direct injection of cells into the BM cavity may not accurately depict the normal seeding patterns that occur during homing thr ough an intact vascular system. As discussed and demonstrated in Chapter 3, BM provides unique challenges in detecti ng single cells. Transverse relaxation rates are extremely short in no rmal adult, or irradiated mouse BM (178) resulting in an increased difficulty in accurately distinguishing dark, SPIO labeled cells aga inst a normally dark background. Th e id eal way of dealing with this dilemma would be by using positive contrast generating agents for cell labeling. However to date, the only reported CA s sensitive enough to enable single cell detection in vivo are iron based CAs that normally do not provide positive contrast. Methods for generating positive contrast from iron based CAs exist (200) but could be proven difficult to use at this location because of the strong susceptibilit y effects generated by the bone itself. Other means of generating positive contrast for single cell tracking at u ltra -high magnetic field strengt hs have yet to be improved. To allow for the imaging of, for instance, darker BM at later time points following irradiation, non -sus ceptibility based CA s sensitive enough for single cell detection would be ideal. To this end, creating contrast by chemica l exchange saturation transfer (CEST) techniques are extremely interesting but so far seem to exhibit too low sensitivity for single cell tracking (137) O ther tissues, exhibiting longer transverse relaxation rates would be ideally su ited for single cell tracking experiments using iron oxide based CA s as reported previously (141) MR active gene reporter systems have recently become an area of much investigation. The advantages of these systems over the traditional ways of cell pre labeling are many. Such a system, as described in Chapters 4 and 5 could potentially be used to track stem cell migration and the functional involvement of their progeny over extended periods of time i n multiple experimental models. While SPIO labeled cancer cells could easily be observed migrating to the
87 brain by MRI in vivo in a model of cancer metastasis (191) The use of S Gal as a cellular in vivo gene reporter m ay potentially suffer from decreased detection sensitivity at lower magnetic field strengths, due to systemic delivery and the resulting dilution of the MR active substrate. On the other hand, it has distinct advantages over using cells pre labeled with SP IOs First, specificity is increased since theoretically only live cells expressing the LacZ transgene will generate contrast. Secondly, issues regarding label dilution within pre -labeled cells due to cell division and subsequent loss of contrast could eff ectively be avoided. Additionally, cells can be re labeled in vivo at subsequent time points to allow for longitudinal noninvasi ve studies. D ue to the fact that the iron based S Gal/LacZ reaction product seems to be cleared faster than SPIOs in vivo (193) the possibility of detecting false contr ast originating from resident macrophages taking up iron label from dead cells, could be reduced (159,193) Finally, specific promoters can be used to drive the gene expression and provide more specific information about the function of the cells of interest. However, in order to uti lize these advantages, great emphasis is put on the use of higher magnetic field strengths to maximize detection sensitivity allowing for the in vivo detection of small amounts of cells. Other methods of creating genetically specific contrast have been proposed previously and have been proven to be very promising (161-163,165,182) However, the use of LacZ has distinct advantages in that, numerous animal models utilizing the LacZ gene reporter has already been developed and together with S -Gal are readily available for use. Additionally, no visible side effects were observed following dail y intravenous injections of 10 mg/kg of S Gal over a period of 14 days In Chapter 5, the detection sensitiv ity of S Gal, following systemic injection, was validated using a model of myeloid cell invasion into growing tumors. BM derived myeloid cells in tumors represent a large amount of cells generating LacZ specific contrast. The use of conditional Tie2 -
88 LacZ e xpression further tested th e in vivo detection sensitivity by reducing the amount of cells generating the contrast. The large amounts of available LacZ expressing cells, together with the availability of leaky tumor vasculature, comprise an ideal situation for the use of this gene reporter system in vivo. Ultimately, it was shown that systemic administration of S Gal during early time points of tumor growth did not result in detectable contrast generation. This prompted the suspicion that the interaction between S -Gal and LacZ expressing cells was dependent on the level of intra -tumoral vascularization. Therefore, this model could serve as a valuable tool in visualizing BM derived myeloid cell involvement in growing tumors, duringand following the angiog enic switch. Potentially, this can be used to predict the onset of malignancy and metastasis as they have both been linked to the degree of intra tumoral TAM/TEM involvement and vascularization (196) Additional experiments aimed at elucidating the roles of BM derived cells in modulating angiogenesis, growth and metastasis of tumors, are poised to benefit from this system as a non invasive, longitudinal readout. This new applicat ion, of an already established gene reporter system presents novel and exciting possibilities for studying transgene expression noninvasively, using MRI Because of the wide spread use of the L acZ gene reporter system, there are many current biological mo dels that could directly benefit from utilizing this new methodology. The efficacy of combining ultra high field MRI with the LacZ gene reporter for real time in vivo imaging could prove to be a useful tool for longitudinal studies of rapidly dividing cell s in real time following transplantation, such as in: hematopoiesis recovery, hematopoietic niche remodeling, cancer metastasis, embryo development and tissue regeneration.
89 LIST OF REFERENCES 1. Wilson A, Trumpp A. Bone -marrow haemato poietic -stem -cell niches. Nat Rev Immunol 2006;6(2):93106. 2. Grove JE, Bruscia E, Krause DS. Plasticity of bone marrow -derived stem cells. Stem Cells 2004;22(4):487500. 3. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126(4):663676. 4. Muguruma Y, Yahata T, Miyatake H, Sato T, Uno T, Itoh J, Kato S, Ito M, Hotta T, Ando K. Reconstitution of the functional human hematopoietic microenvironment derived from huma n mesenchymal stem cells in the murine bone marrow compartment. Blood 2006;107(5):18781887. 5. Moore MA, Metcalf D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br J Haem atol 1970;18(3):279296. 6. Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 1996;86(6):897906. 7. Gekas C, Dieterlen Lievre F, Orkin SH, Mikkola HK. The placenta is a niche for hematopoietic stem cells. Dev Cell 2005;8(3):365375. 8. Mikkola HK, Orkin SH. The journey of developing hematopoietic stem cells. Development 2006;133(19):37333744. 9. Ema H, Nakauchi H. Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood 2000;95(7):22842288. 10. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A 1995;92(22):1030210306. 11. Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution. Cell 2000;100(1):157168. 12. Till JE, Mc CE. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 1961;14:213222. 13. Osawa M, Hanada K, Hamada H, Nakauchi H. Long -term l ymphohematopoietic reconstitution by a single CD34low/negative hematopoietic stem cell. Science 1996;273(5272):242245.
