AMacrophage-Specific Fluorescent Probe for IntraoperativeLymph Node Staging
Jung Sun Yoo1, Sung-Chan Lee2, Zhi Yen Jow2, Pamela Yun Xiang Koh1, and Young-Tae Chang1,2
AbstractSuccessful identification of nodal metastases in patients with cancer is crucial to prescribe suitable treatment
regimens that can improve recurrence-free survival. Although some new imaging technologies for nodal staginghave been developed, such as nanoparticle-enhanced MRI and quantum-dot–based fluorescence imaging, soundtechnologies for intraoperative differentiation of metastatic and inflamed lymph nodes remain lacking. In thisstudy, we illustrate the feasibility of using a macrophage-specific fluorescent probe (MFP) to visualize sentinellymph nodes during surgery, highlighting abnormalities related to inflammation and tumor infiltration withsignal enhancement and reduction methods using this technology. MFP was identified by high-throughputscreening of fluorescent small-molecule libraries synthesized with a diversity-oriented approach. It selectivelyvisualized monocyte andmacrophage cell populations in vitro, by live-cell imaging and flow cytometry, as well asin vivo, for imaging-guided surgery. Collectively, this study provides preclinical proof of concept for anintraoperative imaging platform to accurately assess lymph node status, eliminating the need for invasive nodaldissections that can contribute to complications of cancer therapy. Cancer Res; 74(1); 44–55. �2013 AACR.
IntroductionIntraoperative/interventional fluorescence imaging is an
evolving field of imaging science that offers new ways tohighlight abnormalities in patients in real-time (1–3). Commonradiologic modalities such as MRI and positron emissiontomography (PET) are not suitable for intraoperative/inter-ventional applications due to their high levels of irradiationand lack of integration in the operating room. In contrast,optical imaging is a natural approach in a variety of interven-tional procedures because it closely relates to the doctor'svision during operation, and provides high spatiotemporalresolution and sensitivity. There have been vibrant develop-ments of newfluorescent-probe technologies for labelingmanycellular and subcellular processes intraoperatively (4–6). Thisimposed contrast is now captured with an increasing numberof new optical imaging systems (7) such as multispectralreflectance fluorescence imaging (8, 9) and dual-modalitycatheter (10). Such advances have already started to impactclinical practice by providing real-time feedback on diseasedistribution. Recently, the first-in-human use of intraoperativetumor-specific fluorescence imaging with a folate receptor-
a–targeted fluorescent agent was reported (11). In addition,endoscopic identification of dysplasia using fluorescent lectins(12) or targeted heptapeptides (13) has been demonstrated in apilot study involving patients. Accurate real-time imaging withspecific fluorescent agents is now shifting the paradigm for theoperating suite from blind surgery based on anatomic contrastand tactile information to elaborate imaging-guided surgeryoffering real-time pathology.
One elegant example of intraoperative fluorescence imagingis sentinel lymph node (SLN) biopsy (SLNB) using visualguidance with fluorescent probes. The nodal status of canceris an important prognostic indicator for patients diagnosedwith many forms of solid cancers (14–18). The SLN conceptstates that, if the first lymph node to receive lymphaticdrainage from a tumor site does not contain tumor cells, thetumor is not likely to have metastasized to the lymphaticsystem. Since the initial introduction of SLNB in 1992 (19), ithas been a gold-standard for cancermanagement and has beenwidely used in many countries (20). The identification of theSLN is typically achieved by the use of radionuclide colloids(Technetium-99m) with a g-camera (21, 22) and/or the injec-tion of blue dye (isosulfan blue, methylene blue; refs. 23, 24),subcutaneously in the vicinity of the tumor tissue at the time ofsurgery. However, radiolabeled colloids have limited precisionand show a slow uptake into the lymphatic system of up to 3hours due to their bulky size. Blue chromophores rapidlyattenuate the light penetration depth and show a high rateof false-negative results because the small dye molecules canreadily diffuse through the true SLN to the second- and thethird-tier nodes. Therefore, quantum dots (QD; ref. 25) orfluorescent dyes such as indocyanine green (26, 27) andIRDye800 (28) have emerged as promising alternatives forsensitive and more accurate localization of SLNs. QDs have
Authors' Affiliations: 1Department of Chemistry and MedChem Programof LifeSciences Institute,NationalUniversity ofSingapore; and 2Laboratoryof Bioimaging Probe Development, Singapore Bioimaging Consortium,Agency for Science, Technology and Research, Singapore
