Review Article Advances in Molecular Imaging Strategies ...downloads.hindawi.com/journals/bmri/2016/1946585.pdf · monitoring of cell viability. Proliferation of labeled cells in

Review ArticleAdvances in Molecular Imaging Strategies forIn Vivo Tracking of Immune Cells

Ho Won Lee Prakash Gangadaran Senthilkumar Kalimuthu and Byeong-Cheol Ahn

Department of Nuclear Medicine Kyungpook National University School of Medicine and Hospital Daegu Republic of Korea

Correspondence should be addressed to Byeong-Cheol Ahn abc2000knuackr

Received 27 May 2016 Revised 12 August 2016 Accepted 23 August 2016

Academic Editor Patrizia Rovere-Querini

Copyright copy 2016 HoWon Lee et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Tracking of immune cells in vivo is a crucial tool for development and optimization of cell-based therapy Techniques for trackingimmune cells have been applied widely for understanding the intrinsic behavior of immune cells and include non-radiation-basedtechniques such as optical imaging and magnetic resonance imaging (MRI) radiation-based techniques such as computerizedtomography (CT) and nuclear imaging including single photon emission computerized tomography (SPECT) and positronemission tomography (PET) Each modality has its own strengths and limitations To overcome the limitations of each modalitymultimodal imaging techniques involving two or more imaging modalities are actively applied Multimodal techniques allowintegration of the strengths of individual modalities In this review we discuss the strengths and limitations of currently availablepreclinical in vivo immune cell tracking techniques and summarize the value of immune cell tracking in the development andoptimization of immune cell therapy for various diseases

1 Introduction

Immune cells have been studied extensively to elucidate theirbiological roles under various physiological and patholog-ical conditions Improved understanding of immune cellfunctions can help lay the foundation for safe and efficientapplication of these cells for therapeutic purposes Moreoverimmune cells are being used increasingly as new potentialtherapeutics to treat conditions such as autoimmune diseaseand cancer [1] Noninvasive in vivo cell tracking is an emerg-ing approach for imaging cells in their native environmentMolecular imaging is a rapidly growing field with implica-tions in biology chemistry computer science engineeringand medicine which allows visualizing cellular and subcel-lular processes within living subjects at the molecular andthe anatomical level [2] Dynamic noninvasive imaging candirect proper decision-making processes during preclinicaland clinical studies which are aimed at enhancing efficacyand safety of immune cell therapies Molecular imaging isevolving rapidly and has been facilitated by the developmentof relevant materials such as imaging agents reporter con-structs ligands and probes [3] Various molecular imaging

techniques such as computed tomography (CT) magneticresonance imaging (MRI) bioluminescent imaging (BLI)fluorescence imaging (FLI) single photon emission com-puted tomography (SPECT) and positron emission tomogra-phy (PET) are actively applied for tracking immune and stemcells [4ndash9] AlthoughMRI and CT provide excellent anatom-ical resolution and are easy to translate into clinical applica-tion these modalities are limited by low sensitivity and highinstrumentation cost [10 11] CT is one of the radiologytechnologies applied to track immune cells in the field ofbiomedical imaging [3 12 13] MRI is now emerging andrapidly expanding wings in the field It has the advantages ofsafety high resolution and direct applicability to cell trackingin clinical studies [14 15] Various types of reporter genessuch as those that encode fluorescent and bioluminescentproteins have been used as imaging reporters for visualizationand tracking of immune cells in vivo Application of imagingreporters is facilitated by the development of efficient vectordelivery systems [3 9 16 17] BLI can track migration ofimmune cells to sites of inflammation [18 19] FLI hasbeen used in noninvasive in vivo tracking of dendritic cell(DC) migration into lymph nodes and primary macrophage

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 1946585 10 pageshttpdxdoiorg10115520161946585

2 BioMed Research International

migration toward induced inflammatory lesions [4 20] PETis a sensitive imaging tool for detecting immune cells in var-ious animal models and provides quantitative and temporaldistribution of immune cells by radiolabeling with 18F-FDGor 111In-oxine [3 21ndash25] The above-mentioned molecularimaging techniques are widely exploited for immune cellmonitoring at high resolution in living animals

Molecular imaging is considered the preferred approachfor tracking immune cells in imaging studies in vivo There istherefore a need for researchers to be familiar with proper celllabeling methods and appropriate imaging modalities spe-cific for the particular labelingmethod In this reviewwe pro-vide a general overview and specific examples of in vivo track-ing of immune cells with various imaging modalities for bet-ter understanding of the roles played by immune cells undervarious pathophysiological conditions

2 Advantages and Disadvantages ofEach Molecular Imaging Technology

BLI and FLI are relatively low-cost and high-throughputtechniques but they are limited by the lack of fine spatial reso-lution and difficulty in scaling up for application in larger ani-mals and humans because of inherent depth limitation origi-nating from poor tissue penetration of optical signals [11 26]PET and SPECT have the advantages of high sensitivity andunlimited depth penetration excellent signal-to-backgroundratios and a broad range of clinically applicable probesHowever nuclear images have the disadvantages of highbackground activity and limited anatomical information [27]Multimodal fusion molecular imaging is now widely appliedto overcome the limitations of a single imaging modal-ity Commercially available systems integrate optical PETSPECT CT andMRI imaging in various combinationsThesemultimodal approaches allow different imaging technolo-gies to be combined by simultaneous acquisition and thustogether incorporate the best features and utilities of eachmodality [28]

In vivo imaging strategies in preclinical studies have animportant advantage the same animal can be examinedrepeatedly at different time points thereby decreasing thevariability in study population and reducing the sample size[29 30] To monitor adoptively transferred immune cellsan effective labeling methodology needs to be selected Celllabeling can be classified as either direct or indirect [31]Direct labeling of the imaging moiety of therapeutic cells isthe most commonly used strategy for monitoring cells in liv-ing subjects [32] In direct labeling the cells can be harvestedand labeled with radioisotopes MRI-based contrast agentsor fluorophores thereby allowing cells to be visualized byPETSPECT MRI or FLI respectively This strategy has theadvantages of simple labeling protocols and high sensitivityHowever it has major drawbacks [1] First the extent oflabeling depends on the ability of the signal element in thecells to retain the label Second it does not allow long-termmonitoring of cell viability Proliferation of labeled cells inthe living subject results in diluted signal and persistentsignals are emitted from the labeled cells even after cell death