90 14. Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, Sharkis SJ. Multi organ, multi lineage engraftment by a single bone marrow -derived stem cell. Cell 2001;105(3):369377. 15. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241(4861):5862. 16. Bunting KD. ABC transporters as phenotypi c markers and functional regulators of stem cells. Stem Cells 2002;20(1):1120. 17. Goodell MA, McKinneyFreeman S, Camargo FD. Isolation and characterization of side population cells. Methods Mol Biol 2005;290:343352. 18. Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 2005;121(7):11091121. 19. Morrison SJ, Weissman IL. The long-term repopulating subset of hemat opoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994;1(8):661-673. 20. Christensen JL, Weissman IL. Flk 2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long term stem cells. Proc Natl Acad Sci U S A 2001;98(25):1454114546. 21. Yang L, Bryder D, Adolfsson J, Nygren J, Mansson R, Sigvardsson M, Jacobsen SE. Identification of Lin( -)Sca1(+)kit(+)CD34(+)Flt3 short term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 2005;105(7):27172723. 22. Adolfsson J, Mansson R, Buza -Vidas N, Hultquist A, Liuba K, Jensen CT, Bryder D, Yang L, Borge OJ, Thoren LA, Anderson K, Sitnicka E, Sasaki Y, Sigvardsson M, Jacobsen SE. Identification of Flt3+ lympho-myeloid stem cells lacking erythro megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 2005;121(2):295306. 23. Harris RG, Herzog EL, Bruscia EM, Grove JE, Van Arnam JS, Krause DS. Lack of a fusion requirement for development of bone marrow -derived epithelia. Science 2004;305(5680):9093. 24. Balsam LB, Wagers AJ, Christensen JL, Kofidis T, Weissman IL, Robbins RC. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 2004;428(6 983):668673. 25. Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science 1999;284(5417):11681170.
91 26. Grant MB, May WS, Caballero S, Brown GA, Guthrie SM, Mames RN, Byrne BJ, Vaught T, Spoerri PE, Peck AB, Scott EW. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002;8(6):607612. 27. Schofield R. The relationship between the sp leen colony -forming cell and the haemopoietic stem cell. Blood Cells 1978;4(12):7 25. 28. Moore KA, Lemischka IR. Stem cells and their niches. Science 2006;311(5769):18801885. 29. Kiel MJ, Morrison SJ. Maintaining hematopoietic stem cells in the vascular niche. Immunity 2006;25(6):862864. 30. Taichman RS. Blood and bone: two tissues whose fates are intertwined to create the hematopoietic stem -cell niche. Blood 2005;105(7):26312639. 31. Wilson A, Oser GM, Jaworski M, Blanco -Bose WE, Laurenti E, Adolphe C Essers MA, Macdonald HR, Trumpp A. Dormant and self renewing hematopoietic stem cells and their niches. Ann N Y Acad Sci 2007;1106:6475. 32. Lord BI, Testa NG, Hendry JH. The relative spatial distributions of CFUs and CFUc in the normal mouse femur. Blo od 1975;46(1):6572. 33. Gong JK. Endosteal marrow: a rich source of hematopoietic stem cells. Science 1978;199(4336):14431445. 34. Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, Ross J, Haug J, Johnson T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Li L. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003;425(6960):836841. 35. Taichman RS, Emerson SG. The role of osteoblasts in the hematopoietic microenvironment. Stem Cells 1998;16(1):715. 36. Calvi LM, Adams G B, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP, Schipani E, Divieti P, Bringhurst FR, Milner LA, Kronenberg HM, Scadden DT. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 2003;425(6960):841846. 37. Kinashi T, Springer TA Steel factor and c kit regulate cell -matrix adhesion. Blood 1994;83(4):10331038. 38. Kovach NL, Lin N, Yednock T, Harlan JM, Broudy VC. Stem cell factor modulates avidity of alpha 4 beta 1 and alpha 5 beta 1 integrins expressed on hematopoietic cell lin es. Blood 1995;85(1):159167. 39. Lyman SD, Jacobsen SE. c kit ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities. Blood 1998;91(4):11011134.
92 40. Miyazawa K, Williams DA, Gotoh A, Nishimaki J, Broxmeyer HE, Toyam a K. Membrane -bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c kit gene -encoded protein than its soluble form. Blood 1995;85(3):641649. 41. Nilsson SK, Johnston HM, Whitty GA, Williams B, Webb RJ, Denhardt DT Bertoncello I, Bendall LJ, Simmons PJ, Haylock DN. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells. Blood 2005;106(4):12321239. 42. Visnjic D, Kalajzic Z, Rowe DW, Katavic V, Lo renzo J, Aguila HL. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood 2004;103(9):32583264. 43. Li L, Milner LA, Deng Y, Iwata M, Banta A, Graf L, Marcovina S, Friedman C, Trask BJ, Hood L, Torok Storb B. The human hom olog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1. Immunity 1998;8(1):4355. 44. Varnum -Finney B, Xu L, Brashem Stein C, Nourigat C, Flowers D, Bakkour S, Pear WS, Bernstein ID. Pluripotent cytokine -dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000;6(11):12781281. 45. Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, Ito K, Koh GY, Suda T. Tie2/angiopoietin 1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004;118(2):149161. 46. Adams GB, Chabner KT, Alley IR, Olson DP, Szczepiorkowski ZM, Poznansky MC, Kos CH, Pollak MR, Brown EM, Scadden DT. Stem cell engraftment at the endosteal niche is spec ified by the calcium -sensing receptor. Nature 2006;439(7076):599603. 47. Fliedner TM, Graessle D, Paulsen C, Reimers K. Structure and function of bone marrow hemopoiesis: mechanisms of response to ionizing radiation exposure. Cancer Biother Radiopharm 200 2;17(4):405426. 48. Narayan K, Juneja S, Garcia C. Effects of 5 -fluorouracil or total -body irradiation on murine bone marrow microvasculature. Exp Hematol 1994;22(2):142148. 49. Tavassoli M. Structure and function of sinusoidal endothelium of bone marrow Prog Clin Biol Res 1981;59B:249256. 50. Huber TL, Kouskoff V, Fehling HJ, Palis J, Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 2004;432(7017):625630. 51. Nilsson SK, Johnston HM, Coverdale JA. Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches. Blood 2001;97(8):22932299.