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Author: Young-Tae Chang, National University of Singa-pore, S09-03-02 3 Science Drive 3, Singapore 117543. Phone: 65-6516-7795; Fax: 65-6478-9957; E-mail: [email protected]
exhibited rapid uptakes into the lymphatic system and remainin the SLN owing to their innate lymphotropic properties withhydrodynamic diameters of 10 to 50 nm.Although intraoperative fluorescence imaging approaches,
especially those using fluorescent nanoscale agents (29, 30),enables rapid uptake into the lymphatic system and effectivemapping of the SLN, invasive surgical dissection of the node,followed by time-consuming examination by a pathologist, isstill required (31–33). If the necessity for a painful secondsurgery after pathologic examination of the SLN following theremoval of the primary cancer and SLNB or a delay in thesurgical procedure due to intraoperative histology of the SLNduring the surgery is to be avoided, real-time visual assess-ment of metastatic lymph nodes is needed to guide thesurgeon's scalpel without any unwanted interference withstandard surgical procedures. Recently, a new superparamag-netic iron-oxide nanoparticle (SPION) was developed as anMRI contrast agent to identify tumor-infiltrated lymph nodesin patients with prostate cancer (34). Contrast-enhancedMRI correctly identified all patients with nodal metastasesin whom nodal infiltration of macrophages that had engul-fed the superparamagnetic particles highlighted metastaticregions devoid of signal, and a node-by-node analysis had asignificantly higher sensitivity than conventional MRI. Thispreoperative imaging technology has implicated the possibil-ity of using a synthetic fluorochrome with macrophage-spe-cific binding affinity to provide effective intraoperative guid-ance on nodal involvement in cancer. Therefore, such amacrophage-specific fluorescence imaging modality may bevery helpful to differentiate tumor-induced and inflamma-tion-induced lymph node enlargement based on reduced andenhanced signals.Recently, small-molecule fluorescent probes, in conjunction
with significant advances in in vivo microscopy and interven-tional clinical imaging, have gained particular attention forreliable in vivo labeling (35, 36). Completely defined syntheticvital dyes have considerable potential to rapidly label cells,subcellular compartments, and molecular effectors in livingobjects due to their diverse biologic properties caused byskeletal and stereochemical complexities (37, 38). In this study,we report the development of a macrophage-specific fluores-cent probe (MFP) using a diversity-oriented synthesis (39, 40)for intraoperative lymph node staging. A boron-dipyrro-meth-ane (BODIPY)–based MFP was discovered in a screening of1,153 tailor-made, organic,fluorescent, smallmolecules (41, 42)against different blood-cell lines, includingmacrophages. Here,we show the capability of MFP to target monocytes/macro-phages with high selectivity for live-cell microscopy and flowcytometry. We then demonstrate that the injection of MFPallows sensitive in vivo epifluorescence imaging of the SLNwithinminutes because of active bindingmacrophages, amaincomponent of lymph nodes, and minimizing diffusion. Lymphnode-mapping data in the inflammation and the metastasismouse models showcase the potential of MFP for accuratelyassessing nodal status as a possible alternative to SPION-MRIin surgical guide imaging. Such an intraoperative macrophage-specific imaging with a small-molecule fluorescent probestrategy may be beneficial for improving surgical lymph-node
staging and for making the clinical management of cancermore effective.
Materials and MethodsCell preparation and differentiation
MOLT-4, HL-60, and U-937 cells were obtained from theAmerican Type Culture Collection (ATCC) and were main-tained according to ATCC protocols using RPMI1640 mediumwith 10% FBS, 1� GlutaMAX, 1� NEAA, 100 U/mL penicillin,and 100 mg/mL streptomycin (Life Technologies). For differ-entiation into macrophages, the nonadherent monocyte-likeU-937 cells (2� 105/mL) were induced to differentiate by a 24-hour exposure to 150 nmol/L phorbol-12-myristate-13-acetate(PMA; Sigma). After 24 hours, nonadherent cells were removedby a gentle washing with PBS.
Fresh whole-blood samples were obtained from consentinghealthy human donors. The NUS Institutional Review Board(NUS-IRB Reference code 12-195) approved the studies, andsubjects gave written, informed consent. Flow cytometry withMFP was performed on minimally manipulated whole-bloodsamples. Imaging experiments were carried out with enrichedT lymphocytes, granulocytes, monocytes, and monocyte-derived macrophages using negative magnetic-bead sorting(BD). A detailed description of the preparation and analysis ofblood cells can be found in the Supplementary Figs. S9 and S10and Supplementary Materials.
