METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISIONOFBIOLOGY CALIFORNIAINSTITUTEOFTECHNOLOGY PASADENA,CALIFORNIA FOUNDINGEDITORS Sidney P. Colowick and Nathan O. Kaplan Preface As these volumes were being completed, American Paul C. Lauterbur and Briton Sir Peter Mansfield won the 2003 Nobel Prize for medicine for discoveriesleadingtothedevelopmentofMRI. The Washington Post story on October 6, 2003 announced the accolade, noting: ‘‘Magnetic resonance imaging, or MRI, has become a routine method for medical diagnosis and treatment. It is used to examine almost all organs withoutneedforsurgery,butisespeciallyvaluablefordetailedexaminationof thebrain and spinal cord.’’ Unfortunately,the articleoverlooked thegrowing usefulnessofthistechniqueinbasicresearch. MRI, along with other imaging methods, has made it possible to glance inside the living system. For patients, this may obviate the need for surgery; for researchers, it becomes a noninvasive method that enables the model systemstocontinue‘‘doingwhattheydo’’withoutbeingdisturbed.Thevalue and potential of these techniques is enormous, and that is why these once clinicalmethodsarefindingtheirwaytothelaboratory. Authors have been selected based on research contributions in the area about which they have written and based on their ability to describe their methodologicalcontributionsinaclearandreproducibleway.Theyhavebeen encouragedtomakeuseofgraphicsandcomparisonstoothermethods,andto providetricksandapproachesthatmakeitpossibletoadaptmethodstoother systems. The editor wants to express appreciation to the contributors for providing theircontributionsinatimelyfashion,tothesenioreditorsforguidance,andto thestaffatAcademicPressforhelpfulinput. P. Michael Conn xi Contributors to Volume 386 Articlenumbersareinparenthesesandfollowingthenamesofcontributors. Affiliationslistedarecurrent. Ellen Ackerstaff (1), Department of Kishore K. Bhakoo (14), Stem Cell Ima- Radiology, The Johns Hopkins Univer- ging Group, MRC Clinical Sciences sity School of Medicine, Baltimore, Centre, Faculty of Medicine, Imperial Maryland21205 College London, Hammersmith Hospi- tal,LondonW120HS,UnitedKingdom Shivani Agarwal (2), Department of NeurologicalSurgery,ColumbiaUniver- ZaverM.Bhujwalla(1),Departmentof sity,NewYork,NewYork10032 Radiology, The Johns Hopkins Univer- sity School of Medicine, Baltimore, Noam Alperin (16), Physiologic Imaging Maryland21205 andModelingLab,DepartmentofRadi- ology, The University of Illinois at David A. Bluemke (5), Department of Chicago,Chicago,Illinois60612 Radiology, Johns Hopkins Hospital, The Johns Hopkins University School CarolynJ.Anderson(11),Mallinckrodt ofMedicine,Baltimore,Maryland21287 InstituteofRadiology,WashingtonUni- versity School of Medicine, St. Louis, Jeff W. M. Bulte (13), Department of Missouri63110 Radiology and Institute for Cell Engi- neering, The Johns Hopkins University Ali S. Arbab (13), Laboratory of Diag- SchoolofMedicine,Baltimore,Maryland nostic Radiology Research, Experimen- 21205-2195 tal Neuroimaging Section, National E.SanderConnolly,Jr.(2),Department InstitutesofHealth,Bethesda,Maryland ofNeurologicalSurgery,ColumbiaUni- 20892 versity,NewYork,NewYork10032 DmitriArtemov(1),DepartmentofRadi- I. Jane Cox (14), Imaging Sciences De- ology, The Johns Hopkins University partment,MRCClinicalSciencesCentre, SchoolofMedicine,Baltimore,Maryland Imperial College London, Hammer- 21205 smith Hospital, London W12 0HS, MarkD.Bednarski(10),LucasMRIRe- UnitedKingdom searchCenter,DepartmentofRadiology, AnthonyL.D’Ambrosio(2),Department StanfordUniversity,Stanford,California, ofNeurologicalSurgery,ColumbiaUni- 94305-5488 versity, New York, New York 10032- 2699 Jimmy D. Bell (14), Molecular Imaging Group, MRC Clinical Sciences Centre, MilindY.Desai(5),DivisionofCardiol- ImperialCollegeLondon,Hammersmith