93 52. Ohneda O, Fennie C, Zheng Z, Donahue C, La H, Villacorta R, Cairns B, Lasky LA. Hematopoietic stem cell maintena nce and differentiation are supported by embryonic aorta gonad-mesonephros region derived endothelium. Blood 1998;92(3):908919. 53. Li W, Johnson SA, Shelley WC, Ferkowicz M, Morrison P, Li Y, Yoder MC. Primary endothelial cells isolated from the yolk sac and para aortic splanchnopleura support the expansion of adult marrow stem cells in vitro. Blood 2003;102(13):43454353. 54. Li W, Johnson SA, Shelley WC, Yoder MC. Hematopoietic stem cell repopulating ability can be maintained in vitro by some primary en dothelial cells. Exp Hematol 2004;32(12):12261237. 55. Avecilla ST, Hattori K, Heissig B, Tejada R, Liao F, Shido K, Jin DK, Dias S, Zhang F, Hartman TE, Hackett NR, Crystal RG, Witte L, Hicklin DJ, Bohlen P, Eaton D, Lyden D, de Sauvage F, Rafii S. Chemo kine -mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med 2004;10(1):6471. 56. Rafii S, Mohle R, Shapiro F, Frey BM, Moore MA. Regulation of hematopoiesis by microvascular endotheliu m. Leuk Lymphoma 1997;27(56):375386. 57. Sipkins DA, Wei X, Wu JW, Runnels JM, Cote D, Means TK, Luster AD, Scadden DT, Lin CP. In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment. Nature 2005;435(7044):969973. 58. Kopp HG, Avecilla ST, Hooper AT, Shmelkov SV, Ramos CA, Zhang F, Rafii S. Tie2 activation contributes to hemangiogenic regeneration after myelosuppression. Blood 2005;106(2):505513. 59. Lapidot T, Petit I. Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 2002;30(9):973981. 60. Yin T, Li L. The stem cell niches in bone. J Clin Invest 2006;116(5):11951201. 61. McQuibban GA, Butler GS, Gong JH, Benda ll L, Power C, Clark Lewis I, Overall CM. Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell derived factor 1. J Biol Chem 2001;276(47):4350343508. 62. Levesque JP, Takamatsu Y, Nilsson SK, Haylock DN, Simmons PJ. Vascular cell a dhesion molecule 1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony -stimulating factor. Blood 2001;98(5):12891297. 63. Heissig B, Hattori K, Dias S, Friedrich M, Ferri s B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Rafii S. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP 9 mediated release of kit ligand. Cell 2002;109(5):625637.
94 64. Ponomaryov T, Peled A, Petit I, Taic hman RS, Habler L, Sandbank J, Arenzana Seisdedos F, Magerus A, Caruz A, Fujii N, Nagler A, Lahav M, Szyper -Kravitz M, Zipori D, Lapidot T. Induction of the chemokine stromal -derived factor 1 following DNA damage improves human stem cell function. J Clin I nvest 2000;106(11):13311339. 65. Lapidot T, Kollet O. The essential roles of the chemokine SDF 1 and its receptor CXCR4 in human stem cell homing and repopulation of transplanted immune -deficient NOD/SCID and NOD/SCID/B2m(null) mice. Leukemia 2002;16(10): 19922003. 66. Netelenbos T, van den Born J, Kessler FL, Zweegman S, Merle PA, van Oostveen JW, Zwaginga JJ, Huijgens PC, Drager AM. Proteoglycans on bone marrow endothelial cells bind and present SDF 1 towards hematopoietic progenitor cells. Leukemia 2003;17(1):175184. 67. Netelenbos T, Zuijderduijn S, Van Den Born J, Kessler FL, Zweegman S, Huijgens PC, Drager AM. Proteoglycans guide SDF 1 induced migration of hematopoietic progenitor cells. J Leukoc Biol 2002;72(2):353362. 68. Wright DE, Wagers AJ, Gul ati AP, Johnson FL, Weissman IL. Physiological migration of hematopoietic stem and progenitor cells. Science 2001;294(5548):19331936. 69. Mazo IB, GutierrezRamos JC, Frenette PS, Hynes RO, Wagner DD, von Andrian UH. Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1. J Exp Med 1998;188(3):465474. 70. Peled A, Grabovsky V, Habler L, Sandbank J, Arenzana Seisdedos F, Petit I, Ben -Hur H, Lapidot T, Alon R. The chemokine SDF 1 stimulates integrin -mediated arrest of CD34(+) cells on vascular endothelium under shear flow. J Clin Invest 1999;104(9):11991211. 71. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, Slav MM, Nagler A, Lider O, Alon R, Zipori D, Lapidot T. The chemokine SDF 1 activates the integrins LFA 1, VLA 4, and VLA 5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 2000;95(11):32893296. 72. Folkman J, Shing Y. A ngiogenesis. J Biol Chem 1992;267(16):1093110934. 73. Asahara T, Kawamoto A. Endothelial progenitor cells for postnatal vasculogenesis. Am J Physiol Cell Physiol 2004;287(3):C572579. 74. Vaupel P, Kelleher DK, Hockel M. Oxygen status of malignant tumors: pathogenesis of hypoxia and significance for tumor therapy. Semin Oncol 2001;28(2 Suppl 8):2935. 75. De Palma M, Naldini L. Role of haematopoietic cells and endothelial progenitors in tumour angiogenesis. Biochim Biophys Acta 2006;1766(1):159166.
