Cytotherapy, 2014; 16: 699e710

Magnetic resonance and near-infrared imaging using a novel dual-modality nano-probe for dendritic cell tracking in vivo YU-CHEN CHEN*, SONG WEN*, SONG-AN SHANG, YING CUI, BING LUO & GAO-JUN TENG Jiangsu Key Laboratory of Molecular and Functional Imaging, Department of Radiology, Zhongda Hospital, Medical School, Southeast University, Nanjing, China Abstract Background aims. The effect of cellular-based immunotherapy is highly correlated with the success of dendritic cells (DCs) homing to the draining lymph nodes (LNs) and interacting with antigen-specific CD4þ T cells. In this study, a novel magneto-fluorescent nano-probe was used to track the in vivo migration of DCs to the draining LNs. Methods. A dualmodality nano-probe composed of superparamagnetic iron oxide (SPIO) and near-infrared fluorescent (NIRF) dye (NIR797) was developed, and its magnetic and optical contrasting properties were characterized. DCs generated from mouse bone marrow were co-cultured with the probe at a lower concentration of 10 mg/mL. The cell phenotype and function of DCs were also investigated by fluorescence-activated cell sorting analysis and mixed leukocyte reactivity assay. Labeled DCs were injected into the footpad of C57BL/6 mice. Afterward, magnetic resonance imaging, NIRF imaging, Perls staining and CD11c immunofluorescence were used to observe the migration of the labeled DCs into draining LNs. Results. The synthetic SPIO-NIR797 nano-probe had a desirable superparamagnetic and near-infrared behavior. Perls staining showed perfect labeling efficiency. The cell phenotypes, including CD11c, CD80, CD86 and major histocompatibility complex class II, as well as the T-cell activation potential of the mature DCs were insignificantly affected after incubation (P > 0.05). Labeled DCs migrating into LNs could be detected by both magnetic resonance imaging and NIRF imaging simultaneously, which was further confirmed by Perls staining and immunofluorescence. Conclusions. The novel dual-modality SPIONIR797 nano-probe has highly biocompatible characteristics for labeling and tracking DCs, which can be used to evaluate cancer immunotherapy in clinical applications. Key Words: dendritic cell, dual-modality, immunotherapy, migration

Introduction Dendritic cells (DCs) are antigen-presenting cells with the unique ability to take up and process antigens in the peripheral blood and tissues. They subsequently migrate to draining lymph nodes (LNs), where they present antigen to resting lymphocytes (1,2). DCs are an essential target for generating therapeutic immunity against cancer (3). Development of targeted nano-delivery systems carrying vaccine components, including antigens and adjuvants, to DCs in vivo represents a promising strategy to enhance immune responses (4). For effective immunotherapy, the antigen-loaded DCs must migrate into lymphoid tissues to present their antigens to T cells located within LNs (5e7). Accurate deposition and migration of DCs to target organs remain crucial for the success of immunotherapy.

Evaluating the migration efficiency of DCs requires an effective and non-invasive imaging method. Superparamagnetic iron oxide (SPIO) nanoparticles have been considered excellent contrast agents for magnetic resonance imaging (MRI) and can be used for cell labeling, cell tracking, sorting and purifying procedures (8e10). The SPIO particle has a small diameter and strong penetrating capability into cells, which makes it possible to cause signal change in MRI at a super-low tracer concentration (nanomole level) (11). SPIO has been used for labeling and tracking stem cells, such as mesenchymal stromal cells and endothelial progenitor cells (9,10,12). Several groups have reported the application of SPIO to label and track DCs in vitro and in vivo (13e15), which was successfully used in a clinical trial (16). However, the low sensitivity and

*These authors contributed equally to this work. Correspondence: Gao-Jun Teng, MD, PhD, Department of Radiology, Zhongda Hospital, Southeast University, 87 Dingjiaqiao Road, Nanjing 210009, China. E-mail: [email protected] (Received 22 February 2013; accepted 28 September 2013) ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2013.09.006

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biologic safety were limitations. Near-infrared fluorescent (NIRF) imaging can increase light emission by >100-fold compared with small organic fluorophores at similar wavelengths, which enables realtime observation and has a more sensible and longer visualization for tracking in vivo (17,18). Some groups also exploited NIRF dyes to track the DC migration in vivo (19). However, the poor penetrability and toxicity should be considered. At the present time, numerous dual-modality probes and techniques combining SPIO and NIRF have been developed to monitor cell engraftment, which can provide more information than achieved by using any single-modality approach and can overcome the shortcomings from each method (20,21). However, none of these techniques have been dedicated to tracking the migration process of DCs effectively and non-invasively. In the present study, we developed a novel magneto-fluorescent dual-modality imaging nanoprobe, SPIO-NIR797. We investigated whether SPIO-NIR797 was non-toxic for labeling and tracking DCs and whether magnetic resonance imaging (MRI) and NIRF imaging could enable in vivo surveillance over migration and homing of DCs to the targeted LNs.