ogy, Department of Internal Medicine, Hospital, London W12 0HS, United The Johns Hopkins University School Kingdom ofMedicine,Baltimore,Maryland21287 vii viii contributors to volume 386 T. Douglas (13), Department of Chemis- Joa˜oA.Lima(5),DivisionofCardiology, try and Biochemistry, Montana State Department of Internal Medicine, The University,Bozeman,Montana59717 Johns Hopkins University School of Medicine,Baltimore,Maryland21287 J.A.Frank(13),LaboratoryofDiagnos- tic Radiology Research, Experimental Pengnian Charles Lin (4), Department Neuroimaging Section, National Insti- of Radiation Oncology, Vanderbilt tutes of Health, Bethesda, Maryland University Medical Center, Nashville, 20892 Tennessee37232 BarjorGimi(1),DepartmentofRadiology, Hanli Liu (17), Biomedical Engineering The Johns Hopkins University School Program, The University of Texas at ofMedicine,Baltimore,Maryland21205 Arlington,Arlington,Texas76019 Kristine Glunde (1), Department of Jia Lu (8), Defence Medical Research Institute,Singapore117510 Radiology, The Johns Hopkins Univer- sity School of Medicine, Baltimore, Mark F. Lythgoe (6), RCS Unit of Bio- Maryland21205 physics, Institute of Child Health, Uni- versityCollegeLondon,LondonWC1N Yueqing Gu (17), Biomedical Engineer- 1EH,UnitedKingdom ing Program, The University of Texas atArlington,Arlington,Texas76019 WilliamJ.Mack(2),DepartmentofNeu- rological Surgery, Columbia University, Samira Guccione (10), Lucas MRI Re- NewYork,NewYork10032 search Center, Department of Radiol- ogy, Stanford University School of Pasquina Marzola (7), Anatomy and Medicine, Stanford, California 94305- Histology Section, Department of Mor- 5488 phologicalandBiomedicalSciences,Uni- versityofVerona,Verona37134 Italy ChienHo(3),PittsburghNMRCenterfor Biomedical Research, Department of Ralph P. Mason (17, 18), Department of Radiology, The University of Texas Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania Southwestern Medical Center at Dallas, Dallas,Texas75390-9058 15213 J.Mocco(2),DepartmentofNeurological Lan Jiang (18), The University of Texas Surgery,ColumbiaUniversity,NewYork, Southwestern Medical Center at Dallas, NewYork10032 Dallas,Texas75390 ShabbirMoochhala(8),DefenceMedical Jae G. Kim (17), Biomedical Engineering ResearchInstitute,Singapore117510 Program, The University of Texas at Arlington,Arlington,Texas76019 Arvind P. Pathak (1), Department of Radiology, The Johns Hopkins Univer- RyanG.King(2),DepartmentofNeuro- sity School of Medicine, Baltimore, logical Surgery, Columbia University, Maryland21205 NewYork,NewYork10032 Richard L. Roberts (4), Department of KincC.P.Li(10),DepartmentofRadiol- Pathology, Vanderbilt-Ingram Cancer ogy, Stanford University, Stanford, CA Center, Vanderbilt University Medical 94305-5488 Center,Nashville,Tennessee37232 contributors to volume386 ix KazuyaSato(3),PittsburghNMRCenter John S. Thornton (6), Lysholm Depart- for Biomedical Research, Department ment of Neuroradiology, National Hos- ofBiologicalSciences,CarnegieMellon pital for Neurology and Neurosurgery, University, Pittsburgh, Pennsylvania UCLH NHS Trust, London WC1N 15213 3BG,UnitedKingdom Andrea Sbarbati (7), Anatomy and Louise van der Weerd (6), RCS Unit of Histology Section, Department of Mor- Biophysics, Institute of Child Health, phological and Biomedical Sciences, University College London, London University of Verona, Verona, Italy WC1N1EH,UnitedKingdom 37134 HuaWu(9),DepartmentofNuclearMed- Daniel M. Sforza (15), Department icine, Tongji Hospital, Tongji Medical of Molecular and Medical Pharmaco- College,HuazhongUniversityofScience logy, UCLA School of Medicine, Los andTechnology,Wuhan430030,China Angeles,California90095-1735 Yi-JenL.Wu(3),PittsburghNMRCenter Desmond J. Smith (15), Department forBiomedicalResearch,Departmentof of Molecular and Medical Pharmaco- Biological