95 76. Brigati C, Noonan DM, Albini A, Benelli R. Tumors and inflammatory infiltrates: friends or foes? Clin Exp Metastasis 2002;19(3):247258. 77. de Visser KE, Coussens LM. The inflammatory tumor microenvironment and its impact on cancer development. Contrib Micr obiol 2006;13:118137. 78. de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 2006;6(1):2437. 79. Schmid MC, Varner JA. Myeloid cell trafficking and tumor angiogenesis. Cancer Lett 2007;2 50(1):18. 80. Murdoch C, Giannoudis A, Lewis CE. Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood 2004;104(8):22242234. 81. Sica A, Schioppa T, Mantovani A, Allavena P. Tumour associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti cancer therapy. Eur J Cancer 2006;42(6):717727. 82. Sica A, Bronte V. Altered macrophage differentiation and immune dysfunction in tumor development J Clin Invest 2007;117(5):11551166. 83. Burke B, Tang N, Corke KP, Tazzyman D, Ameri K, Wells M, Lewis CE. Expression of HIF 1alpha by human macrophages: implications for the use of macrophages in hypoxia regulated cancer gene therapy. J Pathol 2002;196(2):204 212. 84. Talks KL, Turley H, Gatter KC, Maxwell PH, Pugh CW, Ratcliffe PJ, Harris AL. The expression and distribution of the hypoxia inducible factors HIF 1alpha and HIF 2alpha in normal human tissues, cancers, and tumor associated macrophages. Am J Pathol 2000;157(2):411421. 85. Lewis C, Murdoch C. Macrophage responses to hypoxia: implications for tumor progression and anti -cancer therapies. Am J Pathol 2005;167(3):627635. 86. Hagemann T, Wilson J, Burke F, Kulbe H, Li NF, Pluddemann A, Charles K Gordon S, Balkwill FR. Ovarian cancer cells polarize macrophages toward a tumor associated phenotype. J Immunol 2006;176(8):50235032. 87. Luo JL, Tan W, Ricono JM, Korchynskyi O, Zhang M, Gonias SL, Cheresh DA, Karin M. Nuclear cytokine activated IKKalp ha controls prostate cancer metastasis by repressing Maspin. Nature 2007;446(7136):690694. 88. De Palma M, Venneri MA, Roca C, Naldini L. Targeting exogenous genes to tumor angiogenesis by transplantation of genetically modified hematopoietic stem cells. Nat Med 2003;9(6):789795.
96 89. De Palma M, Venneri MA, Galli R, Sergi Sergi L, Politi LS, Sampaolesi M, Naldini L. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericy te progenitors. Cancer Cell 2005;8(3):211226. 90. Venneri MA, De Palma M, Ponzoni M, Pucci F, Scielzo C, Zonari E, Mazzieri R, Doglioni C, Naldini L. Identification of proangiogenic TIE2 expressing monocytes (TEMs) in human peripheral blood and cancer. Bl ood 2007;109(12):52765285. 91. Murdoch C, Tazzyman S, Webster S, Lewis CE. Expression of Tie 2 by human monocytes and their responses to angiopoietin2. J Immunol 2007;178(11):74057411. 92. Blann AD, Woywodt A, Bertolini F, Bull TM, Buyon JP, Clancy RM, Haubitz M, Hebbel RP, Lip GY, Mancuso P, Sampol J, Solovey A, Dignat George F. Circulating endothelial cells. Biomarker of vascular disease. Thromb Haemost 2005;93(2):228235. 93. Kopp HG, Ramos CA, Rafii S. Contribution of endothelial progenitors and proa ngiogenic hematopoietic cells to vascularization of tumor and ischemic tissue. Curr Opin Hematol 2006;13(3):175181. 94. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003;9(6):70 2 712. 95. Lyden D, Hattori K, Dias S, Costa C, Blaikie P, Butros L, Chadburn A, Heissig B, Marks W, Witte L, Wu Y, Hicklin D, Zhu Z, Hackett NR, Crystal RG, Moore MA, Hajjar KA, Manova K, Benezra R, Rafii S. Impaired recruitment of bone -marrow -derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7(11):11941201. 96. Nolan DJ, Ciarrocchi A, Mellick AS, Jaggi JS, Bambino K, Gupta S, Heikamp E, McDevitt MR, Scheinberg DA, Benezra R, Mittal V. Bone marrow -der ived endothelial progenitor cells are a major determinant of nascent tumor neovascularization. Genes Dev 2007;21(12):15461558. 97. Li B, Sharpe EE, Maupin AB, Teleron AA, Pyle AL, Carmeliet P, Young PP. VEGF and PlGF promote adult vasculogenesis by enhanc ing EPC recruitment and vessel formation at the site of tumor neovascularization. Faseb J 2006;20(9):14951497. 98. Okamoto R, Ueno M, Yamada Y, Takahashi N, Sano H, Suda T, Takakura N. Hematopoietic cells regulate the angiogenic switch during tumorigenesis. Blood 2005;105(7):27572763. 99. Donovan MJ, Lin MI, Wiegn P, Ringstedt T, Kraemer R, Hahn R, Wang S, Ibanez CF, Rafii S, Hempstead BL. Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabiliz ation. Development 2000;127(21):45314540.