Methods Reagents RPMI-1640 medium (Clonetics, Sunnyvale, CA, USA), penicillin and streptomycin were obtained from Gibco Invitrogen (Grand Island, NY, USA). The following were also used for the experiments: fetal bovine serum (Gibco, Life Technologies, Grand Island, NY, USA), recombinant murine granulocytemacrophage colony-stimulating factor, recombinant murine interleukin-4, tumor necrosis factor-a (TNF-a) (all Peprotech, Rocky Hills, NJ, USA), Perls staining kit (Yuanye Chemical Co, Ltd, Shanghai, China), NIR797-isothiocyanate (molecular weight 880.14 Da), lipopolysaccharide (LPS), poly-L-lysine (PLL) (all Sigma-Aldrich, St Louis, MO, USA), isoflurane (Abbott, Shanghai, China), Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan), CD11c, CD80, CD86, major histocompatibility complex class II (MHC-II) (all eBioscience, San Diego, CA, USA), propidium iodide (Biouniquer; BioStar Pharmaceuticals, Nanjing, China), red blood cell lysis buffer (Beyotime, Shanghai, China), trypan blue staining (Yuanye Chemical Co, Ltd, Shanghai, China), 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Life Technologies, Breda, the Netherlands), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining kit (Roche Inc, Mannheim, Germany), mouse

monoclonal anti-CD11c antibody, Alexa Fluor 488 Donkey Anti-Mouse and normal donkey serum (all Abcam, Hong Kong, China) and Triton-X (Amresco, Solon, OH, USA).

Synthesis and characterization of SPIO-NIR797 The preparation of SPIO-NIR797 is schematically shown in Figure 1A. SPIO-polyacrylic acid (PAA) was a gift from Dr Dong-Fang Liu. Methods for the synthesis of SPIO-PAA have been well described (22,23). SPIO-PAA was diluted with deionized water to a concentration of 1 g/L and reacted with 1% PLL diluted in RPMI-1640 medium on a rotator shaker for 2 h to obtain PLL-capped SPIO-PAA nanoparticles. The corresponding mass ratio of SPIO-PAA to PLL was 1:0.05. PLL-SPIO-PAA nanoparticles and NIR797-isothiocyanate were mixed in a hydrous dimethyl sulfoxide and incubated for 3 h with continuous mixing at room temperature (calculated molar ratio, NIR797:PLL-SPIO-PAA ¼ 1:10). The unconjugated NIR797-isothiocyanate was dialyzed through rapid rotation in double distilled water three times, and the SPIO-NIR797 conjugates were purified and stored at 4 C with the final concentration adjusted to 10 mg/mL for cell labeling experiments. The morphology of SPIONIR797 was characterized by transmission electron microscopy (JEOL 100CX transmission electron microscope; JEOL, Tokyo, Japan), and the particle sizes and size distributions were calculated using at least 400 particles with image analysis software (Image-Pro Plus 5.0; Media Cybernetics, Bethesda, MD, USA). Immediately after synthesis of the nano-probe, the hydrated particle sizes were analyzed by dynamic light scattering (90Plus Particle Size Analyzer; Brookhaven Instruments, Holtsville, NY, USA), and the magnetic properties of the iron oxide nanoparticles were measured using a vibrating sample magnetometer (model number 7407; Lake Shore Cryotronics, Westerville, OH, USA). Dynamic light scattering measurements were performed for 4 weeks. Each measurement was repeated three times to obtain a mean value.

Mice All experimental animal protocols were approved by the animal care committee of Southeast University, Nanjing, China. Male C57BL/6 mice and BALB/c mice, 6e8 weeks old (Shanghai Laboratory Animal Center, Chinese Academy of Science, China), were used in this study and housed under standard conditions at the Central Animal Facility, Southeast University.

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Figure 1. Characterization of the SPIO-NIR797 nano-probe. (A) Schematic diagram of the SPIO-NIR797 nano-probe. (B) Representative transmission electron microscopy image of SPIO-NIR797 nano-probe. Upper insert shows a photograph of the SPIO-PAA (left) and SPIONIR797 (right) nano-probes in PBS (0.3 mg/mL) in 4 C. (C) Dynamic light-scattering diameters of the SPIO-PAA and SPIO-NIR797 nano-probes in PBS. (D) Room temperature magnetization curve of SPIO-NIR797 nano-probe.