In contrast indirect labeling with reporter genes such asluciferase (Luc) green fluorescent protein (GFP) and sodiumiodide symporter (NIS) does not have such limitations andthis approach is therefore preferred for long-term in vivo cellmonitoring [33] In indirect labeling the cells are transfectedwith a vector containing the imaging reporter genes Thereporter genes are integrated into the cell genome and tran-scribed to mRNAs which are translated to reporter proteinsIn stably transfected cells the reporter gene is inherited byboth daughter cells upon cell division This strategy is essen-tial for long-term in vivo tracking of cells and for evaluation ofdivision of labeled cells Despite the advantages of the indirectcell labeling strategy it has its own limitations It is difficult togenerate stably transfected cells because of the low efficiencyof transfection in immune and primary cells Safety concernsarising from genetic modification of cells by indirect labelingare an issue that substantially limits clinical application

3 Relevance of Immune Cell Tracking

Tracking of immune cells such as T cells natural killercells DCs and macrophages is used to develop cell-basedimmunotherapy approaches against various diseases primar-ily malignant diseases [34 35] Most of the information aboutimmune cell tracking was previously obtained using flowcytometry and confocal microscopy [36] Flow cytometryis a good experimental approach for counting transferredimmune cells in an organismHowever this is only applicablein the case of ex vivo samples and does not provide informa-tion about the precise location of the analyzed immune cellsConfocal microscopy can provide information about the spa-tial distribution of cells by using immunostained tissue sec-tions and real-time in vivo distribution of cells in a superficialorgan that can be accessed by a light signalHowever confocalmicroscopy is unsuitable for real-time in vivo monitoring ofthe cells in deep organs

Recent advances in imaging technology in vivo haverevealed the potential of various imaging techniques formon-itoring immune cells The functional changes associated withthe death survival proliferation andmigration of cells can beaccurately assessed [37] Successful application of such in vivoimmune cell tracking tools can potentially optimize image-guided therapeutic options and eventuallymay improve ther-apeutic options or therapeutic outcome In particular the bestroute of administration of therapeutic cells and the optimaldose for cell therapy can be easily determined by imaging

31 Immune Cells

311 Dendritic Cells Dendritic cells (DCs) occupy a centralposition in the immune systemDCs are professional antigen-presenting cells (APCs) that play a critical role in the regula-tion of adaptive immune response [21 38] They arise frombone marrow precursors and are present in immature formsin the peripheral tissues DCs capture and process antigensand then undergomaturation [39]MatureDCs can stimulatehelper and killer T cells in vivo by expressing at high levelsMHC class III molecules costimulatory molecules (B7) andadhesion molecules (ICAM-1 ICAM-3 and LFA-3) [40 41]

BioMed Research International 3

Days after injection of Raw2647effluc1 3 5 7 80

10

times106

(a)

00

20 times 107

40 times 107

60 times 107

80 times 107

Tota

l pho

ton

flux

(ps

)

Day 3 Day 5 Day 7Day 1

(b)

Figure 1 In vivo monitoring of macrophage migration toward inflammatory lesions by using optical imaging modality (a) The right hindlimb of Balbc mice was intramuscularly injected with turpentine oil to induce inflammation Seven days later Raw2647 cells expressing theenhanced firefly luciferase (effluc) gene were intravenously administered to these mice Bioluminescence imaging was undertaken at days 13 5 and 7 after injection of Raw2647effluc cells (b) The bioluminescence signals from Raw2647 cells were used to quantify the migrationof cells toward the inflammatory lesion Data are expressed as the mean plusmn SD

When used to vaccinate cancer patients DCs loaded withtumor-associated antigens are a potentially powerful tool forinducing antitumor immunity [42] Because of these impor-tant DC characteristics many recent studies have trackedDCmigration with various imaging modalities de Vries et almonitored themigration of antigen-pulsed DCs to the lymphnodes in melanoma patients with gamma camera imagingThey isolated DCs from peripheral blood mononuclear cells(PBMCs) and labeled them with 111In-oxine [43] Olasz etal tracked the migration of DCs into the lymph nodes withPET imaging modality in the case of bone marrow-derivedDCs (BMDCs) labeled with 18F-succinimidylfluorobenzoate(SFB) [44] Noh et al studied BMDC migration into thelymph nodes by labeling BMDCs with near-infrared- (NIR-)emitting quantumdots (QD) and tracking the labeled cells upto 3 days after injection by using FLI [4] Kim et al establishedDCs expressing ferritin heavy chain (FTH) as anMR reportergene and monitored DC migration by MRI [45] Xu et alsuccessfully labeled mature BMDC with SPIO nanoparticlesand monitored BMDC migration in vivo toward popliteallymph nodes by clinical 3T MR scanner [46] Lee et al alsodemonstrated DC migration into lymph nodes with BLI and124I PETCT imaging modalities using DCs expressing fireflyluciferase (Fluc) and sodium iodine symporter (NIS) reportergenes [47] (Figure 2) For clinical application another studyperformed evaluation of in vivo labeled DC migration inpatients with melanoma or renal carcinoma They generatedDCs from PBMC and then labeled immature (i) and mature(m) DCs with radioisotopes 99mTc-HMPAO and 111In-oxinerespectively The results showed that mDCs give approxi-mately 6ndash8-fold higher uptake in lymph node than immatureDCs and better migration activity was obtained with intra-dermal administration than with a subcutaneous route [48]Thus these studies using various molecular imaging tech-niques will help evaluate DC-based immunotherapy aimed

at increasing the efficacy of DCmigration and improving thedesign of clinical trials (Table 1)