97 100. Dolcetti L, Marigo I, Mantelli B, Peranzoni E, Zanovello P, Bronte V. Myeloid derived suppressor cell role in tumor -related inflammation. Cancer Lett 2008;267(2):216225. 101. Ardi VC, Kupriyanova TA, Deryugi na EI, Quigley JP. Human neutrophils uniquely release TIMP -free MMP 9 to provide a potent catalytic stimulator of angiogenesis. Proc Natl Acad Sci U S A 2007;104(51):2026220267. 102. Curiel TJ, Cheng P, Mottram P, Alvarez X, Moons L, EvdemonHogan M, Wei S, Zou L, Kryczek I, Hoyle G, Lackner A, Carmeliet P, Zou W. Dendritic cell subsets differentially regulate angiogenesis in human ovarian cancer. Cancer Res 2004;64(16):55355538. 103. McCourt M, Wang JH, Sookhai S, Redmond HP. Proinflammatory mediators st imulate neutrophil -directed angiogenesis. Arch Surg 1999;134(12):13251331; discussion 13311322. 104. Theoharides TC, Kempuraj D, Tagen M, Conti P, Kalogeromitros D. Differential release of mast cell mediators and the pathogenesis of inflammation. Immunol Rev 2007;217:6578. 105. Rando TA. Stem cells, ageing and the quest for immortality. Nature 2006;441(7097):10801086. 106. Askenasy N, Stein J, Farkas DL. Imaging approaches to hematopoietic stem and progenitor cell function and engraftment. Immunol Inves t 2007;36(56):713738. 107. Ciancio SJ, Coburn M, Hornsby PJ. Cutaneous window for in vivo observations of organs and angiogenesis. J Surg Res 2000;92(2):228232. 108. Dewhirst MW, Shan S, Cao Y, Moeller B, Yuan F, Li CY. Intravital fluorescence facilitat es measurement of multiple physiologic functions and gene expression in tumors of live animals. Dis Markers 2002;18(56):293311. 109. Halin C, Rodrigo Mora J, Sumen C, von Andrian UH. In vivo imaging of lymphocyte trafficking. Annu Rev Cell Dev Biol 2005; 21:581603. 110. Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake -Lye S, Fujisaki J, Cote D, Rowe DW, Lin CP, Scadden DT. Live animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 2009;457(7225):9296. 111. Xie Y, Yin T, W iegraebe W, He XC, Miller D, Stark D, Perko K, Alexander R, Schwartz J, Grindley JC, Park J, Haug JS, Wunderlich JP, Li H, Zhang S, Johnson T, Feldman RA, Li L. Detection of functional haematopoietic stem cell niche using real time imaging. Nature 2009;457(7225):97101. 112. Ntziachristos V, Bremer C, Weissleder R. Fluorescence imaging with near infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol 2003;13(1):195208.
98 113. Troy T, Jekic McMullen D, Sambucetti L, Rice B Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models. Mol Imaging 2004;3(1):923. 114. Rice BW, Cable MD, Nelson MB. In vivo imaging of light -emitting probes. J Biomed Opt 2001;6(4):432440. 115. Koransky ML, Ip TK, Wu S, Cao Y, Berry G, Contag C, Blau H, Robbins R. In vivo monitoring of myoblast transplantation into rat myocardium. J Heart Lung Transplant 2001;20(2):188189. 116. Contag CH, Bachmann MH. Advances in in vivo bioluminescence im aging of gene expression. Annu Rev Biomed Eng 2002;4:235260. 117. Mayer -Kuckuk P, Gade TP, Buchanan IM, Doubrovin M, Ageyeva L, Bertino JR, Boskey AL, Blasberg RG, Koutcher JA, Banerjee D. High-resolution imaging of bone precursor cells within the intact bone marrow cavity of living mice. Mol Ther 2005;12(1):3341. 118. Cao YA, Wagers AJ, Beilhack A, Dusich J, Bachmann MH, Negrin RS, Weissman IL, Contag CH. Shifting foci of hematopoiesis during reconstitution from single stem cells. Proc Natl Acad Sci U S A 2004;101(1):221226. 119. Lin Y, Molter J, Lee Z, Gerson SL. Bioluminescence imaging of hematopoietic stem cell repopulation in murine models. Methods Mol Biol 2008;430:295306. 120. Sweeney TJ, Mailander V, Tucker AA, Olomu AB, Zhang W, Cao Y, Negrin RS Contag CH. Visualizing the kinetics of tumor -cell clearance in living animals. Proc Natl Acad Sci U S A 1999;96(21):1204412049. 121. Ntziachristos V, Ripoll J, Wang LV, Weissleder R. Looking and listening to light: the evolution of whole -body photonic i maging. Nat Biotechnol 2005;23(3):313320. 122. Chaudhari AJ, Darvas F, Bading JR, Moats RA, Conti PS, Smith DJ, Cherry SR, Leahy RM. Hyperspectral and multispectral bioluminescence optical tomography for small animal imaging. Phys Med Biol 2005;50(23):5421 5441. 123. Schmidt GP, Schoenberg SO, Schmid R, Stahl R, Tiling R, Becker CR, Reiser MF, Baur Melnyk A. Screening for bone metastases: whole -body MRI using a 32 channel system versus dual -modality PET CT. Eur Radiol 2007;17(4):939949. 124. Contag CH. In vivo pathology: seeing with molecular specificity and cellular resolution in the living body. Annu Rev Pathol 2007;2:277305. 125. Gorantla S, Dou H, Boska M, Destache CJ, Nelson J, Poluektova L, Rabinow BE, Gendelman HE, Mosley RL. Quantitative magnetic resonance and SPECT imaging for macrophage tissue migration and nanoformulated drug delivery. J Leukoc Biol 2006;80(5):11651174.