Isolation of bone marrow-derived DCs DCs were harvested from male C57BL/6 mice, 6e8 weeks old. Bone marrow monocytes were flushed out from the femora and tibiae of mice with RPMI-1640 medium, filtered by a 400-mm filtering mesh and depleted of erythrocytes in red blood cell lysis buffer for 5 min. The cells were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 ng/mL recombinant murine granulocyte macrophage colony-stimulating factor and 1 ng/mL recombinant murine interleukin-4 (24) at 37 C in 5% CO2. On days 3 and 5, half of the media were suctioned,

followed by addition of fresh media. The released non-adherent and loosely adherent cells were collected on day 6 and used as immature DCs. The cells were stimulated with 1 mg/mL LPS for 48 h and collected on day 8 as mature DCs.

Cell labeling and Perls staining SPIO-NIR797 was diluted with RPMI-1640 medium before labeling. Immature and mature DCs were co-cultured with 10 mg/mL SPIO-NIR797 for 12 h, washed three times with phosphate-buffered solution (PBS) and harvested by blowing lightly.

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Labeled cells were fixed through incubation with 4% paraformaldehyde for 30 min, incubated for 30 min with 10% potassium ferrocyanide in 10% hydrochloric acid, washed and counterstained with 0.1% nuclear fast red for 5 min. Finally, the cells were examined with a light microscope (Carl Zeiss, Jena, Germany). Cell viability and fluorescence-activated cell sorting analysis For cytotoxicity analysis, labeled and unlabeled mature DCs were tested using trypan blue staining. For phenotypic analysis, four types of cells were stained at 4 C for 30 min with fluorescein isothiocyanate-conjugated monoclonal antibodies specific for CD11c and phycoerythrin-conjugated monoclonal antibodies specific for CD80, CD86 and MHC-II. After thoroughly washing with PBS, the cells were analyzed with a FACScan instrument (FACSCalibur, FACSCanto; BD Biosciences, Franklin Lakes, NJ, USA) using the CellQuest software (BD Biosciences). The apoptosis rate was measured by cell cycle analysis. Briefly, labeled and unlabeled mature DCs were collected, stained with propidium iodide and washed. The DNA contents in different periods of the cell cycle (G1, S, G2) and the apoptosis rate were also measured by FACS analysis. All experiments were performed in triplicate for each condition, and values were obtained from three separate experiments. Mixed leukocyte reactivity assay CD4þ T cells were acquired from the spleen of the BALB/c mice. Labeled and unlabeled DCs were incubated with the heterogeneous splenic T cells at ratios of 1:1, 1:2, 1:5, 1:10, 1:20, 1:50 and 1:100 in 96-well flat-bottomed plates (Corning, Corning, NY, USA). Each group was repeated in three wells to get a mean value. After 3 days, CCK-8 was used to evaluate cell activation and proliferation, and the absorbance was measured using an enzyme-linked immunosorbent detector (Tecan, Männedorf, Switzerland). Mixed leukocyte reactivity analysis was repeated three times. Cellular MRI analysis in vitro and in vivo Both cells and mice were imaged with a 7.0-tesla micro-MRI scanner (PharmaScan; Bruker, Bremen, Germany) using a 23-mm mouse head circular volume coil. For the in vitro study, 1  104, 1  105 and 1  106 SPIO-NIR797-labeled DCs were pelleted by 1% agarose gel. A turbo rapid acquisition with relaxation enhancement (RARE) T2-weighted sequence