Chemical libraries and high-throughput screenThe combinatorial libraries of 1,153 fluorescent small mole-
cules from diversity-oriented synthesis were screened againstlymphocytes (MOLT-4) and monocytes/macrophages (HL-60,U-937, U-937-DM), which were plated onto 384-well plates at adensity of 7,000 cells/well in a culturemedium. The fluorescentcompounds were added at a final concentration of 0.5 or 1mmol/L in a volume of 100 mL per well containing 0.1%dimethyl sulfoxide (DMSO; v/v). After compound incubationfor 1 hour at 37�C, the cells were counterstained withHoechst33342 and imaged using an automated high-through-put imager (ImageXpress System, Molecular Devices). The hitsthat stained monocytes/macrophages more brightly than lym-phocytes were selected on the basis of the imaging perfor-mance and the intensity histogramwith the aid of high-contentimage analysis software (MetaXpress, Molecular Devices).
Synthesis and characterization of MFPA detailed description of the synthesis of the MFP can be
found in the Supplementary Materials. The synthesized MFPwas characterized using liquid chromatography–mass spec-trometry (LC–MS) as well as both 1H and 13C nuclearmagneticresonance (Supplementary Figs. S2–S4).
Cell line imaging and flow cytometryThe cells were incubated with 0.8 mmol/L and 50 nmol/L
MFP for microscopy and flow cytometry, respectively, in theculture medium for 1 hour at 37�C in a 5% CO2 atmosphere.Following incubation, all cells werewashedwith PBS to removeany probe left in the solution and to optimize the backgroundsignal. U-937-DMs were dissociated and collected using 0.25%
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trypsin–EDTA for flow cytometry. Imaging experiments wereperformed on a LSM5-DUO confocal microscope (Carl Zeiss).All MFP fluorescence images were obtained with identicalmeasurement parameters and contrasts. Flow cytometry wasperformed using a LSR-Fortessa cytometer (BD), anddatawereanalyzed using FlowJo software (Tree Star).
MiceA total of 20 Balb/c nude mice from the Biological Resource
Center (BRC; Biomedical Sciences Institute, A�STAR) wereused for the experiments, and their use was approved by theBRC Institutional Animal Care and Use Committee. For lymphnode metastasis-evaluation experiments, human lung carci-noma NCI-H460 cells (ATCC, 5 � 106) were implanted withMatrigel (BD) on the left dorsal flank of athymicmice. Two to 3weeks after injection, in vivo imaging experiments were per-formed. Lipopolysaccharide (LPS)-induced inflammationmodels were generated by intra-lymph node injection of LPS(5 mg/kg; Sigma) 2 days before the imaging experiments.
In vivo fluorescence imaging of lymph nodeMice were anesthetized by intraperitoneal injection of keta-
mine (150 mg/kg) and xylazine (10 mg/kg). The body temper-ature of the mice was kept constant at 37�C during allprocedures. TheMFPs [50 mmol/L in 50mL of saline containing1% of poly(ethylene glycol)4600(PEG) and 0.1% of Tween 20]were intradermally injected either in the left forepaw, fornormal and inflammation model imaging, or in both theforepaws for tumor model imaging. A detailed description ofreproducible injection technique can be found in the Supple-mentary Materials. An OV100 small animal imaging system(Olympus) was used to take fluorescence images of theaxillary lymph nodes after injection. In vivo imaging datawas analyzed with Matlab software (MathWorks). The SLN'starget-to-background ratio (TBR) was determined as theratio of the mean intensity calculated on the identifiedlymph node region divided by the one calculated in a similararea of surrounding fat and muscle tissue. The regions ofinterest (ROI) were manually chosen over the entire lymphnode, as well as in areas of surrounding tissue and areasoutside the mouse's body. The intensity measurements wererepeated 3 times and averaged, and the mean intensity andSD were used for the TBR calculations. Data are presented asmean � SD. Group comparisons were done by means of theunpaired Student t test.
ImmunohistofluorescenceAfter in vivo fluorescence imaging, the mouse was eutha-
nized and the axillary lymph node was dissected. Afterimmediate ex vivo fluorescence imaging, the lymph nodewas embedded in tissue-freezing medium (Triangle Biomed-ical Sciences), frozen, and consecutively cryo-sectioned in 8-mm segments. The tissue sections were rinsed with PBS andfixed with 4% paraformaldehyde for 20 minutes at roomtemperature. After an additional series of washes with PBS,the tissue sections were cleared with 3% sodium deoxycho-late solution for 2 hours at room temperature, blocked with20% normal goat serum in 1% BSA-PBS for 2h at 37�C,
incubated with the primary antibodies at 4�C overnight(>17 hours), diluted in 1% BSA–PBS, rinsed 3 times withPBS, and incubated with secondary antibodies at 4�C for 2hours. Then, the slides were washed with PBS several times,counterstained with Hoechst33342, and mounted with Pro-Long Gold antifade reagent (Life Technologies). The imageswere captured with an Eclipse Ti-E microscope (Nikon).