99 126. Benveniste H, Blackband SJ. Translational neuroscience and magnetic resonance microscopy. Lancet Neurol 2006;5(6):536544. 127. Maronpot RR, Sills RC, Johnson GA. Applications of magnetic resonance microscopy. Toxicol Pathol 2004;32 Suppl 2:42 48. 128. Ciobanu L, Seeber DA, Pennington CH. 3D MR microscopy with resolution 3.7 microm by 3.3 microm by 3.3 microm. J Magn Reson 2002;158(12):178182. 129. Tyszka JM, Fraser SE, Jacobs RE. Magnetic resonance microscopy: recent advances and applications. Curr Opin Biotechnol 2005;16(1):9399. 130. Hoehn M, Kustermann E, Blunk J, Wiedermann D, Trapp T, Wecker S, Focking M, Arnold H, H escheler J, Fleischmann BK, Schwindt W, Buhrle C. Monitoring of implanted stem cell migration in vivo: a highly resolved in vivo magnetic resonance imaging investigation of experimental stroke in rat. Proc Natl Acad Sci U S A 2002;99(25):1626716272. 131. Smirnov P, Gazeau F, Beloeil JC, Doan BT, Wilhelm C, Gillet B. Single -cell detection by gradient echo 9.4 T MRI: a parametric study. Contrast Media Mol Imaging 2006;1(4):165174. 132. Beck B, Plant DH, Grant SC, Thelwall PE, Silver X, Mareci TH, Benveniste H, Smith M, Collins C, Crozier S, Blackband SJ. Progress in high field MRI at the University of Florida. Magma 2002;13(3):152157. 133. Caravan P, Ellison JJ, McMurry TJ, Lauffer RB. Gadolinium(III) Chelates as MRI Contrast Agents: Structure, Dynamics, an d Applications. Chem Rev 1999;99(9):22932352. 134. Vander Elst L, Zhang S, Sherry AD, Laurent S, Botteman F, Muller RN. Dy -complexes as high field T2 contrast agents: influence of water exchange rates. Acad Radiol 2002;9 Suppl 2:S297299. 135. Vander Elst L, Roch A, Gillis P, Laurent S, Botteman F, Bulte JW, Muller RN. Dy DTPA derivatives as relaxation agents for very high field MRI: the beneficial effect of slow water exchange on the transverse relaxivities. Magn Reson Med 2002;47(6):11211130. 136. Carav an P, Greenfield MT, Bulte JW. Molecular factors that determine Curie spin relaxation in dysprosium complexes. Magn Reson Med 2001;46(5):917922. 137. Geraldes CF, Laurent S. Classification and basic properties of contrast agents for magnetic resonance ima ging. Contrast Media Mol Imaging 2009;4(1):123. 138. Bulte JW, Kraitchman DL. Iron oxide MR contrast agents for molecular and cellular imaging. NMR Biomed 2004;17(7):484499.
100 139. Dunning MD, Kettunen MI, Ffrench Constant C, Franklin RJ, Brindle KM. Magne tic resonance imaging of functional Schwann cell transplants labelled with magnetic microspheres. Neuroimage 2006;31(1):172180. 140. Kraitchman DL, Bulte JW. Imaging of stem cells using MRI. Basic Res Cardiol 2008;103(2):105113. 141. Shapiro EM, Sharer K Skrtic S, Koretsky AP. In vivo detection of single cells by MRI. Magn Reson Med 2006;55(2):242249. 142. Modo M, Hoehn M, Bulte JW. Cellular MR imaging. Mol Imaging 2005;4(3):143164. 143. Dodd CH, Hsu HC, Chu WJ, Yang P, Zhang HG, Mountz JD, Jr., Zinn K Forder J, Josephson L, Weissleder R, Mountz JM, Mountz JD. Normal T -cell response and in vivo magnetic resonance imaging of T cells loaded with HIV transactivator -peptide -derived superparamagnetic nanoparticles. J Immunol Methods 2001;256(12):89105. 144. Hauger O, Frost EE, van Heeswijk R, Deminiere C, Xue R, Delmas Y, Combe C, Moonen CT, Grenier N, Bulte JW. MR evaluation of the glomerular homing of magnetically labeled mesenchymal stem cells in a rat model of nephropathy. Radiology 2006;238(1):200210. 145. Walczak P, Kedziorek DA, Gilad AA, Lin S, Bulte JW. Instant MR labeling of stem cells using magnetoelectroporation. Magn Reson Med 2005;54(4):769774. 146. Watson DJ, Walton RM, Magnitsky SG, Bulte JW, Poptani H, Wolfe JH. Structure specific pattern s of neural stem cell engraftment after transplantation in the adult mouse brain. Hum Gene Ther 2006;17(7):693704. 147. Arbab AS, Yocum GT, Wilson LB, Parwana A, Jordan EK, Kalish H, Frank JA. Comparison of transfection agents in forming complexes with fe rumoxides, cell labeling efficiency, and cellular viability. Mol Imaging 2004;3(1):2432. 148. Frank JA, Anderson SA, Kalsih H, Jordan EK, Lewis BK, Yocum GT, Arbab AS. Methods for magnetically labeling stem and other cells for detection by in vivo magnetic resonance imaging. Cytotherapy 2004;6(6):621625. 149. Arbab AS, Bashaw LA, Miller BR, Jordan EK, Bulte JW, Frank JA. Intracytoplasmic tagging of cells with ferumoxide s and transfection agent for cellular magnetic resonance imaging after cell transplantation: methods and techniques. Transplantation 2003;76(7):11231130. 150. Anderson SA, Glod J, Arbab AS, Noel M, Ashari P, Fine HA, Frank JA. Noninvasive MR imaging of ma gnetically labeled stem cells to directly identify neovasculature in a glioma model. Blood 2005;105(1):420425.