312 Macrophages Macrophages play crucial and distinctroles in host defense They are strategically located through-out the body tissues where they ingest and process for-eign materials dead cells and debris and recruit additionalmacrophages in response to inflammatory signals [65ndash67]There are twomajormacrophage subsets classically activatedmacrophages (M1) and alternatively activated macrophages(M2 or tumor-associated macrophages TAMs) The M1macrophages secrete proinflammatory cytokines such as IL-1120573 TNF-120572 IL-6 and IL-12 as well as nitric oxide (NO)Theyhave various functions including boosting inflammationdebris removal sterilization and apoptotic cell removal Thealternatively activated M2 macrophages can be classifiedinto subtypes M2a M2b M2c and M2d which are involvedin tissue repairwound healing and immunoregulatoryand immunosuppressive activities [68 69] Monitoring ofmacrophages is necessary to understand inflammatory dis-eases and tumormicroenvironments thereforemacrophageshave been widely investigated using various molecular imag-ing techniques Several studies reported successful in vivomonitoring of macrophages transfected with reporter genessuch as Fluc or NIS in animal models with inflammatorylesions or tumors (Figure 1) [51ndash53] Lee et al investigatedthe recruitment of iron oxide-labeled primary macrophagesto the inflammatory lesion in a mouse model using MRI(Figure 3) [70] Kang et al tracked migration of primarymacrophages toward carrageenan-induced inflammatorylesions by both FLI and MR with NIR fluorescent magneticnanoparticles [20] Gramoun et al demonstrated tracking ofSPION-labeled macrophages using MR to assess treatmenteffects in a mouse model of rheumatoid arthritis by usingMR [50] TAMs have been successfully monitored with


Table 1 Immune cell tracking imaging strategies

Types of cells Labelingstrategy

Imagingmodality Labeling method Subject Duration

of tracking Purpose Clinicaltranslation Reference

DC Direct

FLI NIR-QD Mouse 3 days Tracking study Limited [4]PET 18F-SFB Mouse 4 h Tracking study Yes [44]

SPECT 111Indium Human 24ndash48 h Tracking study Yes [43]

SPECT111Indium99mTc-

HMPAO Human 48ndash72 h Tracking study Yes [48]

DC IndirectBLI Fluc Mouse 4 days Tracking study Limited [47]PET NIS124I Mouse 4 days Tracking study Yes [47]MRI FTH Mouse 48 h Tracking study Yes [45]

Macrophage Direct

FLI NIR nanoparticle Mouse 3ndash24 h Tracking toinflammation Limited [20]

MRI SPIO Mouse 24 h Tracking toinflammation Yes [49]

MRI Magnetic nanoparticle Mouse 3ndash24 h Tracking toinflammation Yes [20]

MRI SPIO Mouse 6ndash13 daysTracking torheumatoidarthritis

Yes [50]

Macrophage Indirect

BLI Fluc Mouse 0ndash21 days Tracking toinflammation Limited [51]

BLI Fluc Mouse 1ndash4 days Colon tumortargeting Limited [52]

PET NIS124I Mouse 7 days Tracking toinflammation Yes [53]

PET NIS124I Mouse 8ndash21 days Tracking toinflammation Yes [51]

PETCT 18F-FB Mouse 3 h Tracking to lungcarcinoma Yes [54]

T cells Direct MRI IOPC-NH2 Rat 24ndash48 h Tracking study Yes [49]MRI PFPE19F Mouse 48 h Tracking study Yes [55]

T cells Indirect

BLI Fluc Mouse 24 h Tracking study Limited [56]

BLI Fluc Mouse 10 days Tracking to lungcarcinoma Limited [57]

PETCT sr39tk18F-FHBG Mouse 1ndash21 days Melanomatumor targeting Yes [58]

B cells DirectFLI NIR nanoparticle Mouse 1ndash15 days Tracking study Limited [59]

PETCT 89Zr-anti-B220 Mouse 15ndash72 h Biodistributionstudy Yes [60]

B cells Indirect MRI SPIO Mouse 1ndash15 days Tracking study Yes [60]

NK Direct

FLI NIR dye Rat 24 h Tracking study Limited [61]PET 11C Mouse 05ndash1 h Tracking study Yes [62]

SPECT 111In Human 05ndash144 h Tracking andtherapy study Yes [63]

SPECT 111In Human 6 days Biodistributionstudy Yes [64]

SPECT 111In Human 6ndash96 h Tracking study Yes [63]DC dendritic cell NK natural killer cell FLI fluorescence imaging PET positron emission tomography SPECT single photon emission computerizedtomography BLI bioluminescence imaging MRI magnetic resonance imaging CT computed tomography NIR near infrared QD quantum dot SFBfluorobenzoate NIS sodium iodide symporter HMPAO hexamethylpropyleneamineoxime SPIO superparamagnetic iron oxide Fluc firefly luciferaseFB fluorobenzene IOPC iron oxide nanoparticles coated PFPE perfluoropolyethers sr39tk mutant type of HSV-thymidine kinase and FHBGfluorohydroxymethyl butyl guanine

various imaging modalities Choi et al reported evaluationof TAM migration into tumor lesions and the modulationof tumor progression using multimodal optical reporter geneimaging [52] Blykers et al tracked TAMs using PETCTwith

18F-labeled camelid single-domain antibody fragments totarget mannose receptor-expressing macrophages usingPETCT [54] Daldrup-Link et al showed that SPIO with2T MRI could be applied to track TAMs in a mouse model


Day 1 Day 4

2

times106

16

PET

CTBL

I

600

0

(a)

DCNF-injected footpadDC-injected footpad

0000

0005

0010

0015

0020

ID

cc

00

20 times 108

40 times 108

60 times 108

80 times 108

Tota

l pho

ton

flux

(ps

)

Day 4Day 1Day after injection of DC cells

Day 4Day 1Day after injection of DCNF cells

(b)

Figure 2 Visualization of DC migration into the lymph node in vivo using multimodal imaging DC24 or DC24 cells expressing NIS andeffluc genes (DCNF) were injected in the left or right mouse footpad respectively (a) Signals were observed in the lymph node by both BLIand 124I PETCT imaging (b) Quantification of BLI signals and radioiodine uptake in the lymph node Data are expressed as the mean plusmn SD

Before Hour after injection of 1 CG solution

T2lowast

6 h 24 h

Figure 3 In vivo tracking of peritoneal macrophage migration toward CG-induced inflammatory lesion by MRI Peritoneal macrophageswere isolated from C57BL6 mice at day 4 after injection with 3 thioglycollate medium and then labeled with magnetic nanoparticles MRIwas obtained before or after injection of 1 carrageenan by 47TMRIArrow indicates hypointense signal ofmigrated peritonealmacrophages

of mammary carcinogenesis [49] Improved understandingof the roles of macrophage migration in inflammation andtumor formation can offer useful clues to modulate macro-phage activity by developing and evaluating anti-inflam-matory or antitumor compounds