Sciences, Carnegie Mellon logy, UCLA School of Medicine, Los University, Pittsburgh, Pennsylvania Angeles,California90095-1735 15213 PeterM.Smith-Jones(12),NuclearMed- Qing Ye (3), Pittsburgh NMR Center for icineService,DepartmentofRadiology, Biomedical Research, Department of Memorial Sloan-Kettering Cancer Cen- Biological Sciences, Carnegie Mellon ter,NewYork,NewYork10021 University, Pittsburgh, Pennsylvania DavidB.Solit(12),DepartmentofMed- 15213 icine, Memorial Sloan-Kettering Cancer XuemeiZhang(9),DepartmentofNucle- Center,NewYork,NewYork10021 ar Medicine, Tongji Hospital, Tongji Michael E. Sughrue (2), Department of Medical College, Huazhong University NeurologicalSurgery,ColumbiaUniver- of Science and Technology, Wuhan sity,NewYork,NewYork10032 430030,China Xiankai Sun (11), Mallinckrodt Institute Zhenwei Zhang (9), Department of Nu- of Radiology, Washington University clear Medicine, Tongji Hospital, Tongji SchoolofMedicine,St.Louis,Missouri Medical College, Huazhong University 63110 of Science and Technology, Wuhan SimonD.Taylor-Robinson(14),Depart- 430030,China ment of Medicine, Imperial College London, Hammersmith Hospital, Dawen Zhao (18), The University of LondonW120HS,UnitedKingdom Texas Southwestern Medical Center at Dallas,Dallas,Texas75390 David L. Thomas (6), Wellcome Trust High Field MR Research Laboratory, Ming Zhao (9), Department of Nuclear Department of Medical Physics and Medicine,TongjiHospital,TongjiMed- Bioengineering, University College ical College, Huazhong University of London, London WC1E6JA, United Science and Technology, Wuhan Kingdom 430030,China [1] molecularand functional imagingof cancer 3 [1] Molecular and Functional Imaging of Cancer: Advances in MRI and MRS By Arvind P. Pathak, Barjor Gimi, Kristine Glunde, Ellen Ackerstaff, Dmitri Artemov, and Zaver M. Bhujwalla Introduction Cancer is a disease that exhibits a degree of multiplicity and redun- dancy of pathways almost protean in nature. To understand and exploit molecular pathways in cancer for therapeutic strategies, it is essential not onlytodetectandimagetheexpressionofthese pathways, butalso tode- termine the impact of this expression on function at the cellular level, as wellaswithinthecomplexsystem, whichis atumor.Multiparametricmo- lecularandfunctionalimagingtechniqueshaveseveralkeyrolestoplayin cancertreatment,suchasrevealingkeytargetsfortherapy,visualizingde- livery of the therapy, and assessing the outcome of treatment. As a tech- nique, magnetic resonance (MR) has a formidable array of capabilities to characterize function. Noninvasive multinuclear magnetic resonance im- aging (MRI) and MR spectroscopic imaging (MRSI) provide a wealth of spatial and temporal information on tumor vasculature, metabolism, and physiology.MRisthereforeparticularlyapplicabletoinvestigatingacom- plex disease such as cancer. Several of the MRI techniques are also trans- latable into the clinic, and are therefore compatible with ‘‘bench to bedside’’applications. Tumor vasculature plays an important role in growth, treatment, and metastatic dissemination. The first section in this chapter therefore de- scribes the use of MRI techniques and the underlying assumptions and mechanisms in characterizing tumor vasculature. Recent advances in the development of targeted contrast agents have significantly increased the versatilityofMRformolecularimaging.AlthoughMRtechniquesprovide a wealth of structural and functional information, MR suffers from poor sensitivity.Thesecondsectiondiscussestheuseoftargetedcontrastagents and amplification strategies to increase the sensitivity of detection of mo- lecular targets in MR molecular imaging of cancer. Technical strategies to improve the signal to noise ratio (SNR) for applications of MR mi- croscopyincancerarealsoincludedinthissection.BecauseMRspectros- copy(MRS)andMRSIprovideinformationonmetabolismandpH,MRS