Molecular Imaging and Biology

, Volume 14, Issue 2, pp 183–196

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

  • Cristina Martelli
  • Manuela Borelli
  • Luisa Ottobrini
  • Veronica Rainone
  • Anna Degrassi
  • Micaela Russo
  • Umberto Gianelli
  • Silvano Bosari
  • Carlo Fiorini
  • Daria Trabattoni
  • Mario Clerici
  • Giovanni Lucignani
Research Article

DOI: 10.1007/s11307-011-0496-0

Cite this article as:
Martelli, C., Borelli, M., Ottobrini, L. et al. Mol Imaging Biol (2012) 14: 183. doi:10.1007/s11307-011-0496-0

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.

Key words

Dendritic cellsMRISPECTIn vivo imagingVaccine protocolBreast cancer

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

Supplementary material

11307_2011_496_MOESM1_ESM.pdf (73 kb)
Fig. S1MNP 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)
11307_2011_496_MOESM2_ESM.pdf (68 kb)
Fig. S2Kinetics 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)
11307_2011_496_MOESM3_ESM.pdf (81 kb)
Fig. S3Kinetics 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)
11307_2011_496_MOESM4_ESM.pdf (122 kb)
Fig. S4Perl’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)

Copyright information

© Academy of Molecular Imaging and Society for Molecular Imaging 2011

Authors and Affiliations

  • Cristina Martelli
    • 1
    • 2
  • Manuela Borelli
    • 3
  • Luisa Ottobrini
    • 1
    • 2
  • Veronica Rainone
    • 3
  • Anna Degrassi
    • 4
  • Micaela Russo
    • 4
  • Umberto Gianelli
    • 5
  • Silvano Bosari
    • 5
  • Carlo Fiorini
    • 6
  • Daria Trabattoni
    • 3
  • Mario Clerici
    • 2
    • 7
    • 8
  • Giovanni Lucignani
    • 1
    • 2
  1. 1.Department of Biomedical Sciences and Technologies, Section of Radiological SciencesUniversity of MilanMilanItaly
  2. 2.Centre of Molecular and Cellular Imaging – IMAGOUniversity of MilanSegrateItaly
  3. 3.Department of Clinical Sciences, Chair of ImmunologyUniversity of MilanMilanItaly
  4. 4.Pharmacology Department, BU OncologyNerviano Medical SciencesNervianoItaly
  5. 5.Pathology Unit, Department of Medicine, Surgery and DentistryUniversity of Milan Medical School, IRCCS Ca’ Granda – Ospedale Maggiore Policlinico FoundationMilanItaly
  6. 6.Electronic and Information Technology DepartmentPolitecnico di MilanoMilanItaly
  7. 7.Department of Biomedical Sciences and TechnologiesUniversity of MilanSegrateItaly
  8. 8.IRCCS Don C. Gnocchi FoundationMilanItaly