313 T Cells T cells are lymphocytes that play crucialroles in cell-mediated immunity They have unique surfaceproteins known as T-cell receptors (TCRs) a complex ofintegral membrane proteins that recognize antigens when theantigen is presented on the surface of antigen-presenting cells


including macrophages B cells and DCs [71ndash73] Activationof T cells is induced by the interaction between TCR andantigen peptide There are two main classes of T cells helperT cells (Ths or CD4+ T cells) and cytotoxic T cells (CTLs orCD8+ T cells)TheThs recognize the peptides bound toMHCclass II molecules They not only help to stimulate B cellsto release antibodies and macrophages to destroy ingestedmicrobes but also help to activate CTLs [74 75] On the otherhand CTLs are able to recognize peptides presented byMHCclass I molecules and then release cytolytic mediators such asperforin and granzyme which subsequently induce apoptosisin tumor cells and virus-infected cells [76 77] Although Tcells possess remarkable potential as a component of immunecell therapy the fate of the infused T cells and the inter-mediate steps between cell migration and therapy outcomeare not well understood Many researchers have attemptedin vivo tracking of the infused T cells with various imagingmodalities to determine their biodistribution viability andfunctionality Chewning et al generated transgenic mice (T-Lux) in which the luciferase gene is expressed by T cellsT cells were isolated from splenocytes of the T-Lux modelmice Using the BLI imaging system they visualized T-cellmigration to secondary lymphoid tissues within 24 h of adop-tive transfer of T-Lux T cells [56] Kim et al investigated thetargetedmovement of CTLs into B-cell lymphomas using BLI[57] T cells labeled with nanosized MRI contrast agent wereobserved by MRI to be involved in the rejection of allograft-transplanted hearts and lungs [78] T-cell migration intomelanomas with or without antigen-pulsedDCs was success-fully imaged using reporter gene technology combined withPETCT acquisition showing increased uptake by the spleenand lymph node with combined immunotherapy comparedto the control [58] Srinivas et al visualized T-cell homingbehavior in an adoptive transfer model of an autoimmunediseaseThey labeled T cells isolated from splenocytes of TCRtransgenicmicewith perfluoropolyether (PFPE) nanoparticletracer agent and were able to demonstrate in vivo T-cell hom-ing to pancreas in a murine diabetes model by 19F MRI [55]Overall tracking of T cells in vivo is useful to understand T-cell biology in various pathophysiological conditions such asautoimmune disorders cancer allergy and transplantationT-cell tracking will help optimize adoptively transferred T-cell therapy for various disorders

314 B Cells B cells play a vital role in the adaptive immuneresponse to infectious diseases by producing specific antibod-ies to the antigens expressed by invading pathogens [79 80]Antigen-specific interactions require antigens either free-floating or presented by APCs to first be internalized by theB-cell receptors (BCR) followed by triggering of signalingcascades that initiate the activation of B cells into antibody-secreting effector cells [81ndash83]There are five isotypes of anti-bodies (IgA IgD IgE IgG and IgM) based on the C-terminalregions of heavy chains Antibodies can neutralize infectiouspathogens and activate macrophages and other immune cells[84 85] Beneficial functions apart B cells also play a patho-logical role in allergic and autoimmune diseases includingasthma rheumatoid arthritis systemic lupus erythematosusand vasculitis [86ndash88] The use of imaging modality-based

tracking of B cells is still in its infancy when comparedto tracking of other immune cells Only a few studiesusing radioisotopes and magnetic nanoparticles have beenreported to date Walther et al monitored B cells in thespleen lymph nodes testes and joints by using PETCT afterinjecting 89Zr-labeled anti-B-cell antibody [60] Thorek et altracked the migration of primary murine B cells toward thespleen by using FLI and MRI with fluorescent and magneticnanoparticles respectively [59] These results can lead toimproved understanding of B-cell-related diseases and effec-tive treatment regimens for such diseases

315 Natural Killer Cells Natural killer (NK) cells are lym-phocytes of the innate immune system that control severaltypes of tumors andmicrobial infections [89 90] NK cells areregulated by inhibitoryactivating receptors which decide thefate of NK cell [91 92] NK cells are activated by interferons(INF-120572 -120573 and -120574) or macrophage-derived cytokines (IL-12 and IL-18) which results in secretion of cytotoxic granuleproteins (perforingranzyme) that induce apoptosis in targetcells [93ndash95] Unlike T cells the non-MHC-restricted cyto-toxicity of NK cells renders them appealing for investigationas potential effectors of immunotherapy Although manystudies have investigated the therapeutic effects of NK cell-based immunotherapy for various cancers alteration of NKcell functions and cytokine imbalance reduce the therapeuticpotency of cell therapy [63] Using NK cells to target malig-nant cells is another key factor for successful therapy Thetracking of NK cells with various imaging modality tech-niques can provide information about the presence quantityand distribution of administered NK cells in living subjectsFor tracking NK cells with nuclear imaging modalities NKcells were labeled with 18F or 11C for PET imaging and with111In for SPECT and the signals emitted from labeledNKcellswere observed in lung spleen liver and tumor lesions [6264 96ndash98] NK cell tracking with optical imaging modalitieswas also successfully performed by labeling the NK cellswith fluorescent dyes or transfecting with GFP or luciferasereporter genes [61 99] Daldrup-Link et al observedincreased fluorescent signal in tumors 24 h after inject-ing NK cells labeled with DiD (111015840-dioctadecyl-333101584031015840-tetramethylindodicarbocyanine) fluorescent dye [61] TotrackNK cells usingMRI Daldrup-Link et al transducedNKcells with scFV (FRP5)-zeta and then labeled themwith feru-carbotran The genetically engineered NK cells were injectedinto NIH 3T3 HER2neu receptor positive tumor bearingmice and the group demonstrated increased tumor targetingof the genetically engineered NK cells by 15T MR scanner[100] NK cell tracking might be invaluable for improvingthe efficacy of NK cell-based immunotherapy by modulatingthe therapeutic protocols used in translational and clinicalapproaches

4 Conclusion

In vivo tracking of immune cells (DCs macrophages T cellsB cells and NK cells) using various imaging techniques con-tinues to contribute to improved understanding of the roleof each immune cell type as well as aiding the development


of therapy using or targeting immune cells Cell labelinga prerequisite for cell tracking can be achieved directly orindirectly Direct labeling strategies using clinically approvedmaterials and methods hold great potential for clinical appli-cation Meanwhile indirect labeling strategies with reportergenes can assist long-term study of cell survival proliferationand activation of immune cells However none of the avail-able cell labeling strategies meets all requirements thereforean appropriate specific labeling strategy should be selected foreach experimental setting