ResultsScreening identifies the unique fluorescent smallmolecule, MFP
Several classes of fluorescent small molecules (MW < 1,000Da) were synthesized based on a diversity-oriented approachin order to combine the properties of conjugatability andbiocompatibility. Libraries comprising 1,153 compounds werescreened against different human blood cell lines, that is,MOLT-4, HL-60, U-937, and U-937-derived macrophages (U-937-DM; Supplementary Fig. S1). We were particularly inter-ested in the selection of fluorochromes that could discriminatebetween lymphoid (MOLT-4) and myeloid cells (HL-60, U-937,U-937-DM). As a final hit, an MFP was discovered and showedthe most preferential staining for all myeloid lineage cells,compared with lymphoid cells, based on imaging performanceand intensity histogram analyses (Supplementary Fig. S1).
For further studies, we scaled up the synthesis of MFP andperformed characterization as summarized in SupplementaryFigs. S2–S4. MFP (lex/lem ¼ 562/589 nm; Supplementary Fig.S5) has a molecular weight of 505.75 Da, and displays excellentphotophysical properties with a high fluorescent quantumyield (0.15) and a high extinction coefficient (7.61 � 104
mol/L per centimeter). Fig. 1A shows an apparent preferenceof MFP for primitive human myeloid cells (HL-60), monocytes(U-937), and macrophages (PMA-stimulated U-937 cells), aswell asmurinemacrophages (RAW264.7), when using confocalmicroscopy. In addition, a flow cytometric analysis confirmedthe selectivity of the probe (Fig. 1B). The quantification shows agreater than 3-fold difference in intensity signal betweenmyeloid and lymphoid cells for both microscopy and flowcytometry (Fig. 1C and D). Differentiation of U-937-DMs wasconfirmed with differentiation markers (CD68, CD36) byimmunocytofluorescence and flow cytometry (SupplementaryFig. S6). MFP proved to be robust for live-cell imaging byprobing for monocytes/macrophages selectively at variousconcentrations (100–800 nmol/L) and incubation times (1–4hours; Supplementary Fig. S7). In addition, structural selectiv-ity was tested with several negative analogs of MFP (Supple-mentary Fig. S13). Even at a high concentration of MFP (25mmol/L) for 24 hours, the adverse effects of the probe on cellviability were minimal (Supplementary Fig. S8).
White blood cell phenotyping by MFPWe next tested peripheral blood taken from healthy human
donors to further evaluate the feasibility of using MFP forwhite-blood-cell phenotyping. Primary human T lymphocytes,granulocytes, monocytes, andmonocyte-derivedmacrophages(Supplementary Figs. S9 and S10) with macrophage colony-stimulating factor from three independent donors were allprobed with MFP in duplicates. Fig. 2A depicts representative
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fluorescence images demonstrating MFP as a compoundpreferred for primary monocytes and macrophages. All MFPfluorescence images had identical exposure times and sameoverall contrast settings to measure relative intensity differ-ence. Phenotypic characterization of nucleated blood cells inflow cytometry was feasible when employing MFP in conjunc-tionwithminimallymanipulatedwhole-blood samples (Fig. 2Band 2C) although competition for compound uptake wasincreased, thus specificity formonocytes was reduced in wholeblood environment compared with separated blood cell test(Fig. 2A). As shown in Fig. 2B and D, monocytes, granulocytesand lymphocytes—the three main components—can be repre-sented as MFPbright, MFPintermediate, MFPdim, respectively, ontheflowdot plot andhistogram. The quantification shows 1.73-and 13.2-fold difference in the fluorescence signal for mono-cytes as compared with those for granulocytes and lympho-cytes, respectively (Fig. 2C).To showcase the capability of MFP to isolate each of
these cell populations, we performed fluorescence-activatedcell sorting (FACS) for human leukocytes in MFPbright,MFPintermediate, MFPdim populations. Immunofluorescencestaining was performed with cytospin preparations for eachpopulation. Figure 2E shows a high expression of themonocyte
marker CD14 in MFPbright cells, a high expression of thegranulocyte marker CD66b in MFPintermediate cells, and a highexpression of the T lymphocyte marker CD3e and the Blymphocyte marker CD19 in MFPdim cells, indicative of suc-cessful isolation of the three main components of peripheralblood.