(repetition time/echo time ¼ 2500 ms/11 ms, slice ¼ 5, number of averages ¼ 4, matrix ¼ 256  256, RARE factor ¼ 8) was used. For the in vivo study, C57BL/6 mice (n ¼ 10) were pre-injected with TNFa in the footpads of both hind legs (30 ng/leg) (25) for 24 h. The mice were injected in the right footpad with 1  106 SPIO-NIR797-labeled DCs in 50-mL PBS solution (n ¼ 10) as well as in the contralateral footpad with either 50 mL unlabeled DCs (n ¼ 5) or 50 mL PBS solution (n ¼ 5). After injection, mice were anesthetized with 1e2% isoflurane delivered in O2 2.5 L/min and placed on a warming pad maintaining body temperature near 37 C throughout the scan. Axial fast low-angle shot-multi-slice T2*weighted sequences with fat suppression were used for evaluation of the inguinal and popliteal LNs (repetition time ¼ 333.1 ms, echo time ¼ 5.0 ms, slice thickness ¼ 0.48 mm, slices ¼ 25, number of averages ¼ 6, matrix ¼ 512  512). Dynamic MRI was performed every 24 h for 5 days. Images were analyzed for LN volume, signal void volume and fractional signal loss, which was described previously (26). NIRF imaging in vitro and in vivo Both cells and mice were imaged with the Maestro system (CRi, Woburn, MA, USA). For in vitro study, 1  104, 1  105 and 1  106 SPIO-NIR797-labeled DCs in 300 mL of PBS solution were placed into 300-mL tubes. The tubes were placed into the imaging chamber. White light and NIRF images were obtained using the Maestro in vivo imaging system with exposure times of 0.5e4 s. Optimized parameters of excitation and emission filters were used to obtain a maximized fluorescence signal and low background. The excitation filter is 684e729 nm, whereas a 745-nm-long pass was used for the emission filter. For the in vivo study, DC-injected mice were anesthetized with 1e2% isoflurane and supplementary oxygen after each MRI scan; the fur overlying the abdomen and legs was shaved in accordance with the animal care committee protocol guidelines. Histomorphometry and immunofluorescence After injection of DCs for 48 h, mice were euthanized, and the bilateral inguinal and popliteal LNs were removed to analyze the distribution of SPIONIR797-labeled DCs. LNs were fixed with 4% paraformaldehyde in PBS overnight, embedded in optimal cutting temperature compound (Sakura FineTechnical, Tokyo, Japan) and frozen at 80 C. Cryo-sections (10 mm) were cut using a cryostatic microtome and transferred to glass slides for Perls

A novel dual-modality nano-probe for tracking dendritic cells staining, NIRF imaging and CD11c immunofluorescence. For Perls staining, the procedure was the same as that of the labeled cell staining. In addition, the slides were dehydrated in 95% ethanol. The iron distribution was confirmed by a light microscope. For CD11c immunofluorescence, 10-mm-thick cryosections were prepared and fixed through incubation with 4% paraformaldehyde for 30 min. The sections were blocked in 0.1% Triton-X and 10% normal donkey serum for 1 h and incubated with mouse anti-CD11c monoclonal antibodies for 1 h at room temperature. After washing in PBS solution three times, the sections were incubated with Alexa Fluor 488 Donkey Anti-Mouse for 1 h. The distribution of the labeled DCs in the draining LNs was investigated using a multi-channel fluorescent microscope (BX53; Olympus, Hamburg, Germany). The nearinfrared and CD11c immunofluorescence images were merged using the post-processing QImaging software (QImaging, Surrey, BC, Canada). We stained LN sections with an in situ cell death detection kit-TUNEL staining to assess apoptotic cells in the popliteal LNs. Quantitative analysis of migration capacity Both unlabeled and SPIO-NIR797-labeled DCs (1  106) were additionally stained with 1 mmol/L CFSE for 10 min at 37 C and washed extensively. We injected SPIO-NIR797-labeled CFSEþ DCs into right hind footpad and unlabeled CFSEþ DCs into the contralateral hind footpad of the mice with TNF before injection. After 24 h, popliteal LNs were dissected, and single-cell suspensions were prepared and measured by FACS analysis. We performed the measurements at 24 h, 48 h, 3 days, 4 days and 5 days after injection in accordance with dynamic MRI observation. All the measurements were performed in triplicate for each time point. Statistical analysis The results were presented as mean  standard deviation. Data were analyzed using the Statistical Package for Social Sciences (version 17.0; SPSS Inc, Chicago, IL, USA). Differences between the control and test groups were assessed by one-way analysis of variance and two-tailed Student t test. For all statistical analyses, P < 0.05 was considered significant. Results Characterization of SPIO-NIR797 nano-probe Figure 1A shows the schematic diagram of the SPIONIR797 nano-probe. As shown in the transmission