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

H W Lee is the first author

Acknowledgments

This study was supported by the National Nuclear RampDProgram through theNational Research Foundation of Korea(NRF) funded by the Ministry of Education Science andTechnology (no 2012M2A2A7014020) by a grant from theKorea Health Technology RampD Project Ministry of Healthamp Welfare Republic of Korea (HI16C1501) by a grant fromthe Medical Cluster RampD Support Project of Daegu Gyeong-buk Medical Innovation Foundation (DGMIF) funded bythe Ministry of Health amp Welfare Republic of Korea(HT13C0002) and by a grant from the Korea Health Tech-nology RampD Project through the Korea Health IndustryDevelopment Institute (KHDI) funded by the Ministry ofHealth ampWelfare Republic of Korea (HI15C0001)This workwas also supported by a National Research Foundation ofKorea (NRF) grant funded by the Korea government (MSIP)(no NRF-2015M2A2A7A01045177)

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[3] H Youn and K-J Hong ldquoIn vivo non invasive molecularimaging for immune cell tracking in small animalsrdquo ImmuneNetwork vol 12 no 6 pp 223ndash229 2012

[4] Y-WNoh Y T Lim and BH Chung ldquoNoninvasive imaging ofdendritic cell migration into lymph nodes using near-infraredfluorescent semiconductor nanocrystalsrdquo FASEB Journal vol22 no 11 pp 3908ndash3918 2008

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[9] J E Kim S Kalimuthu and B-C Ahn ldquoIn Vivo cell trackingwith bioluminescence imagingrdquo Nuclear Medicine and Molecu-lar Imaging vol 49 no 1 pp 3ndash10 2015

[10] S N Histed M L Lindenberg E Mena B Turkbey P LChoyke and K A Kurdziel ldquoReview of functionalanatomicalimaging in oncologyrdquo Nuclear Medicine Communications vol33 no 4 pp 349ndash361 2012

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[14] E T Ahrens and J W M Bulte ldquoTracking immune cells in vivousing magnetic resonance imagingrdquo Nature Reviews Immunol-ogy vol 13 no 10 pp 755ndash763 2013

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[27] A A Neves and K M Brindle ldquoAssessing responses to cancertherapy using molecular imagingrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1766 no 2 pp 242ndash261 2006

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[33] J H Kang and J-K Chung ldquoMolecular-genetic imaging basedon reporter gene expressionrdquo Journal of Nuclear Medicine vol49 supplement 2 pp 164Sndash179S 2008

[34] M Schuster A Nechansky and R Kircheis ldquoCancer immuno-therapyrdquo Biotechnology Journal vol 1 no 2 pp 138ndash147 2006

[35] T Hinz C J Buchholz T van der Stappen K Cichutekand U Kalinke ldquoManufacturing and quality control of cell-based tumor vaccines a scientific and a regulatory perspectiverdquoJournal of Immunotherapy vol 29 no 5 pp 472ndash476 2006

[36] M D Cahalan I Parker S H Wei and M J Miller ldquoTwo-photon tissue imaging seeing the immune system in a freshlightrdquo Nature Reviews Immunology vol 2 no 11 pp 872ndash8802002

[37] S Mandl C Schimmelpfennig M Edinger R S Negrin andC H Contag ldquoUnderstanding immune cell trafficking patternsvia in vivo bioluminescence imagingrdquo Journal of Cellular Bio-chemistry vol 39 pp 239ndash248 2002

[38] E Vacchelli I Vitale A Eggermont et al ldquoTrial watchdendritic cell-based interventions for cancer therapyrdquo OncoIm-munology vol 2 no 10 Article ID e25771 2013

[39] R M Steinman K Inaba S Turley P Pierre and I MellmanldquoAntigen capture processing and presentation by dendriticcells recent cell biological studiesrdquoHuman Immunology vol 60no 7 pp 562ndash567 1999

[40] M Cella F Sallusto and A Lanzavecchia ldquoOrigin maturationand antigen presenting function of dendritic cellsrdquo CurrentOpinion in Immunology vol 9 no 1 pp 10ndash16 1997

[41] R M Steinman ldquoThe dendritic cell system and its role inimmunogenicityrdquo Annual Review of Immunology vol 9 no 1pp 271ndash296 1991

[42] N Burdin and P Moingeon ldquoCancer vaccines based on den-dritic cells loaded with tumor-associated antigensrdquo Cell Biologyand Toxicology vol 17 no 2 pp 67ndash75 2001

[43] I J M de Vries D J E B Krooshoop N M Scharenborg et alldquoEffective migration of antigen-pulsed dendritic cells to lymphnodes in melanoma patients is determined by their maturationstaterdquo Cancer Research vol 63 no 1 pp 12ndash17 2003

[44] E B Olasz L Lang J Seidel M V Green W C Eckelmanand S I Katz ldquoFluorine-18 labeledmouse bonemarrow-deriveddendritic cells can be detected in vivo by high resolution pro-jection imagingrdquo Journal of Immunological Methods vol 260no 1-2 pp 137ndash148 2002

[45] H S Kim J Woo J H Lee et al ldquoIn vivo tracking of dendriticcell using MRI reporter gene ferritinrdquo PLoS ONE vol 10 no 5article e0125291 2015

[46] Y Xu C Wu W Zhu et al ldquoSuperparamagnetic MRI probesfor in vivo tracking of dendritic cell migration with a clinical 3T scannerrdquo Biomaterials vol 58 pp 63ndash71 2015

[47] H W Lee S Y Yoon T D Singh et al ldquoTracking of dendriticcell migration into lymph nodes using molecular imaging withsodium iodide symporter and enhanced firefly luciferase genesrdquoScientific Reports vol 5 article 9865 2015

[48] R Ridolfi A Riccobon R Galassi et al ldquoEvaluation of in vivolabelled dendritic cell migration in cancer patientsrdquo Journal ofTranslational Medicine vol 2 no 1 article 27 2004

[49] H E Daldrup-Link D Golovko B Ruffell et al ldquoMRI oftumor-associated macrophages with clinically applicable ironoxide nanoparticlesrdquo Clinical Cancer Research vol 17 no 17 pp5695ndash5704 2011

[50] A Gramoun L A Crowe L Maurizi et al ldquoMonitoring theeffects of dexamethasone treatment by MRI using in vivo ironoxide nanoparticle-labeled macrophagesrdquo Arthritis ResearchandTherapy vol 16 no 3 article R131 2014