applications in cancer are reviewed in the third section. One of the most exciting aspects of MR is the ability to perform multiparametric imaging. Copyright2004,ElsevierInc. Allrightsreserved. METHODSINENZYMOLOGY,VOL.386 0076-6879/04$35.00 4 disease models [1] In the fourth section, we present two examples of the use of multipara- metricimaginginunderstandingcancercellinvasionandincharacterizing the relationship between tumor vasculature and metabolism. Vascular Imaging of Tumors with MRI MRI techniques can be used to characterize several aspects of tumor vasculature. Tumor vasculature is typified by structural and functional anomalies that include alterations in hemodynamics, blood rheology, permeability,anddrainage,andplaysacriticalroleincancergrowth,treat- ment,andmetastasis.VascularMRmethodsarethereforeusefulincancer treatment and management. An overview of the endogenous and exoge- nousMRcontrastmechanismsutilizedincharacterizingtumorvasculature is presented inthis section. MR Relaxation Mechanisms and the Basis of Contrast Everycontrastmechanismforprobingthetumorvasculature,including the use of exogenous MR contrast agents, is in some way a result of changes in the MR signal intensity brought about by changes in tissue re- laxation times (T , T , or T *). Briefly, T , the spin–lattice or longitudinal 1 2 2 1 relaxationtime,isthetimeconstantthatcharacterizestheexponentialpro- cessbywhichthemagnetizationreturnsor‘‘relaxes’’toitsequilibriumpo- sition. It does so by exchanging energy with its surroundings, or lattice, at theLarmorfrequency.T relaxationoccursatthemolecularlevelthrough 1 several pathways, including interactions between protons in tissue water and those on macromolecules or proteins, and by interactions with para- magneticsubstances(i.e.,substanceswithunpairedelectronsintheirouter- most shells). T -based MR contrast results from differences in T 1 1 dominating the MR signal intensity. For example, tissues with short T s 1 (such as fat) appear bright in T -weighted MRI, since the transverse mag- 1 netizationrecoverstoequilibriumrapidlycomparedwithtissueswithlong T s (such ascerebrospinal fluid). 1 Microscopic magnetic field heterogeneities inthe main field, aswell as variations in local magnetic susceptibility due to the physiologic microen- vironment,causespinscontributingtothetransversemagnetizationtolose phase coherence. The process through which this occurs is known as T * 2 relaxation.Thelossintransversecoherenceattributabletostaticmagnetic fieldheterogeneitiescanberecoveredusingaspin–echosequenceorare- focusing pulse. However, as protons diffuse through the microscopic field inhomogeneities, they also lose phase coherence due to their Brownian random walks through the magnetic field gradients, which result in phase [1] molecularand functional imagingof cancer 5 dispersionthatcannotbereversedbytheapplicationofarefocusingpulse. ThisprocessisknownasT relaxation.InT -weightedMRimages,tissues 2 2 with short T s, such as the liver, appear dark due to the rapid decay of 2 transverse magnetization compared with those with long T s, such as fat. 2 Similarly, in T *-weighted images, regions with large susceptibility gradi- 2 ents, such as air–tissue interfaces of the inner ear or orbits of the eye, or large veins carryingdeoxygenated blood, appear hypointense. In general, the addition of a paramagnetic solute causes an increase in the 1/T and 1/T of solvent nuclei. The diamagnetic and paramagnetic 1 2 contributions to the relaxation rates of such solutions are additive and areexpressed as1: (cid:1) (cid:2) (cid:1) (cid:2) (cid:1) (cid:2) 1 1 1 ¼ þ i¼1;2 (1) Ti Ti Ti obs d p where(1/Ti) istheobservedsolventrelaxationrateinthepresenceofa obs paramagnetic species (e.g., contrast agent), (1/Ti) is the (diamagnetic) d solvent relaxation