MFP SLN mappingDue to the specific macrophage-targeting ability of MFP, we
explored whether MFP could be used for lymphatic mappingbecause macrophages are the major cell type in lymph nodes.After an intradermal injection in the lymphatic-rich front pawof a mouse, MFPs entered the lymphatic collecting vessels andmigrated within minutes to an axillary node that could bedetected by epifluorescence imaging (Fig. 3A and B). Thefluorescent deposits became progressively denser, makingthe draining lymph node perceptible, and remained fixed inthe node for hours, with minimum diffusion to the next node.Owing to the outstanding photophysical properties of theMFPs, we could follow lymphatic flow toward the regionalnode from the injected forepaw in real time with high sensi-tivity and then dissect the node following quick identificationof its position (Fig. 3C and D), which mimics the SLNB
Figure 1. Selective staining of myeloid cells by MFP. A, fluorescence live imaging of blood-originating cells after incubation with 800 nmol/L of MFP for1 hour at 37�C. (scale bars, 10 mm). B, flow cytometry diagram of MOLT-4, HL-60, U-937, and U-937-DMs after incubation with 50 nmol/L of MFP for1 hour at 37�C. DMSO was added for unstained control cells. The plots for each pure-cell population are overlaid. Inset, chemical structure of MFP.C, quantitative image analysis from three separate experiments (mean� SD). All MFP fluorescence images are normalized to the peak signal to calculate themean intensity. D, quantitative flow cytometric analysis from three separate experiments (mean � SD).
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procedure in cancer surgery. Amagnified intraoperative imageand an ex vivo image (Fig. 3C and D) showed that the fluo-rescence signal associated with the SLN, but not the adjacentfat tissue, indicated preferential accumulation of MFPs in theSLN.
Lymph node inflammation imagingHaving shown the ability to detect the SLN with MFPs, we
proceeded to determine the ability of the MFP to distinguish amacrophage-rich, inflamed lymph node from a normal node.We first generated inflammation models (n ¼ 3) by intra–lymph-node injection of LPS into the left axillary node. Twodays after LPS injection, both the inflammation model and thecontrol group received an intradermal injection of MFPs in theleft forepaw. One hour after MFP injection, mice were imagedin vivo, and the node TBRs were calculated for the acquiredfluorescence images. In the inflammation-induced miceinjected with MFPs, intense signals in the axillary node were
observedwith in vivo fluorescence imaging, indicating elevatedmacrophage content (left in Fig. 4A and D). In contrast, theMFP-injected control mice demonstrated lower fluorescencesignals (right, Fig. 4A and B). In the MFP-injected mice, the invivo node TBRwasmore than 200% higher in the inflammationgroup than in the normal group (TBR: 5.8� 0.2 vs. 2.5� 1.5, P¼0.02; Fig. 4C).
In vivo imaging data were verified by immunohistofluores-cence of lymph nodes harvested 1 hour after injection of theMFP. Using several markers specific for lymph-node–consist-ing cells, wewere able to identify the characteristics of theMFPlocalization (Fig. 5). As shown in the first row in Fig. 5A and B,MFP fluorescence localized in the scattered large cells withinthe subcapsular sinus (SCS) and the medullary sinus—areasthat are well-known locations for lymph node macrophages.MFP-binding cells located beneath the SCS floor were typicallyin contactwith the lymph compartment that contains B cells inthe underlying follicles (Fig. 5A and B, the second bottom row),
Figure 2. MFP enables separation of human white blood populations by targeting peripheral monocytes. A, fluorescence live-cell imaging of enrichedperipheral leukocytes following Ficoll–Paque density gradient centrifugation after incubation with 800 nmol/L of MFP for 1 hour at 37�C. Scale bars, 100 mm.B, flow cytometry diagram of minimally manipulated whole-blood samples. The three main components, peripheral blood lymphocytes, monocytes, andgranulocytes, are represented on the scattering dot plot (left) based on their size and the cell's complexity. Owing to their different intrinsic affinities toMFP, the three distinct populations can bemore clearly seen (right) after incubationwithMFP at 50 nmol/L for 1 hour at 37�C. (SSC, side scatter channel; FSC,forward scatter channel). C, quantitative flow cytometric analysis from three independent donors (mean� SD). D, human peripheral leukocytes were sortedusing FACS into MFPbright, MFPintermediate, and MFPdim populations. E, immunofluorescence staining of the sorted cell slides prepared by cytocentrifugation.MFPbright, MFPintermediate, and MFPdim populations were identified as CD14-positive monocytes, CD66b-positive granulocytes, andCD3e-positive or CD19-positive lymphocytes, respectively. Scale bars, 50 mm.