[51] H W Lee Y H Jeon M-H Hwang et al ldquoDual reporter geneimaging for tracking macrophage migration using the humansodium iodide symporter and an enhanced firefly luciferase ina murine inflammation modelrdquoMolecular Imaging and Biologyvol 15 no 6 pp 703ndash712 2013

[52] Y J Choi S-G Oh T D Singh et al ldquoVisualization of thebiological behavior of tumor-associated macrophages in livingmice with colon cancer using multimodal optical reporter geneimagingrdquo Neoplasia vol 18 no 3 pp 133ndash141 2016

[53] J H Seo Y H Jeon Y J Lee et al ldquoTrafficking macrophagemigration using reporter gene imaging with human sodiumiodide symporter in animal models of inflammationrdquo Journalof Nuclear Medicine vol 51 no 10 pp 1637ndash1643 2010

[54] A Blykers S Schoonooghe C Xavier et al ldquoPET imagingof macrophage mannose receptor-expressing macrophages intumor stroma using 18F-radiolabeled camelid single-domainantibody fragmentsrdquo Journal of Nuclear Medicine vol 56 no 8pp 1265ndash1271 2015

[55] M Srinivas P A Morel L A Ernst D H Laidlaw and E TAhrens ldquoFluorine-19 MRI for visualization and quantificationof cell migration in a diabetes modelrdquo Magnetic Resonance inMedicine vol 58 no 4 pp 725ndash734 2007


[56] J H Chewning K J Dugger T R Chaudhuri K R Zinn andC T Weaver ldquoBioluminescence-based visualization of CD4 Tcell dynamics using a T lineage-specific luciferase transgenicmodelrdquo BMC Immunology vol 10 article 44 2009

[57] H Kim G Peng J M Hicks et al ldquoEngineering human tumor-specific cytotoxic T cells to function in a hypoxic environmentrdquoMolecular Therapy vol 16 no 3 pp 599ndash606 2008

[58] C J Shu C G Radu S M Shelly et al ldquoQuantitative PETreporter gene imaging of CD8+ T cells specific for a melanoma-expressed self-antigenrdquo International Immunology vol 21 no 2pp 155ndash165 2009

[59] D L JThorek P Y Tsao V Arora L Zhou R A Eisenberg andA Tsourkas ldquoIn vivo multimodal imaging of B cell distributionand response to antibody immunotherapy in micerdquo PLoS ONEvol 5 no 5 Article ID e10655 2010

[60] M Walther P Gebhardt P Grosse-Gehling et al ldquoImplemen-tation of 89Zr production and in vivo imaging of B-cells inmice with 89Zr-labeled anti-B-cell antibodies by small animalPETCTrdquoApplied Radiation and Isotopes vol 69 no 6 pp 852ndash857 2011

[61] H E Daldrup-Link S Tavri P Jha et al ldquoOptical imagingof cellular immunotherapy against prostate cancerrdquo MolecularImaging vol 8 no 1 pp 15ndash26 2009

[62] R J Melder A L Brownell T M Shoup G L Brownell andR K Jain ldquoImaging of activated natural killer cells in mice bypositron emission tomography preferential uptake in tumorsrdquoCancer Research vol 53 no 24 pp 5867ndash5871 1993

[63] L Zamai C Ponti P Mirandola et al ldquoNK cells and cancerrdquoThe Journal of Immunology vol 178 no 7 pp 4011ndash4016 2007

[64] B Meller C Frohn J-M Brand et al ldquoMonitoring of a newapproach of immunotherapy with allogenic 111In-labelled NKcells in patients with renal cell carcinomardquo European Journal ofNuclear Medicine andMolecular Imaging vol 31 no 3 pp 403ndash407 2004

[65] B Burke S Sumner N Maitland and C E Lewis ldquoMacro-phages in gene therapy cellular delivery vehicles and in vivotargetsrdquo Journal of Leukocyte Biology vol 72 no 3 pp 417ndash4282002

[66] D Orlic J Kajstura S Chimenti et al ldquoBone marrow cellsregenerate infarcted myocardiumrdquo Nature vol 410 no 6829pp 701ndash705 2001

[67] M Feldmann and L Steinman ldquoDesign of effective immuno-therapy for human autoimmunityrdquo Nature vol 435 no 7042pp 612ndash619 2005

[68] A Chawla ldquoControl of macrophage activation and function byPPARsrdquo Circulation Research vol 106 no 10 pp 1559ndash15692010

[69] S Gordon and P R Taylor ldquoMonocyte and macrophageheterogeneityrdquo Nature Reviews Immunology vol 5 no 12 pp953ndash964 2005

[70] J S Lee H J Kang G Gong et al ldquoMR imaging of in vivorecruitment of iron oxide-labeled macrophages in experimen-tally induced soft-tissue infection in micerdquo Radiology vol 241no 1 pp 142ndash148 2006

[71] J Sloan-Lancaster B D Evavold and P M Allen ldquoInduction ofT-cell anergy by altered T-cell-receptor ligand on live antigen-presenting cellsrdquo Nature vol 363 no 6425 pp 156ndash159 1993

[72] K-H Lee A D Holdorf M L Dustin A C Chan P M AllenandA S Shaw ldquoT cell receptor signaling precedes immunologi-cal synapse formationrdquo Science vol 295 no 5559 pp 1539ndash15422002

[73] A Viola and A Lanzavecchia ldquoT cell activation determined byT cell receptor number and tunable thresholdsrdquo Science vol273 no 5271 pp 104ndash106 1996

[74] T R Mosmann H Cherwinski andMW Bond ldquoTwo types ofmurine helper T cell clone I Definition according to profilesof lymphokine activities and secreted proteinsrdquo Journal ofImmunology vol 136 no 7 pp 2348ndash2357 1986

[75] S L Swain L M Bradley M Croft et al ldquoHelper T-cell subsetsphenotype function and the role of lymphokines in regulatingtheir developmentrdquo Immunological Reviews vol 123 no 1 pp115ndash144 1991

[76] M L Albert B Sauter and N Bhardwaj ldquoDendritic cellsacquire antigen from apoptotic cells and induce class I-restricted CTLSrdquo Nature vol 392 no 6671 pp 86ndash89 1998