rate in the absence of a paramagnetic species, and (1/Ti) representstheadditionalparamagneticcontribution.Intheabsence p of any solute–solute interactions, the solvent relaxation rates (in solution) are linearly dependent on the concentration of the paramagnetic species [M], and if (1/Ti) or the relaxivity R, is defined as the slope of this i dependencein mM(cid:3)1 s(cid:3)1, we may write Eq. (1)as: (cid:1) (cid:2) (cid:1) (cid:2) 1 1 ¼ þR½M(cid:5) i¼1;2 (2) i Ti Ti obs d All molecules, large and small, are in a constant state of motion, tum- bling and colliding with other molecules. Intramolecular motion, as well as interaction with nearby molecules, produces fluctuations in the local magnetic field experienced by a proton. It turns out that these magnetic interactions can promote both T and T relaxation, but whether they do 1 2 so depends on the rate at which their magnetic fields fluctuate. For example,asmallmoleculesuchaswatermovesquickly,sothatitproduces rapid magnetic fluctuations. A large molecule such as a protein moves moreslowlyandproducesmagneticfluctuationsatacorrespondinglylower rate. From the relaxation theory described by Solomon–Bloembergen,2,3 three primary factors that regulate the dipole–dipole interactions respon- sible for both T and T relaxation are: (1) the strength of the magnetic 1 2 1R.B.Lauffer,Chem.Rev.87,901(1987). 2I.Solomon,Phys.Rev.99,559(1955). 3N.Bloembergen,J.Chem.Phys.27,572(1957). 6 disease models [1] moment, (2) the separation between the two dipoles, and (3) the relative motion of the two dipoles. Intrinsic or Endogenous Contrast Probing tumor vasculature using intrinsic contrast produced by deoxy- hemoglobinintumormicrovesselsisbasedonthebloodoxygenationlevel dependent (BOLD) contrast mechanism first proposed by Ogawa.4 The concentration of endogenous paramagnetic deoxyhemoglobin is one of the primary determinants of the eventual image contrast observed. The presence of deoxyhemoglobin in a blood vessel causes a susceptibility dif- ferencebetweenthevesselanditssurroundingtissue,inducingmicroscopic magnetic field gradients that cause dephasing of the MR proton signal, leadingtoareductioninthevalueofT *(Fig.1).Becauseoxyhemoglobin 2 is diamagnetic and does not produce the same dephasing, changes in oxy- genation of the blood can be observed as signal changes in T *-weighted 2 images. The functional dependence of T * on oxygenation in a tissue is 2 expressed as: 1 /ð1(cid:3)YÞb (3) T * 2 where Y is the fraction of oxygenated blood and b the fractional blood volume. In hypoxic tumors where 0 < Y < 0.2, the contrast produced by themethodisprimarilydependentonb.Thismethodworksbestinpoorly oxygenatedtumorssuchassubcutaneousmodels,andinhumanxenografts withrandomorientationofsproutingcapillaries,anditprovidesafastand noninvasive measurement of tumor fractional blood volume because ex- ogenous contrast is not required. However, the method cannot provide quantitativemeasurementsoftumorvascularvolume,vascularpermeabil- ity, or blood flow. Nonetheless, this technique has been used to detect changes in tumor oxygenation and vascularization following induction of angiogenesis by external angiogenic agents,5 as well as to obtain maps of the ‘‘functional’’ vasculature in genetically modified HIF-1 (þ/þ and (cid:3)/(cid:3)) animal models.6 BOLD contrast is not solely related to the oxy- genationstatusofblood,butisalsoaffectedbyfactorssuchasoxygensat- uration, the hematocrit, blood flow, blood volume, vessel orientation, and 4S.Ogawa,Magn.Reson.Med.14,68(1990). 5R. Abramovitch, H. Dafni, E. Smouha, L. Benjamin, and M. Neeman, Cancer Res. 59, 5012(1999). 6P. Carmeliet, Y. Dor, J.-M. Herbert, D. Fukumura, K. Brusselmans, M. Dewerchin, M.Neeman,F.Bono,R.Abramovitch,P.Maxwell,C.J.Koch,P.Ratcliffe,L.Moons, R.K.Jain,D.Collen,andE.Keshet,Nature394,485(1998).