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thus showing structural features ofmacrophages. Indeed,mostMFPþ cells expressed macrophage markers, including CD68,CD11b, and F4/80, showing selective macrophage targetingof MFP in the SCS and medulla (Fig. 5 and SupplementaryFig. S11). The MFP signal, however, did not colocalize withLYVE-1þ lymphatic endothelial cells surrounding medullarysinuses, Gr-1þ granulocytes and B220þ follicular B cells. Thehigher content of MFP-binding cells stained with macrophagemarkers in an inflamed node (Fig. 5A) compared with that in anormal node (Fig. 5B) in accordance with in vivo fluorescenceimaging.
Detection of metastases in draining lymph nodeRecently, a SPION was developed as an MRI contrast agent
to identify tumor-infiltrated lymph nodes and was successfullyapplied in clinics (34). This technology relies on an indirectidentification of metastasis in which nodal infiltration ofmacrophages that have engulfed the magnetic particles high-light metastatic regions devoid of signals. On the basis of itshighly specific targeting ability for macrophages, we hypoth-esized that MFP should enable in vivo assessment of lymphaticmetastasis, thus allowing for an excellent changeover fromSPION-MRI to intraoperative fluorescence imaging.
To test the hypothesis, we generated xenograftmodels of lungcancer (n ¼ 4) by left dorsal injection of NCI-H460 cells topromotemetastasis in the left axillarynode. Two to3weeks afterinjection, in vivo imaging experiments were performed afterintradermal injection of the MFP. Fig. 6 shows the diagnosticcapability of the MFP to detect lymph node metastasis using adeficient fluorescence signal caused by the replacement ofmacrophages by disseminated tumor cells. Injecting the MFPinto both forepaws produced a reduced signal in the ipsilateralaxillary node, but an enhanced signal in the contralateral axillarynode (Figs. 6A–6F). Intraoperative fluorescence imaging welldelineated the precise location of the nodal metastasis. Thecontralateral node's TBR with in vivo fluorescence images was21.9� 2.3. In contrast, the ipsilateral node exhibited low signals(180%–200% decrease), with a node TBR of 10.6� 3.1 (P¼ 0.001)indicating the replacement of macrophages by tumor lesions.Moreover, the intraoperative TBR was more than 360% lowerin the ipsilateral node than in the contralateral node (TBR: 6.8�3.1 vs. 24.8� 2.0, P¼ 0.00007). Ex vivofluorescence imaging afterimage-guided surgical resection of the respective lymph nodesconfirmed the significant intensity difference between the ipsi-lateral and the contralateral lymph nodes, which were consid-ered as macrometastatic and micro-/nonmetastatic nodes,respectively. The extensive tumor-cell infiltration in the
Figure 3. MFP rapidly targets theSLN by in vivo fluorescenceimaging. A, fluorescencereflectance images of axillary SLNs(arrows) attained in vivo afterintradermal injection of MFP in theleft paw of the mouse (scale bars,10 mm). A strong fluorescencesignal was detected at the SLNfrom 5 minutes postinjection. B,expanded views of A (scale bars,5 mm). C, images of the expandedsurgical field of B (scale bars,2mm). The fluorescence signalreflecting MFP putatively capturedby macrophage does notcolocalize with adjacent adiposeregions (asterisks) but colocalizeswith lymphatic vessels (dottedarrows) and lymph nodes (arrows).D, ex vivo fluorescence reflectanceimages of dissected SLN (scalebars, 2 mm). The left columnpresents white light color imagesshowing anatomical structures.The middle column presentsfluorescence reflectance images.The right column presents overlaidfluorescence and color images.The color lookup table applies to allfluorescence white-light fusionimages.
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ipsilateral node was confirmed by the node's enlarged size (Figs.6C and F) and immunohistofluorescent identification with atumor-specific HER2 antibody (Fig. 7).
The paw-directed injection of MFP in the tumor modelresulted in rapid dispersion to the draining axillary node andselective lymph node macrophage labeling. Immunohisto-fluorescence of frozen lymph node serial sections (Fig. 7 andSupplementary Fig. S12) revealed a specificity of theMFP to themacrophages that was similar to the specificity obtained fromthe inflamed-node analysis. All of theMFPs bound to CD68þ orCD11bþ or F4/80þ macrophages, but not to LYVE-1þ endo-thelial cells, Gr-1þ granulocytes, CD11cþ dendritic cells, B220þ
B lymphocytes or HER2þ cancer cells (Fig. 7 and Supplemen-tary Fig. S12). As predicted with in vivo imaging, large macro-scopic metastatic lesions with a high density of HER2þ cellswere present in the left axillary node, whereas onlymicroscopicinfiltration of HER2þ tumor cells was detected in the rightnode.
