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. 2024 Jun 19;16(24):30860-30873.
doi: 10.1021/acsami.4c04920. Epub 2024 Jun 11.

Magnetic Particle Imaging Reveals that Iron-Labeled Extracellular Vesicles Accumulate in Brains of Mice with Metastases

Victoria A Toomajian  1   2 , Anthony Tundo  1 , Evran E Ural  1   2 , Emily M Greeson  1   3 , Christopher H Contag  1   2   3 , Ashley V Makela  1
Affiliations

Magnetic Particle Imaging Reveals that Iron-Labeled Extracellular Vesicles Accumulate in Brains of Mice with Metastases

Victoria A Toomajian et al. ACS Appl Mater Interfaces. .

Abstract

The incidence of breast cancer remains high worldwide and is associated with a significant risk of metastasis to the brain that can be fatal; this is due, in part, to the inability of therapeutics to cross the blood-brain barrier (BBB). Extracellular vesicles (EVs) have been found to cross the BBB and further have been used to deliver drugs to tumors. EVs from different cell types appear to have different patterns of accumulation and retention as well as the efficiency of bioactive cargo delivery to recipient cells in the body. Engineering EVs as delivery tools to treat brain metastases, therefore, will require an understanding of the timing of EV accumulation and their localization relative to metastatic sites. Magnetic particle imaging (MPI) is a sensitive and quantitative imaging method that directly detects superparamagnetic iron. Here, we demonstrate MPI as a novel tool to characterize EV biodistribution in metastatic disease after labeling EVs with superparamagnetic iron oxide (SPIO) nanoparticles. Iron-labeled EVs (FeEVs) were collected from iron-labeled parental primary 4T1 tumor cells and brain-seeking 4T1BR5 cells, followed by injection into the mice with orthotopic tumors or brain metastases. MPI quantification revealed that FeEVs were retained for longer in orthotopic mammary carcinomas compared to SPIOs. MPI signal due to iron could only be detected in brains of mice bearing brain metastases after injection of FeEVs, but not SPIOs, or FeEVs when mice did not have brain metastases. These findings indicate the potential use of EVs as a therapeutic delivery tool in primary and metastatic tumors.

Keywords: brain metastasis; breast cancer; exosome; extracellular vesicles; magnetic particle imaging.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Iron-labeled EV characterization. Iron-labeled EVs (FeEVs) (a,c, 4T1L2 and 4T1BR5-L2G, respectively) and unlabeled EVs (b,d, 4T1L2 and 4T1BR5-L2G, respectively) were visualized via TEM. Both free iron (red arrowheads) and EVs associated with iron (red arrows) can be seen in the FeEV samples. FeEVs and EVs both display the classical cup shape (yellow arrows) and round shape (yellow arrowhead). Nanoparticle tracking analysis determined the peak FeEV size in 4T1L2 (e) and 4T1BR5-L2G (h) cells. Abundance of cytosolic and membrane proteins typically associated with EVs were quantified in 4T1L2 (f,g) and 4T1BR5-L2G (i,j) EVs and FeEVs as detected by mass spectrometry. Scale bars = 250 nm. *p < 0.05, **p < 0.01.
Figure 2
Figure 2
SPIO associated with EVs (FeEVs) accumulate more than SPIO alone in 4T1BR5-L2G cells in culture. After 24 h incubation, there was more SPIO in 4T1BR5-L2G cells when introduced as FeEVs (a) versus SPIO alone (c). The 4T1BR5-L2G cells are identified by blue (DAPI = nuclei) and green (GFP). The SPIO was tagged with a far-red fluorophore (magenta), and FeEVs or SPIO were dyed with PKH26 (yellow) to identify membranes. In the samples which were incubated with FeEVs (b, zoomed in from area identified in a), there are both regions with PKH26 and far-red SPIO fluorescence in the same spatial location (thick arrows, b), as well as PKH26 without far-red SPIO signal (arrowheads, b). In the samples that were incubated with SPIO alone, there was an extracellular far-red SPIO signal noted (dashed outline, d) and far-red SPIO appearing within the cells (thin arrow, d).
Figure 3
Figure 3
Increased retention of FeEVs in the location of the primary tumor compared to SPIO. Magnetic particle imaging (MPI) and microCT images (a,b, representative images, 72 h post-injection) were overlaid to localize iron signal after intratumoral injection of FeEVs (a) or SPIO (b) with cropped 2D MPI images of the tumor at 0, 24 and 72 h located below. Location of tumors is noted by white arrowhead. Tumor iron quantification (c) at 0, 24, and 72 h postinjection for FeEV- and SPIO-injected tumors (normalized to iron quantified at 0 h). Fluorescence microscopy (d) identifies SPIO (far-red, magenta), CD47 tumor cells (PE, green), and F4/80+ cells (AF647, red). SPIO and CD47+ cells in the same spatial location (arrowheads) and SPIO and F4/80+ cells within the same region (arrow). *p < 0.05, **p < 0.01, ***p < 0.0001.
Figure 4
Figure 4
Magnetic particle imaging detects and quantifies iron in the heads of mice. BLI (a) was used to confirm brain metastasis in the heads of mice which received intracardiac administration of 4T1BR5-L2G cells. MPI and brightfield images were overlaid to localize MPI signal. MPI signal was located in the liver (representative image, b) in all groups. White arrowheads note the location of iron fiducials. MPI signal was only located in the head of mice with brain metastasis which were injected with FeEVs (c, BM + FeEV). There was no signal located in mice which did not have brain metastasis and received FeEVs (d, Healthy + FeEVs) or mice which had brain metastasis and received SPIO (e, BM + SPIO). MPI was used to quantify the iron in the heads of mice from each group (f). n.d. = not detected.
Figure 5
Figure 5
Histology locates iron present in brain sections. Sections were stained with PPB for iron (left column) and DAPI to visualize nuclei (middle column). Iron was pseudocolored magenta to overlay with DAPI (blue, right column). Sections from the group that had brain metastasis and received FeEVs (a) had regions where there was iron within metastasis, iron outside of metastasis, and metastasis not associated with iron. Mice that did not have metastasis and received FeEVs (b) did have some iron visualized. Mice which had metastasis and received iron (SPIO) had metastasis visualized without iron and iron within regions of no metastases.
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