101 151. Baklanov DV, Demuinck ED, Thompson CA, Pearlman JD. Novel double contrast MRI technique for intramyocardial detection of percutaneously tra nsplanted autologous cells. Magn Reson Med 2004;52(6):14381442. 152. Daldrup -Link HE, Rudelius M, Oostendorp RA, Jacobs VR, Simon GH, Gooding C, Rummeny EJ. Comparison of iron oxide labeling properties of hematopoietic progenitor cells from umbilical cord blood and from peripheral blood for subsequent in vivo tracking in a xenotransplant mouse model XXX. Acad Radiol 2005;12(4):502510. 153. Daldrup -Link HE, Rudelius M, Piontek G, Metz S, Brauer R, Debus G, Corot C, Schlegel J, Link TM, Peschel C, Rummeny E J, Oostendorp RA. Migration of iron oxide -labeled human hematopoietic progenitor cells in a mouse model: in vivo monitoring with 1.5 T MR imaging equipment. Radiology 2005;234(1):197205. 154. Lee JH, Huh YM, Jun YW, Seo JW, Jang JT, Song HT, Kim S, Cho EJ, Yoon HG, Suh JS, Cheon J. Artificially engineered magnetic nanoparticles for ultra -sensitive molecular imaging. Nat Med 2007;13(1):9599. 155. Ottobrini L, Lucignani G, Clerici M, Rescigno M. Assessing cell trafficking by noninvasive imaging techniques: applications in experimental tumor immunology. Q J Nucl Med Mol Imaging 2005;49(4):361366. 156. Wang X, Rosol M, Ge S, Peterson D, McNamara G, Pollack H, Kohn DB, Nelson MD, Crooks GM. Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging. Blood 2003;102(10):34783482. 157. van den Bos EJ, Wagner A, Mahrholdt H, Thompson RB, Morimoto Y, Sutton BS, Judd RM, Taylor DA. Improved efficacy of stem cell labeling for magnetic resonance imaging studies by the use of ca tionic liposomes. Cell Transplant 2003;12(7):743756. 158. Kostura L, Kraitchman DL, Mackay AM, Pittenger MF, Bulte JW. Feridex labeling of mesenchymal stem cells inhibits chondrogenesis but not adipogenesis or osteogenesis. NMR Biomed 2004;17(7):513517. 159. Metz S, Bonaterra G, Rudelius M, Settles M, Rummeny EJ, Daldrup -Link HE. Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro. Eur Radiol 2004;14(10):18511858. 160. Weissleder R, Moore A, Mahmood U, Bhorade R, Be nveniste H, Chiocca EA, Basilion JP. In vivo magnetic resonance imaging of transgene expression. Nat Med 2000;6(3):351355. 161. Artemov D, Mori N, Okollie B, Bhujwalla ZM. MR molecular imaging of the Her 2/neu receptor in breast cancer cells using targete d iron oxide nanoparticles. Magn Reson Med 2003;49(3):403408.
102 162. Cohen B, Dafni H, Meir G, Harmelin A, Neeman M. Ferritin as an endogenous MRI reporter for noninvasive imaging of gene expression in C6 glioma tumors. Neoplasia 2005;7(2):109117. 163. Gen ove G, DeMarco U, Xu H, Goins WF, Ahrens ET. A new transgene reporter for in vivo magnetic resonance imaging. Nat Med 2005;11(4):450454. 164. Deans AE, Wadghiri YZ, Bernas LM, Yu X, Rutt BK, Turnbull DH. Cellular MRI contrast via coexpression of transferr in receptor and ferritin. Magn Reson Med 2006;56(1):5159. 165. Louie AY, Huber MM, Ahrens ET, Rothbacher U, Moats R, Jacobs RE, Fraser SE, Meade TJ. In vivo visualization of gene expression using magnetic resonance imaging. Nat Biotechnol 2000;18(3):3213 25. 166. Kodibagkar VD, Yu J, Liu L, Hetherington HP, Mason RP. Imaging beta galactosidase activity using 19F chemical shift imaging of LacZ gene -reporter molecule 2 -fluoro 4 nitrophenol beta D -galactopyranoside. Magn Reson Imaging 2006;24(7):959962. 167. Arbab AS, Yocum GT, Kalish H, Jordan EK, Anderson SA, Khakoo AY, Read EJ, Frank JA. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI. Blood 2004;104(4):12171223. 168. Arbab AS, Yocum GT, Rad AM, Khakoo AY, Fellowes V, Read EJ, Frank JA. Labeling of cells with ferumoxides protamine sulfate complexes does not inhibit function or differentiation capacity of hematopoietic or mesenchymal stem cells. NMR Biomed 2005;18(8):553559. 169. Proulx ST, Kwok E, You Z, P apuga MO, Beck CA, Shealy DJ, Calvi LM, Ritchlin CT, Awad HA, Boyce BF, Xing L, Schwarz EM. Elucidating bone marrow edema and myelopoiesis in murine arthritis using contrast-enhanced magnetic resonance imaging. Arthritis Rheum 2008;58(7):20192029. 170. Ja mes AJ, GB), Armstrong, Lyle (Ashington, GB); IDG (UK) Limited (Manchester, GB), assignee. Esculetin derivatives. United States. 1999. 171. Heuermann K, Cosgrove J. S Gal: an autoclavable dye for color selection of cloned DNA inserts. Biotechniques 2001;30(5):11421147. 172. Kishigami S, Komatsu Y, Takeda H, Nomura -Kitabayashi A, Yamauchi Y, Abe K, Yamamura K, Mishina Y. Optimized beta -galactosidase staining method for simultaneous detection of endogenous gene expression in early mouse embryos. Genesis 2006;44(2):5765. 173. Riemer J, Hoepken HH, Czerwinska H, Robinson SR, Dringen R. Colorimetric ferrozine based assay for the quantitation of iron in cultured cells. Anal Biochem 2004;331(2):370375.
103 174. Nolan GP, Fiering S, Nicolas JF, Herzenberg LA. Fluores cence activated cell analysis and sorting of viable mammalian cells based on beta D -galactosidase activity after transduction of Escherichia coli lacZ. Proc Natl Acad Sci U S A 1988;85(8):26032607. 175. Gong H, Zhang B, Little G, Kovar J, Chen H, Xie W, S chutz Geschwender A, Olive DM. beta Galactosidase activity assay using far -red -shifted fluorescent substrate DDAOG. Anal Biochem 2009;386(1):5964. 176. Chute JP. Stem cell homing. Curr Opin Hematol 2006;13(6):399406. 177. Daldrup HE, Link TM, Blasius S, Strozyk A, Konemann S, Jurgens H, Rummeny EJ. Monitoring radiation induced changes in bone marrow histopathology with ultra -small superparamagnetic iron oxide (USPIO) -enhanced MRI. J Magn Reson Imaging 1999;9(5):643652. 178. Sugimura H, Kisanuki A, Tamura S, Kihara Y, Watanabe K, Sumiyoshi A. Magnetic resonance imaging of bone marrow changes after irradiation. Invest Radiol 1994;29(1):3541. 179. Yao B, Li TQ, Gelderen P, Shmueli K, de Zwart JA, Duyn JH. Susceptibility contrast in high field MRI of human brain as a function of tissue iron content. Neuroimage 2009;44(4):12591266. 180. Mills PH, Ahrens ET. Theoretical MRI contrast model for exogenous T2 agents. Magn Reson Med 2007;57(2):442447. 181. Gossuin Y, Gillis P, Muller RN, Hocq A. Relaxation by clus tered ferritin: a model for ferritin induced relaxation in vivo. NMR Biomed 2007;20(8):749756. 182. Zurkiya O, Chan AW, Hu X. MagA is sufficient for producing magnetic nanoparticles in mammalian cells, making it an MRI reporter. Magn Reson Med 2008;59(6): 12251231. 183. Frank JA, Miller BR, Arbab AS, Zywicke HA, Jordan EK, Lewis BK, Bryant LH, Jr., Bulte JW. Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents. Radiology 2003;228(2):480487. 184. Simon GH, Bauer J, Saborovski O, Fu Y, Corot C, Wendland MF, Daldrup -Link HE. T1 and T2 relaxivity of intracellular and extracellular USPIO at 1.5T and 3T clinical MR scanning. Eur Radiol 2006;16(3):738745. 185. Edison ES, Bajel A, Chandy M. Iron homeostasis: new players, newer insights. Eur J Haematol 2008;81(6):411424. 186. Ganz T. Cellular iron: ferroportin is the only way out. Cell Metab 2005;1(3):155157. 187. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the p romotion of tumour angiogenesis. Nat Rev Cancer 2008;8(8):618631.