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electron microscopy image and distribution map (Figure 1B), both the SPIO-PAA and the SPIONIR797 nano-probes were well dispersed in the PBS solution. The SPIO-NIR797 nano-probe had a higher hydrated diameter than the SPIO-PAA (50.57  4.78 nm versus 26.90  4.07 nm) (Figure 1C). The saturation magnetization value of the SPIONIR797 nano-probe was 61.1 emu/g iron at 25 C (Figure 1D). The SPIO-PAA and SPIO-NIR797 nano-probes presented limited ( 0.05). To determine whether DC phenotypes would be influenced after SPIO-NIR797 labeling, a panel of monoclonal antibodies specific for CD11c, CD80, CD86 and MHC-II was used to analyze the cell phenotype through immunostaining assay followed by FACS. As shown in Figure 3, after stimulation by LPS, 82.84% of the cells were CD11cþ; CD11c is considered a major cell phenotype of DCs. The expression of CD80, CD86 and MHC-II significantly increased in both labeled immature and mature DCs in contrast to unlabeled immature cells. The labeled and unlabeled mature DCs were similar in terms of the phenotypic changes of CD11c, CD80, CD86 and MHC-II, with no significant difference between groups (P > 0.05). To examine any effect of the SPIO-NIR797 nano-probe on cell apoptosis, the apoptosis rates of both labeled and unlabeled mature DCs were measured by FACS. The results indicated that although there was slight increase of apoptotic cells after labeling, most DCs were unaffected (Figure 4, Table I).

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Figure 2. SPIO-NIR797 nano-probe labeling of DCs. (A) Unlabeled mature DCs (200) and enlarged morphology (400). (B) SPIONIR797-labeled DCs without staining. (C) Labeled DCs with Perls staining. (D) Trypan blue cell viability assay reveals no significant difference between unlabeled cells and SPIO-NIR797-labeled cells. (Scale bar ¼ 10 mm.)

Mixed leukocyte reactivity assay To investigate the ability of labeled DCs to induce T-cell proliferation, labeled and unlabeled DCs were co-cultured with prepared splenic CD4þ T cells of BALB/c mice at DC-to-T cell ratios of 1:1, 1:2, 1:5, 1:10, 1:20, 1:50 and 1:100. The ability of DCs to stimulate the proliferation of CD4þ T cells was assessed by the CCK-8 assay. With decreased ratio from 1:1 to 1:100, the activation

and proliferation of CD4þ T cells gradually decreased with no significance. The absorbance of LPS-stimulated mature DCs or SPIO-NIR797labeled DCs was significantly higher than absorbance of immature DCs (P < 0.05). Both no labeling and labeling with the SPIO-NIR797 nanoprobe had minimal effect on the potential of the DCs to stimulate CD4þ T cells at various ratios (Figure 5).

Figure 3. Phenotypic changes of DCs after labeling with the SPIO-NIR797 nano-probe through FACS analysis. iDC, immature dendritic cell; mDC, mature dendritic cell.

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Figure 4. DNA contents during different periods of the cell cycle. (A) Unlabeled mature DCs (mDCs). (B) SPIO-NIR797-labeled mDCs. Dip, diploid; FL2-A, fluorescence 2-area.

In vitro dual-modality imaging of labeled DCs To observe the DC labeling efficiency through the dual-modality imaging technique, labeled DCs were diluted at various concentrations ranging from 1  104 to 1  106 cells. NIRF imaging and T2weighted MRI were performed using a near-infrared optical imaging system and a 7.0-tesla MRI scanner, respectively. For in vitro imaging, the detectable NIRF signal intensities gradually increased, with concentrations ranging from 1  104 to 1  106 cells (Figure 6A), whereas the T2-weighted signal intensities gradually decreased with increasing concentrations (Figure 6B).

In vivo tracking of labeled DCs through dual-modality imaging To determine whether the SPIO-NIR797 nano-probe can be used for the in vivo tracking of the migration of DCs to the draining LNs, a near-infrared optical imaging system and a 7.0-tesla MRI scanner were used. After dynamic imaging for 48 h, a decrease in signal intensity (darkening) in the right popliteal LN was observed to confirm the presence of SPIO-NIR797labeled DCs within the margin of the draining LN (red arrows in Figure 7A), whereas no signal was detected in the contralateral popliteal LN (white arrows in Figure 7A) and in both the inguinal LNs (data not shown). This observation suggested that the labeled DCs had successfully migrated to the popliteal LN from the injection region. MRI images were Table I. DNA contents during different periods of the cell cycle in unlabeled and labeled mature DCs. DNA contents

Mature DCs

Apoptosis (%) Diploid G1 (%) Diploid S (%) Diploid G2 (%)

1.90 94.86 1.24 3.23

*P < 0.05.