DiscussionRecent technologic evolutions in optical imaging and the
increasing list of fluorescent probes have driven the rapidprogression of fluorescence imaging into interventional clin-
ical procedures. Fluorescent guidance for SLNB has receivedparticular attention due to the significance of lymph nodes asthe first clinically observed site of most cancer metastasis. Themost common approach for SLN visualization iswith the use ofnonspecific nanoscale fluorescent agents with hydrodynamicdiameters of 10 to 50 nm, which are in the preferred range forlymphatic uptake. Although these techniques have improvedsurgical practice for sensitive and accurate identification ofSLNs, lymph node staging for metastasis can only be deter-mined by preoperative SPION-MRI or postoperative histopath-ologic analysis followed by an invasive lymphadenectomy. Inthis proof-of-concept study, we investigated the potential valueof intraoperative macrophage-specific fluorescence imagingfor pinpointing a suspicious lymph node to improve surgicaloutcomes for patientswith cancer using a xenograftmetastasismodel.
An essential component of this technology is the develop-ment of MFPs through a high-throughput screen of syntheticvital-dyes prepared by small-molecule chemistry with a pro-cess compatible with good manufacturing practices. Weshowed that, in mice, MFPs injected in the interstitial spaceof the paw were readily drained via lymphatic vessels todownstream axillary lymph node. Once the MFP had entered
Figure 4. MFP provides in vivofluorescence signal enhancementin inflamed lymph nodes. A and B,comparison of MFP signals for theLPS-injected inflammation model(left) and the normal mouse (right).In vivo fluorescence imagesshowed a higher signal, indicatinga larger macrophage content in theinflamed lymph node (dottedarrows) than in a normal node(arrows). Shown are representative(n¼ 3mice per group) color images(left column) and fluorescenceimages (right column, color lookuptable). The boxed area in A isenlarged in B. Scale bars are 10mm for A and 5 mm for B. C, TBRsof the LPS-injected and the normalmice after intradermal injection ofMFP were calculated using the invivo fluorescence images. Thevalues of the TBR in the LPS-injected animals were significantlyhigher than those in the normalanimals for in vivo fluorescenceimaging (�, P < 0.01, unpairedStudent t test). Data are reported asmean � SD (n ¼ 3 each).
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intranodal lymph conduits, it specifically binds strongly withthe SCS and medullary macrophages, but not with any othernode-constituting cells, thereby minimizing diffusion to thenext node and highlighting nodal inflammation. In addition,we found that lymph node metastases can be accuratelydetected by a node-by-node comparison of MFP uptake. MFPsselectively bound in macrophages of normal or micrometas-tastic lymph nodes, increasing the nodal fluorescence signalintensity. Nodes that are completely replaced by tumor tissuelack uptake of MFPs and generally show a low fluorescencesignal.
MFP binding in lymph nodes is reduced in the context oftumor deposit, whereas ideally one might select a directimaging agent that shows an increased signal from metastaticlesions compared to normal nodes. There is, however, apotential benefit to using an indirect contrast agent, such asan MFP for intraoperative lymph node imaging. The MFP iscapable of detecting not only nodal metastases but also localinflammation. The capability of the MFP to differentiatebetween metastatic and inflammatory lymph nodes will pro-vide important functional information for surgical guidancebecause inflammation- and tumor-induced lymph node
Figure 5. Histologic assessment of MFP targeting of inflamed lymph node. Shown are frozen lymph node serial sections stained with monoclonal antibodies(mAbs; green) to CD68 (macrophages), Gr-1 (granulocytes), B220 (B lymphocytes), and LYVE-1 (lymphatic vessels) with the presence of MFP positive cells(red). The axillary nodes in A and B were isolated from the mice depicted in the left and the right parts of Fig. 4A and B, respectively. The first row showswhole lymph-node section images, and the other rows depict representative magnified images. All sections were counterstained with Hoechst33342(blue). Arrowheads denote representative coexpressed cells with CD68 andMFP. The second row shows amagnified view of the boxed region in the first row.The scale bars represent 500 mm in the first row and 100 mm in all the other rows.
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enlargement can be misinterpreted, leading to invasive dis-section of benign nodes (43). Moreover, macrophage-specificfluorescence imaging can be used in most cancer surgeries,regardless of specific tumor type, because displacement ofnodal macrophages by tumor cells is a general phenomenon ofmost cancers (44). Together with these advantages, additionalstudies to establish standards of injection and image analysiscan make this technique more attractive for clinical use.Especially, recent multispectral reflectance imaging (8) oroptoacoustic technology (45) could be employed together forquantitative nodal staging by suppressing tissue-autofluores-cence and correcting light-attenuation.