[77] J A Trapani and M J Smyth ldquoFunctional significance of theperforingranzyme cell death pathwayrdquo Nature Reviews Immu-nology vol 2 no 10 pp 735ndash747 2002

[78] L Liu Q Ye Y Wu et al ldquoTracking T-cells in vivo with a newnano-sized MRI contrast agentrdquo Nanomedicine Nanotechnol-ogy Biology and Medicine vol 8 no 8 pp 1345ndash1354 2012

[79] P J Maglione and J Chan ldquoHow B cells shape the immuneresponse againstMycobacterium tuberculosisrdquoEuropean Journalof Immunology vol 39 no 3 pp 676ndash686 2009

[80] J R Dunkelberger and W-C Song ldquoComplement and its rolein innate and adaptive immune responsesrdquo Cell Research vol20 no 1 pp 34ndash50 2010

[81] A Rot and U H Von Andrian ldquoChemokines in innate andadaptive host defense basic chemokinese grammar for immunecellsrdquoAnnual Review of Immunology vol 22 pp 891ndash928 2004

[82] K L Calame ldquoPlasma cells finding new light at the end of B celldevelopmentrdquo Nature Immunology vol 2 no 12 pp 1103ndash11082001

[83] B Heyman ldquoRegulation of antibody responses via antibodiescomplement and Fc receptorsrdquo Annual Review of Immunologyvol 18 pp 709ndash737 2000

[84] H W Schroeder Jr and L Cavacini ldquoStructure and function ofimmunoglobulinsrdquo Journal of Allergy and Clinical Immunologyvol 125 no 2 pp S41ndashS52 2010

[85] TW LeBien andT F Tedder ldquoB lymphocytes how they developand functionrdquo Blood vol 112 no 5 pp 1570ndash1580 2008

[86] P Engel J A Gomez-Puerta M Ramos-Casals F Lozanoand X Bosch ldquoTherapeutic targeting of B cells for rheumaticautoimmune diseasesrdquo Pharmacological Reviews vol 63 no 1pp 127ndash156 2011

[87] C C Mok and C S Lau ldquoPathogenesis of systemic lupuserythematosusrdquo Journal of Clinical Pathology vol 56 no 7 pp481ndash490 2003

[88] J L Browning ldquoB cells move to centre stage novel opportuni-ties for autoimmune disease treatmentrdquo Nature Reviews DrugDiscovery vol 5 no 7 pp 564ndash576 2006

[89] E Vivier E Tomasello M Baratin T Walzer and S UgolinildquoFunctions of natural killer cellsrdquo Nature Immunology vol 9no 5 pp 503ndash510 2008

[90] T Walzer M Dalod S H Robbins L Zitvogel and E VivierldquoNatural-killer cells and dendritic cells lsquolrsquounion fait la forcerdquordquoBlood vol 106 no 7 pp 2252ndash2258 2005

[91] E O Long H S Kim D Liu M E Peterson and SRajagopalan ldquoControlling natural killer cell responses integra-tion of signals for activation and inhibitionrdquo Annual Review ofImmunology vol 31 no 1 pp 227ndash258 2013


[92] H J PegramDMAndrewsM J Smyth P KDarcy andMHKershaw ldquoActivating and inhibitory receptors of natural killercellsrdquo Immunology and Cell Biology vol 89 no 2 pp 216ndash2242011

[93] R M Welsh ldquoNatural killer cells and interferonrdquo CriticalReviews in Immunology vol 5 no 1 pp 55ndash93 1984

[94] T A Fehniger M H Shah M J Turner et al ldquoDifferentialcytokine and chemokine gene expression by human NK cellsfollowing activation with IL-18 or IL-15 in combination withIL-12 implications for the innate immune responserdquo Journal ofImmunology vol 162 no 8 pp 4511ndash4520 1999

[95] B R Lauwerys J-C Renauld and F A Houssiau ldquoSynergisticproliferation and activation of natural killer cells by interleukin12 and interleukin 18rdquoCytokine vol 11 no 11 pp 822ndash830 1999

[96] J-M Brand B Meller K Von Hof et al ldquoKinetics and organdistribution of allogeneic natural killer lymphocytes transfusedinto patients suffering from renal cell carcinomardquo StemCells andDevelopment vol 13 no 3 pp 307ndash314 2004

[97] L Matera A Galetto M Bello et al ldquoIn vivo migrationof labeled autologous natural killer cells to liver metastasesin patients with colon carcinomardquo Journal of TranslationalMedicine vol 4 article 49 2006

[98] R Meier M Piert G Piontek et al ldquoTracking of [18F]FDG-labeled natural killer cells to HER2neu-positive tumorsrdquoNuclear Medicine and Biology vol 35 no 5 pp 579ndash588 2008

[99] M Edinger Y-A Cao M R Verneris M H Bachmann C HContag andR S Negrin ldquoRevealing lymphoma growth and theefficacy of immune cell therapies using in vivo bioluminescenceimagingrdquo Blood vol 101 no 2 pp 640ndash648 2003

[100] H E Daldrup-Link R Meier M Rudelius et al ldquoIn vivo track-ing of genetically engineered anti-HER2neu directed naturalkiller cells to HER2neu positive mammary tumors with mag-netic resonance imagingrdquo European Radiology vol 15 no 1 pp4ndash13 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


migration toward induced inflammatory lesions [4 20] PETis a sensitive imaging tool for detecting immune cells in var-ious animal models and provides quantitative and temporaldistribution of immune cells by radiolabeling with 18F-FDGor 111In-oxine [3 21ndash25] The above-mentioned molecularimaging techniques are widely exploited for immune cellmonitoring at high resolution in living animals

Molecular imaging is considered the preferred approachfor tracking immune cells in imaging studies in vivo There istherefore a need for researchers to be familiar with proper celllabeling methods and appropriate imaging modalities spe-cific for the particular labelingmethod In this reviewwe pro-vide a general overview and specific examples of in vivo track-ing of immune cells with various imaging modalities for bet-ter understanding of the roles played by immune cells undervarious pathophysiological conditions