   

0.02 0.76 0.17 0.18

Labeled mature DCs

P value

   

0.006* 0.009* 0.004* 0.001*

2.00 92.28 1.91 5.63

0.03 0.56 0.10 0.40

analyzed for LN volume, signal void volume and fractional signal loss (Figure 7DeF). The popliteal LNs that received either unlabeled or labeled DCs were significantly larger than the LNs that received PBS at 48 h after injection (P < 0.05). The signal void volume and fractional signal loss at 48 h were also significantly larger for labeled DCs than before injection or 24 h after injection (P < 0.05), but there was no significant increasing trend after 48 h. The NIRF image showed strong NIRF signals in the right footpad and the right popliteal LN (red arrow and white light in Figure 7B), which was in line with the MRI results. The enlarged view of the right popliteal LN clearly showed the aggregation of the labeled DCs (Figure 7C). Histologic observation To confirm the migration of SPIO-NIR797-labeled DCs into the LNs as the in vivo imaging showed, the LNs were sectioned and subjected to Perls staining for iron detection and fluorescein isothiocyanate-conjugated anti-CD11c antibody staining for cell detection. Anti-CD11c antibody staining (Figure 8A), NIRF imaging (Figure 8B) and blue staining (Figure 8D) were detected in the cortical area of the popliteal LN. The merged image of the CD11c immunofluorescence and NIRF also showed the distribution of the labeled DCs in the popliteal LN (Figure 8C). In comparison, no blue staining or fluorescence was detected on the contralateral side and bilateral inguinal LNs. Fewer TUNEL-positive cells could be seen in both the right popliteal LN and the contralateral side, which revealed that most labeled DCs within the draining LN survived and few cells died (see supplementary Figure 2). Quantitative analysis of migrated labeled DCs To elucidate the impact of SPIO-NIR797 labeling on the migratory capacity of DCs in vivo and to measure the migration efficiency, we used FACS analysis to

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Figure 5. Mixed leukocyte reactivity assay of unlabeled DCs and SPIO-NIR797-labeled DCs with splenic CD4þ T cells. Unlabeled DCs and SPIO-NIR797-labeled DCs were co-cultured with splenic CD4þ T cells for 3 days at ratios of 1:1, 1:2, 1:5, 1:10, 1:20, 1:50 and 1:100, and proliferation of CD4þ T cells was measured by the CCK-8 method (450 nm). *P < 0.05. iDC, immature dendritic cell; mDC, mature dendritic cell, O.D., optical density.

measure the migrated cell number into the popliteal LN by CFSE staining. Figure 8E showed that detection of migrated unlabeled and SPIO-NIR797labeled CFSEþ DCs into the popliteal LNs reached a maximum at 48 h and declined thereafter because of the lower sensitivity and quenching effect. The mean percentage of migrated labeled DCs into the popliteal LN was about 3.5%. Unlabeled and SPIONIR797-labeled CFSEþ DCs had similar levels of migration efficiency. Discussion DCs are the most proficient antigen-presenting cells that are poised to sample the environment and transmit the gathered information to cells of the adaptive immune system (27,28). The aim of DCbased anti-cancer vaccination is to induce tumorspecific effective T cells that can reduce the tumor

mass specifically (3). Accurate delivery of antigens to DCs and the effective immune activation of the pathway play the most important roles in developing DC-based anti-cancer vaccines and achieving successful immunotherapy. However, where and if the cells can migrate after injecting DCs subcutaneously and what the cellular viability and function are in vivo still have not been known accurately with effective and non-invasive techniques. In this study, we are the first to develop a novel dual-modality nanoprobe, SPIO-NIR797, for tracking migration of DCs in vivo and detecting them through a non-invasive combined approach. SPIO is a nano-sized iron oxide particle that can be biologically degraded, metabolized and integrated into a serum iron pool to form hemoglobin or to enter other metabolic processes (11). It has been an excellent MRI contrast agent for monitoring cell migration and homing (9,29). Some researchers reported that SPIO could be easily delivered into DCs and tracked by MRI (13,14). MRI with SPIO can provide additional anatomic information and more detailed visualization but has a low sensitivity. To overcome this limitation, in addition to SPIO cell labeling, we employed an optical imaging agent, NIR797. Previous results have shown that in vivo NIRF imaging appears promising for dynamically tracking cell migration because of several advantages, such as high sensitivity, high spatial resolution and relatively simple operation (17). A multi-modal cell tracking method can compensate for the disadvantages of each modality. SPIO-PAA can be readily phagocytosed by macrophages and would remain mostly in the cytoplasm (23). PLL has been known to be non-toxic, biocompatible and biodegradable, with low immunogenicity and anti-bacterial properties (30). In our study, the SPIO-NIR797 nano-probe was composed

Figure 6. Dual-functional properties of SPIO-NIR797-labeled DCs in vitro assay. (A) Signal intensity of labeled DCs at different concentrations by NIRF imaging. (B) Signal intensity of labeled DCs at different concentrations by MRI.