104 188. Wels J, Kaplan RN, Rafii S, Lyden D. Migratory neighbors and distant invaders: tumor associated niche cells. Genes Dev 2008;22(5):559574. 189. Kimura YN, Watari K, Fotovati A, Hosoi F Yasumoto K, Izumi H, Kohno K, Umezawa K, Iguchi H, Shirouzu K, Takamori S, Kuwano M, Ono M. Inflammatory stimuli from macrophages and cancer cells synergistically promote tumor growth and angiogenesis. Cancer Sci 2007;98(12):20092018. 190. Hart LS, El D eiry WS. Invincible, but not invisible: imaging approaches toward in vivo detection of cancer stem cells. J Clin Oncol 2008;26(17):29012910. 191. Heyn C, Ronald JA, Ramadan SS, Snir JA, Barry AM, MacKenzie LT, Mikulis DJ, Palmieri D, Bronder JL, Steeg PS, Yoneda T, MacDonald IC, Chambers AF, Rutt BK, Foster PJ. In vivo MRI of cancer cell fate at the single -cell level in a mouse model of breast cancer metastasis to the brain. Magn Reson Med 2006;56(5):10011010. 192. Bernas LM, Foster PJ, Rutt BK. Magnetic resonance imaging of in vitro glioma cell invasion. J Neurosurg 2007;106(2):306313. 193. Chen IY, Greve JM, Gheysens O, Willmann JK, Rodriguez -Porcel M, Chu P, Sheikh AY, Faranesh AZ, Paulmurugan R, Yang PC, Wu JC, Gambhir SS. Comparison of optical biolum inescence reporter gene and superparamagnetic iron oxide MR contrast agent as cell markers for noninvasive imaging of cardiac cell transplantation. Mol Imaging Biol 2009;11(3):178187. 194. Fiering SN, Roederer M, Nolan GP, Micklem DR, Parks DR, Herzenberg LA. Improved FACS -Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs. Cytometry 1991;12(4):291301. 195. Ohno S, Ohno Y, Suzuki N, Kamei T, Koike K, Inagawa H, Kohchi C, Soma G, Inoue M. Correlation of histological localization of tumor associated macrophages with clinicopathological features in endometrial cancer. Anticancer Res 2004;24(5C):33353342. 196. Lin EY, Li JF, Gnatovskiy L, Deng Y, Zhu L, Grzesik DA, Qian H, Xue XN, Pollard JW. Macrophages r egulate the angiogenic switch in a mouse model of breast cancer. Cancer Res 2006;66(23):1123811246. 197. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, Pollard JW. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer mo del provides a reliable model for human diseases. Am J Pathol 2003;163(5):21132126. 198. Gu J, Yamamoto H, Ogawa M, Ngan CY, Danno K, Hemmi H, Kyo N, Takemasa I, Ikeda M, Sekimoto M, Monden M. Hypoxia induced up regulation of angiopoietin2 in colorectal cancer. Oncol Rep 2006;15(4):779783.
105 199. Stroh A, Faber C, Neuberger T, Lorenz P, Sieland K, Jakob PM, Webb A, Pilgrimm H, Schober R, Pohl EE, Zimmer C. In vivo detection limits of magnetically labeled embryonic stem cells in the rat brain using high-fi eld (17.6 T) magnetic resonance imaging. Neuroimage 2005;24(3):635645. 200. Cunningham CH, Arai T, Yang PC, McConnell MV, Pauly JM, Conolly SM. Positive contrast magnetic resonance imaging of cells labeled with magnetic nanoparticles. Magn Reson Med 2005; 53(5):9991005.
106 BIOGRAPHICAL SKETCH Niclas Emanuel Bengtsson was born on December 1978, in Boras, Sweden. After graduating from Sven Eriksson Gymnasiet (High School) in 1994, he enlisted in military service as a Mine Clearance Diver in The Royal Swedis h Navy. Following the completion of military service he attended The Royal Institute of Technology in Stockholm, Sweden, where he earned his Master of Science degree in chemical/b iomedical engineering in December of 2003. Niclas enrolled into the Universi ty of Floridas College of Medicine Interdisciplinary Program in Biomedical Sciences in August of 2004. After joining the laboratory of Dr Edward Scott, Niclas began his research of investigating the use Magnetic Resonance Imaging for non -invasive tracking of bone marrow derived cells in a number of different transplantation models. His scientific achievements include a firstauthor article published in Magnetic Resonance in Medicine in October of 2009, a secondauthor article published Chemistry of Materia ls in October 2008 and two additional first author articles currently in submission. He is also listed as a co inventor on two pending patent applications and presented parts of his work at the annual International Society for Magnetic Resonance Imaging in Medicine (ISMRM) meeting in Toronto, Canada, in May 2008.