The lymph node is not only a clinically important site forcancer prognosis but also a valuable organ to illuminate theimmune mechanism. In vivo live-cell imaging reveals lymphnode cellular dynamics, and is becoming increasingly usefulin the investigation of adaptive and innate immunity (46).Recently, lymph node macrophages were identified as crucial
gatekeepers to prevent fatal viral invasion (47–49). Because ofthe specific and stable macrophage targeting ability, MFP-mediated live-cell imaging to monitor the behavior of lymphnode macrophages will have certain benefits in the study ofhow they respond to pathogens in native physiologic settings.One interesting application of the lymph node is its use as anectopic transplantation site for multiple tissues and organregeneration (50). To make it realistic, the immune reactionswithin lymph nodes must be tested when healthy donor cellsare injected. The MFP live-cell imaging technique can then beused to observe interactions of macrophages with engraftedcells inside lymph nodes to validate immunogenicity. Overall,live imaging of lymph node macrophages with an MFP canshed new light on the in vivo biology of the immune system,providing measurements of cellular interactions and responsedynamics.
In summary, we present the targeted fluorescent agent MFPas a valuable research tool for in vivo behaviors ofmacrophages
Figure 6. SLNmapping with MFP allows in situ evaluation of nodal metastases. A–F, in vivo fluorescence imaging of the MFP-injected mouse with NCI-H460tumor cells grafted on the left dorsal flank to promote metastasis to the left axillary lymph node. Shown are representative (n ¼ 4) fluorescence reflectanceimages (right column, color lookup table) and their corresponding white-light images (left column) for a mouse injected with 50 mL of 50-mmol/L MFPin both forepaws4weeks after tumor cell implantation. The ipsilateral lymphnode (the left axillary lymphnode; dotted arrows inA–C) shows a low fluorescencesignal intensity with a nonhomogeneous pattern compared with the contralateral lymph node (the right axillary lymph node; arrows in D–F), indicating alack of macrophages due to extensive infiltration by tumor cells. The boxed regions in A and D are enlarged in B and E, respectively. The scale bars are10mm for A andD, and 5mm for B, C, E, and F. G, ex vivo fluorescence (right) and color (left) images of the resected sentinel axillary lymph nodes. Scale bars,2 mm. H and I, in vivo (H) and intraoperative (I) TBRs of the ipsilateral and the contralateral lymph nodes from four animals (mean þ SD).
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Cancer Res; 74(1) January 1, 2014 Cancer Research52
in lymph nodes, as well as a clinical navigation to aid surgeonsin debulking efforts by providing real-time information onadenopathy related to inflammation and caused by infiltrationof tumor cells. Intraoperative macrophage-specific fluores-cence imaging offers a unique opportunity for in situ detectionof lymph node metastases during cytoreductive surgery with-out the need for a preoperative lymph node biopsy or forintraoperative histology. Therefore, we consider this study tobe of clinical relevance and anticipate it to have considerableinfluence on the development of a new surgical oncologicimaging platform for lymph node staging.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: J.S. Yoo, Z.Y. Jow, Y.-T. ChangDevelopment of methodology: J.S. Yoo, S.-C. Lee, Z.Y. JowAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.S. Yoo, Z.Y. JowAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.S. Yoo, S.-C. Lee, Z.Y. Jow, P.Y.X. Koh, Y.-T. Chang
Writing, review, and/or revision of the manuscript: J.S. Yoo, S.-C. Lee, Z.Y.Jow, Y.-T. ChangAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.-C. Lee, Z.Y. Jow, P.Y.X. KohStudy supervision: Y.-T. Chang
AcknowledgmentsThe authors thank Drs. P.W. Kuchel and J. Kim for assistance with the human
blood cell experiments, and H.S. Cheng for management of tumor mouse. Theauthors also thank Drs. C.L. Teoh and R.E. Ericksen for critical review, and Drs.K-S. Soh, E. Kim, and M.H. Kim for scientific discussion. Microscopy data forimmunohistofluorescence were acquired and analyzed in the SBIC-Nikon Imag-ing Centre at Biopolis.
Grant SupportThis study was financially supported by intramural funding from the A�STAR
(Agency for Science, Technology and Research, Singapore) Biomedical ResearchCouncil and the SingaporeMinistry of Education Academic Research FundTier 2(MOE2010-T2-2-030).
The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received July 29, 2013; revised October 30, 2013; accepted October 31, 2013;published OnlineFirst December 9, 2013.
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2014;74:44-55. Published OnlineFirst December 9, 2013.Cancer Res Jung Sun Yoo, Sung-Chan Lee, Zhi Yen Jow, et al. Node StagingA Macrophage-Specific Fluorescent Probe for Intraoperative Lymph
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