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Figure 7. SPIO-NIR797-labeled DCs homing to the draining LNs in vivo assay. (A) Labeled DCs homing to the right popliteal LN by MRI. Red arrow indicates the location of signal loss within the popliteal LN. White arrow shows no signal changes within the control side. (B) Labeled DCs homing to the popliteal LN by NIRF imaging. Red arrow indicates NIRF in the popliteal LN. (C) Enlarged pseudo-color image of labeled DCs within the popliteal LN. (D) LN volume was measured from images acquired before injection (0 h) and 24 h, 48 h, 3 days, 4 days and 5 days after injection of PBS, unlabeled DCs or labeled DCs. (E) Signal void volume and (F) fractional signal loss were measured from images acquired before injection (0 h) and 24 h, 48 h, 3 days, 4 days and 5 days after injection of labeled DCs. *P < 0.05.

of synthetic SPIO-PAA and PLL with tethered NIR797 (Figure 1A). The nano-probe was better for enhancing cytomembrane permeability. DCs are a particularly important cell type in the study of

SPIO-NIR797 properties, such as biocompatibility, cellular uptake, localization and molecular imaging. After synthesizing the SPIO-NIR797 nano-probe, we first investigated its influence on the cell phenotype

Figure 8. Histomorphometry of SPIO-NIR797-labeled DCs within the LN. (A) CD11c immunofluorescence (green color) of LN cryosections. (B) The NIRF imaging (red color) of the LN cryo-sections. (C) Merged imaging of CD11c immunofluorescence (green color) and NIRF (red color). Yellow color indicated the existence of labeled DCs. (D) Perls staining of LN cryo-sections. (Scale bar ¼ 20 mm.) (E) Percentage of CFSEþ DCs within the popliteal LNs by FACS analysis at 24 h, 48 h, 3 days, 4 days and 5 days after injection of unlabeled or SPIO-NIR797-labeled DCs.

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and viability of the labeled DCs in vitro. Owing to the properties of SPIO-NIR797 and the negative cytomembrane potentials and phagocytotic properties of DCs (6), iron particles permeated the cytoplasm of almost all the DCs through Perls staining (Figure 2C). Trypan blue staining showed no significant cell death after labeling (Figure 2D). We presumed that the SPIO-NIR797 nano-probe was phagocytosed as a kind of external antigen by immature DCs, which induced early maturation of the DCs. The expression of CD80, CD86 and MHC-II significantly increased in labeled immature DCs. SPIO-NIR797 had no significant impact on the phenotypes of labeled mature DCs (Figure 3). Additionally, the cell apoptosis rates had been little influenced by SPIO-NIR797 (Figure 4). SPIONIR797-labeled mature DCs did not influence the function of inducing the proliferation of T cells by mixed leukocyte reactivity assay (Figure 5). Mackay et al. (31) developed a transfecting agent-coated hybrid imaging nano-probe, which was composed of near-infrared light emitting quantum dots tethered to the SPIO nanoparticles and applied to imaging of the migration of DCs. However, quantum dots had a greater toxicity if allowed to interact with the cellular environment (32). On MRI and NIRF imaging in vitro, the detectable NIRF signal intensities gradually increased, whereas the T2-weighted MRI signal intensities gradually decreased with ranging cell concentrations (Figure 6), which indirectly showed that SPIO particles had no interaction with the NIRF signal after combining SPIO with NIR797. According to these results, we could label SPIO-NIR797 on mature DCs with minimal impairment and higher labeling efficiency. In our study, for safety considerations, we cocultured the mature DCs and the nano-probe at an optimal concentration of 10 mg/mL, which was lower than the average concentrations in some reports on iron particle-labeled or fluorescent dye-labeled DCs. The labeling efficiency acquired in this study almost equals the efficiency reported in the published articles (15,19,31,33). From our results, we can conclude that this low concentration has a higher labeling efficiency with minimum perturbation on cellular functionality. Because of the high spatial resolution and sensitivity of non-invasive imaging, we used MRI and NIRF to explore whether SPIO-NIR797 labeling affects DC function in vivo. The number of DCs migrating from the hypodermis to the draining LNs was low, and TNF-a was injected beforehand into the footpads to improve migration ability (25). Through dynamic MRI quantitative analysis for 5 days (Figure 7), the time to detect significant increasing signal changes was found to be 48 h, which was

