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In Vivo Imaging of Lymph Node Migration of MNP- and 111In-Labeled Dendritic Cells in a Transgenic Mouse Model of Breast Cancer (MMTV-Ras)

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Abstract

Purpose

The authors present a protocol for the in vivo evaluation, using different imaging techniques, of lymph node (LN) homing of tumor-specific dendritic cells (DCs) in a murine breast cancer model.

Procedures

Bone marrow DCs were labeled with paramagnetic nanoparticles (MNPs) or 111In-oxine. Antigen loading was performed using tumor lysate. Mature DCs were injected into the footpads of transgenic tumor-bearing mice (MMTV-Ras) and DC migration was tracked by magnetic resonance imaging (MRI) and single-photon emission computed tomography (SPECT). Ex vivo analyses were performed to validate the imaging data.

Results

DC labeling, both with MNPs and with 111In-oxine, did not affect DC phenotype or functionality. MRI and SPECT allowed the detection of iron and 111In in both axillary and popliteal LNs. Immunohistochemistry and γ-counting revealed the presence of DCs in LNs.

Conclusions

MRI and SPECT imaging, by allowing in vivo dynamic monitoring of DC migration, could further the development and optimization of efficient anti-cancer vaccines.

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Abbreviations

DC:

dendritic cell

DC-LAMP:

dendritic cell-lysosomal associated membrane glycoprotein

FOV:

field of view

GM-CSF:

granulocyte macrophage colony-stimulating factor

iDC:

immature DC

IL-4:

interleukin-4

LN:

lymph node

LPS:

lipopolysaccharide

mDC:

mature DC

MHC:

major histocompatibility complex

MMTV:

murine mammary tumor virus

MNPs:

paramagnetic nanoparticles

MRI:

magnetic resonance imaging

SDD:

silicon drift detector

SPECT:

single photon emission tomography

T2:

transverse relaxation time

TE:

echo time

TEM:

transmission electron microscopy

TNFα:

tumor necrosis factor α

TR:

repetition time

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Acknowledgements

The authors thank Dr. Fabio Corsi and Mr. Raffaele Allevi (Centro di microscopia elettronica per lo sviluppo delle nanotecnologie applicate alla medicina, University of Milan) for TEM analysis, Mrs. Delfina Tosi (University of Milan) for immunohistochemistry technical support and Dr. Gemma Texido (Nerviano Medical Sciences) for providing the MMTV-Ras founder animals. This work is supported by the FP6 funded HI-CAM project (LSHC-CT-2006-037737), PRIN (20082NHWH9) and AIRC (IG2009-9311). The authors are grateful to Ms Catherine Wrenn for her advice and skilful editorial support.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. Department of Biomedical Sciences and Technologies, Section of Radiological Sciences, University of Milan, via di Rudinì 8, 20142, Milan, Italy

    Cristina Martelli, Luisa Ottobrini & Giovanni Lucignani

  2. Centre of Molecular and Cellular Imaging – IMAGO, University of Milan, via Fratelli Cervi 93, 20090, Segrate, Milan, Italy

    Cristina Martelli, Luisa Ottobrini, Mario Clerici & Giovanni Lucignani

  3. Department of Clinical Sciences, Chair of Immunology, University of Milan, via GB Grassi 74, Milan, Italy

    Manuela Borelli, Veronica Rainone & Daria Trabattoni

  4. Pharmacology Department, BU Oncology, Nerviano Medical Sciences, viale Pasteur 10, 20014, Nerviano, Milan, Italy

    Anna Degrassi & Micaela Russo

  5. Pathology Unit, Department of Medicine, Surgery and Dentistry, University of Milan Medical School, IRCCS Ca’ Granda – Ospedale Maggiore Policlinico Foundation, via Sforza, 28, 20122, Milan, Italy

    Umberto Gianelli & Silvano Bosari

  6. Electronic and Information Technology Department, Politecnico di Milano, via Golgi 40, 20133, Milan and INFN, Milan Division, Milan, 20133, Italy

    Carlo Fiorini

  7. Department of Biomedical Sciences and Technologies, University of Milan, via Fratelli Cervi 93, 20090, Segrate, Milan, Italy

    Mario Clerici

  8. IRCCS Don C. Gnocchi Foundation, Via Capecelatro 66, 20148, Milan, Italy

    Mario Clerici

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  1. Cristina Martelli
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Corresponding author

Correspondence to Mario Clerici.

Electronic Supplementary Material

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Fig. S1

MNP labeling: dose–response and incubation-time study. a Relaxometric analysis of labeled cells in the dose–response study shows a decrease in T2 time, due to the presence of iron in the cells, that is proportional to the increase in the amount of iron used (R2 = 0.984). b Analysis of cell viability in the dose–response study by means of the Trypan Blue Exclusion Test shows that MNP labeling influences cell viability only for the highest dose (p < 0.01 vs ctrl). c Relaxometric analysis of labeled cells in the incubation-time study shows a decrease in T2 time, due to the presence of iron in the cells, in relation to the increase in the incubation time. The increase in T2 time at 48 h is probably a consequence of release of iron from dead cells (the dispersed, as opposed to clustered, MNPs in the cytoplasmic vesicles are associated with the formation of a weaker magnetic field, as described in the text [42]). d Analysis of cell viability in the incubation-time study by means of the Trypan Blue Exclusion Test shows that MNP labeling influences cell viability only for the longest incubation time (p < 0.01 vs ctrl) (PDF 73 kb)

Fig. S2

Kinetics of DC migration to popliteal LNs, visualized by MRI and SPECT imaging. a MSME images and b SPECT imaging of MNP-labeled or 111In-labeled DCs, respectively, at popliteal LN level after DC injection into hind limb footpads. The hypointense signal can be detected 4 h after cell injection, and remains detectable at 24 and 48 h. In SPECT images, the dashed lines identify the field of view (FOV) of the SPECT instrument. The light blue arrows identify the injection site, and the dark blue arrows indicate the subiliac LN, where no signal was detected; K = kidneys (PDF 67 kb)

Fig. S3

Kinetics of DC migration to axillary LNs, visualized by MRI and SPECT imaging. a FLASH images of MNP-labeled DCs at accessory axillary LN level after DC injection into forelimb footpads. The hypointense signal can be detected 24 h after cell injection, and is still detectable at 48 h. No iron signal can be observed in the LNs before cell injection (white arrows). b SPECT imaging of 111In-labeled DCs at the level of both axillary LNs after DC injection into forelimb footpads. The hypointense signal can be detected 4 h after cell injection, and is still detectable at 24 h. At 48 h the signal is no longer visible due to radiotracer decay, as can be observed at the level of the injection site. In SPECT images, the dashed lines identify the field of view (FOV) of the SPECT instrument (PDF 81 kb)

Fig. S4

Perl’s staining demonstrates, in the collected LNs, the presence of iron in the cytoplasm of migrated cells. Consecutive optical enlargement of Perl’s staining in LNs showed that labeled cells localized in the cortical and paracortical areas of the lymph nodes. In the control LNs (untreated), we observed no presence of iron (PDF 121 kb)

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Martelli, C., Borelli, M., Ottobrini, L. et al. In Vivo Imaging of Lymph Node Migration of MNP- and 111In-Labeled Dendritic Cells in a Transgenic Mouse Model of Breast Cancer (MMTV-Ras). Mol Imaging Biol 14, 183–196 (2012). https://doi.org/10.1007/s11307-011-0496-0

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