2 Advantages and Disadvantages ofEach Molecular Imaging Technology

BLI and FLI are relatively low-cost and high-throughputtechniques but they are limited by the lack of fine spatial reso-lution and difficulty in scaling up for application in larger ani-mals and humans because of inherent depth limitation origi-nating from poor tissue penetration of optical signals [11 26]PET and SPECT have the advantages of high sensitivity andunlimited depth penetration excellent signal-to-backgroundratios and a broad range of clinically applicable probesHowever nuclear images have the disadvantages of highbackground activity and limited anatomical information [27]Multimodal fusion molecular imaging is now widely appliedto overcome the limitations of a single imaging modal-ity Commercially available systems integrate optical PETSPECT CT andMRI imaging in various combinationsThesemultimodal approaches allow different imaging technolo-gies to be combined by simultaneous acquisition and thustogether incorporate the best features and utilities of eachmodality [28]

In vivo imaging strategies in preclinical studies have animportant advantage the same animal can be examinedrepeatedly at different time points thereby decreasing thevariability in study population and reducing the sample size[29 30] To monitor adoptively transferred immune cellsan effective labeling methodology needs to be selected Celllabeling can be classified as either direct or indirect [31]Direct labeling of the imaging moiety of therapeutic cells isthe most commonly used strategy for monitoring cells in liv-ing subjects [32] In direct labeling the cells can be harvestedand labeled with radioisotopes MRI-based contrast agentsor fluorophores thereby allowing cells to be visualized byPETSPECT MRI or FLI respectively This strategy has theadvantages of simple labeling protocols and high sensitivityHowever it has major drawbacks [1] First the extent oflabeling depends on the ability of the signal element in thecells to retain the label Second it does not allow long-termmonitoring of cell viability Proliferation of labeled cells inthe living subject results in diluted signal and persistentsignals are emitted from the labeled cells even after cell death

In contrast indirect labeling with reporter genes such asluciferase (Luc) green fluorescent protein (GFP) and sodiumiodide symporter (NIS) does not have such limitations andthis approach is therefore preferred for long-term in vivo cellmonitoring [33] In indirect labeling the cells are transfectedwith a vector containing the imaging reporter genes Thereporter genes are integrated into the cell genome and tran-scribed to mRNAs which are translated to reporter proteinsIn stably transfected cells the reporter gene is inherited byboth daughter cells upon cell division This strategy is essen-tial for long-term in vivo tracking of cells and for evaluation ofdivision of labeled cells Despite the advantages of the indirectcell labeling strategy it has its own limitations It is difficult togenerate stably transfected cells because of the low efficiencyof transfection in immune and primary cells Safety concernsarising from genetic modification of cells by indirect labelingare an issue that substantially limits clinical application

3 Relevance of Immune Cell Tracking

Tracking of immune cells such as T cells natural killercells DCs and macrophages is used to develop cell-basedimmunotherapy approaches against various diseases primar-ily malignant diseases [34 35] Most of the information aboutimmune cell tracking was previously obtained using flowcytometry and confocal microscopy [36] Flow cytometryis a good experimental approach for counting transferredimmune cells in an organismHowever this is only applicablein the case of ex vivo samples and does not provide informa-tion about the precise location of the analyzed immune cellsConfocal microscopy can provide information about the spa-tial distribution of cells by using immunostained tissue sec-tions and real-time in vivo distribution of cells in a superficialorgan that can be accessed by a light signalHowever confocalmicroscopy is unsuitable for real-time in vivo monitoring ofthe cells in deep organs

Recent advances in imaging technology in vivo haverevealed the potential of various imaging techniques formon-itoring immune cells The functional changes associated withthe death survival proliferation andmigration of cells can beaccurately assessed [37] Successful application of such in vivoimmune cell tracking tools can potentially optimize image-guided therapeutic options and eventuallymay improve ther-apeutic options or therapeutic outcome In particular the bestroute of administration of therapeutic cells and the optimaldose for cell therapy can be easily determined by imaging

31 Immune Cells

311 Dendritic Cells Dendritic cells (DCs) occupy a centralposition in the immune systemDCs are professional antigen-presenting cells (APCs) that play a critical role in the regula-tion of adaptive immune response [21 38] They arise frombone marrow precursors and are present in immature formsin the peripheral tissues DCs capture and process antigensand then undergomaturation [39]MatureDCs can stimulatehelper and killer T cells in vivo by expressing at high levelsMHC class III molecules costimulatory molecules (B7) andadhesion molecules (ICAM-1 ICAM-3 and LFA-3) [40 41]



10

times106

(a)

00

20 times 107

40 times 107

60 times 107

80 times 107

Tota

l pho

ton

flux

(ps

)


(b)










DC Direct






Macrophage Direct





Yes [50]

Macrophage Indirect







T cells Indirect







NK Direct








Day 1 Day 4

2

times106

16

PET

CTBL

I

600

0

(a)


0000

0005

0010

0015

0020

ID

cc

00

20 times 108

40 times 108

60 times 108

80 times 108

Tota

l pho

ton

flux

(ps

)



(b)



T2lowast

6 h 24 h









4 Conclusion




Competing Interests




Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















10

times106

(a)

00

20 times 107

40 times 107

60 times 107

80 times 107

Tota

l pho

ton

flux

(ps

)


(b)










DC Direct






Macrophage Direct





Yes [50]

Macrophage Indirect







T cells Indirect







NK Direct








Day 1 Day 4

2

times106

16

PET

CTBL

I

600

0

(a)


0000

0005

0010

0015

0020

ID

cc

00

20 times 108

40 times 108

60 times 108

80 times 108

Tota

l pho

ton

flux

(ps

)



(b)



T2lowast

6 h 24 h









4 Conclusion




Competing Interests




Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















DC Direct






Macrophage Direct





Yes [50]

Macrophage Indirect







T cells Indirect







NK Direct








Day 1 Day 4

2

times106

16

PET

CTBL

I

600

0

(a)


0000

0005

0010

0015

0020

ID

cc

00

20 times 108

40 times 108

60 times 108

80 times 108

Tota

l pho

ton

flux

(ps

)



(b)



T2lowast

6 h 24 h









4 Conclusion




Competing Interests




Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Day 1 Day 4

2

times106

16

PET

CTBL

I

600

0

(a)


0000

0005

0010

0015

0020

ID

cc

00

20 times 108

40 times 108

60 times 108

80 times 108

Tota

l pho

ton

flux

(ps

)



(b)



T2lowast

6 h 24 h









4 Conclusion




Competing Interests




Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















4 Conclusion




Competing Interests




Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests




Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Review Article Advances in Molecular Imaging Strategies ...downloads.hindawi.com/journals/bmri/2016/1946585.pdf · monitoring of cell viability. Proliferation of labeled cells in