similar to some studies (15,34), although 12 h or 24 h had also been reported (35,36). The differences may be due to the different cell densities and the cytotoxicity of the nano-probe. Strong MRI and NIRF signals could be detected only in the right popliteal LN, which was in accordance with the histomorphometry results (Figure 8AeD), indicating that no labeled DCs migrated into the non-draining and secondary draining LNs. Perls staining and the merged image of NIRF and CD11c immunofluorescence showed that SPIO-NIR797-labeled DCs existed in the margin of the LN, mostly in the subcapsular sinus area, which was reported as the first gathering place. DCs then migrated to the medulla to stimulate T cells (37). The optimal numbers of DC per LN for adequate immune response in clinical studies have not been demonstrated. In this study, we chose a density of 1  106 cells for in vivo experiments owing to a relatively good signal intensity of MRI and NIRF. The quantitative analysis of migrated DCs revealed that SPIO-NIR797 labeling did not change the migration capacity of DCs. The mean percentage of migrated DCs into the popliteal LN was about 3.5% (Figure 8E), which was similar to other reports (25,38). The minimum cell number that was necessary for in vivo detection was not exactly the same because of cell viability and MRI sensitivity. Generally, at least 2  103 labeled DCs could be observed in draining LNs through MRI or fluorescent imaging according to several researchers (15,38). A previous report showed that a limitation of cell density was crucial for improving migration of DCs to LNs in vivo (39). Most DCs died at the injection site, but even minimal numbers of DC could cause a great amount of T-cell activation and proliferation to induce specific immunologic response (25,40). The cell viability, not the injected cell number, plays a crucial role in stimulating T cells in the regional LN. During this study, we found an unanticipated result from two mice. A more obvious region of MRI signal void in the popliteal LN than others was observed, whereas no significant signal changes were detected in the ipsilateral inguinal LN from both MRI and NIRF imaging. However, histomorphometry results revealed the existence of some labeled DCs within the inguinal LN (data not shown). We also found hemorrhage surrounding the dissected popliteal LN, which may lead to an expanded signal decrease. However, we cannot distinguish whether the SPIONIR797-labeled DCs caused the hemorrhage or the hemorrhage was undetected before the experiment. This phenomenon suggested that inflammation such as hemorrhage might recruit DCs to capture and present the antigens through the lymphatic tract (41) and cause an interaction between the labeled DCs and

A novel dual-modality nano-probe for tracking dendritic cells the DCs from the secondary LNs, which may facilitate some labeled DC migration to the inguinal LN. However, the migrated number was too small to be detected by MRI or NIRF. An inflammation model would be established to confirm the unclear reasons in the further study. Our study has several limitations. First, the number of animals in our study was small. Second, the dynamic MRI observation time was limited to 5 days owing to no significant increasing trend after observing for 2 days. The long-term fate of the SPIONIR797-labeled DCs within LNs is still unknown. We did not make a dynamic NIRF quantitative analysis. The relationship between MRI and NIRF imaging might better reflect the characteristics of the nano-probe. Also, if a merged image of MRI and NIRF with good quality was acquired, we could differentiate between the labeled DCs and artifacts better. Finally, although we were able to distinguish the injected DCs from DCs within recipient animals by SPIO-NIR797 labeling, we could not measure the migration capacity of labeled DCs accurately. Perhaps we may use DCs expressing some reporter genes to solve the problems in our future study.

Conclusions We successfully developed a novel dual-modality nano-probe, SPIO-NIR797, and cultured, labeled and tracked DCs by MRI and NIRF imaging. SPIONIR797 labeling had almost no adverse effect on cell viability, cell proliferation and migration capacity. The high-performance MRI/NIRF dual-modality imaging nano-probe may be applied in many fields of molecular imaging, including tracking other types of cells such as mesenchymal stromal cells or endothelial progenitor cells and imaging the biologic targets in different types of disease models, such as inflammation, atherosclerosis and tumor. Acknowledgments The authors acknowledge the excellent technical contribution of Dr Dong-Fang Liu at the Jiangsu Key Laboratory of Molecular and Functional Imaging. This work was supported by a grant from the Major State Basic Research Development Program of China (973 Program) (2013CB733800) and National Natural Science Foundation of China (NSFC 81230034, 30910103905, 81101139). All authors were responsible for study design or analysis and interpretation of data, writing the article or revising it critically for important intellectual content and final decision to submit the paper for publication.

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Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jcyt.2013. 09.006.