Magnetic resonance imaging of mesenchymal stem cells homing to pulmonary metastases using biocompatible magnetic nanoparticles

Abstract

The ability of mesenchymal stem cells (MSC) to specifically home to tumors has suggested their potential use as a delivery vehicle for cancer therapeutics. MSC integration into tumors has been shown in animal models using histopathologic techniques after animal sacrifice. Tracking the delivery and engraftment of MSCs into human tumors will need in vivo imaging techniques. We hypothesized that labeling MSCs with iron oxide nanoparticles would enable in vivo tracking with magnetic resonance imaging (MRI). Human MSCs were labeled in vitro with superparamagnetic iron oxide nanoparticles, with no effect on differentiation potential, proliferation, survival, or migration of the cells. In initial experiments, we showed that as few as 1,000 MSCs carrying iron oxide nanoparticles can be detected by MRI one month after their coinjection with breast cancer cells that formed subcutaneous tumors. Subsequently, we show that i.v.- injected iron-labeled MSCs could be tracked in vivo to multiple lung metastases using MRI, observations that were confirmed histologically. This is the first study to use MRI to track MSCs to lung metastases in vivo. This technique has the potential to show MSC integration into human tumors, allowing early-phase clinical studies examining MSC homing in patients with metastatic tumors.

Figure 1. MSCs take up superparamagnetic iron oxide (SPIO) nanoparticles without affecting their phenotype

A) (i) MSCs in culture (scale bar 20μm), (ii) Prussian blue staining of MSCs after 24 hours of culture with SPIO nanoparticles (scale bar 20μm), (iii) EM image showing cytoplasmic location of SPIO nanoparticles (scale bar 1μm), (iv) Confocal image showing co-localization of SPIO nanoparticles (yellow), DiI (red) and the nuclear counterstain DAPI (blue) (scale bar 3μm), (v) Differentiation to osteoblasts, Alizarin Red S staining (scale bar 40μm), (vi) Differentiation to adipocytes, Oil-Red-O staining (scale bar 5μm). B) (i) MSCs stained on the underside of the transwell membrane (scale bar 50μm at ×4mag, 10μm at ×20mag). (ii) The SPIO-loaded MSCs (Fe) migrate towards MDAMB231 breast cancer cells through a transwell membrane at the same rate as non-labeled MSCs (non Fe). C) The SPIO-loaded MSCs proliferate at the same rate as control MSCs. D) There is no increase in death and apoptosis of the SPIO-loaded MSCs compared to the non-labeled MSCs.

Figure 2. Superparamagnetic iron oxide (SPIO)-loaded MSCs can be visualized by MRI in tumors at low concentrations

A) 2×10⁶ MDAMB231 cells were coinjected with i) 1×10⁵, ii) 1×10⁴, iii) 1×10³, iv) 100 SPIO-loaded MSCs, and visualized using a 9.4T MRI scanner 28 days later. The subcutaneous tumors can be seen in all mice (asterix). SPIO-loaded MSCs are visualized (arrow) when as few as 1×10³ cells were originally injected. There were no hypointesities on MRI with v) 1×10⁴ dead SPIO-loaded MSCs, or vi) free iron particle injection (100ng) (n=2 in all groups). B) Prussian blue histochemistry (i,iii) and DiI (red) immunofluoresence (with DAPI nuclear counterstain – blue) (ii,iv) corresponding to the coinjection of 1×10⁵ (i-ii) and 1×10³ (iii-iv) SPIO-loaded MSCs confirming the iron stain colocalizes with the DiI-labeled MSCs. Scale bar 50μm at ×4mag, 20μm at ×10mag.

Figure 3. Intravenously delivered, superparamagnetic iron oxide (SPIO)-loaded MSCs localise to lung metastases and can be visualized by MRI

A) Representative coronal MRI sections (n=4 mice) of a normal mouse lung (Normal), mouse lung with metastases 35 days after intravenous delivery of MDAMB231 cells (pre MSC), and the same mouse lung one hour after SPIO-loaded MSC injection (post MSC). The metastases (circled) are visualized as focal regions of increased signal. These areas correspond to metastases on H&E histological sections (scale bar 100μm). One hour post SPIO-loaded MSC injection, there is a decrease in signal intensity cause by the iron oxide in MSCs. (+ ribcage, * trachea, ^ diaphragm with upper abdomen below, ~ fissue separating lobes). B) The reduction in signal intensity secondary to the SPIO-loaded MSCs one hour and 24 hours post MSC injection was further confirmed and quantified by comparing signal-to-noise between the lung parenchyma and the deltoid muscle in three consecutive MR slices in 3 mice; there was a significant (p=0.005) reduction in signal-to-noise ratio across all 4 radiological areas (left upper (LU), lower (LL), right upper (RU), lower (RL). C) Tumor histology from mice harvested at day 35, one hour after SPIO-loaded MSC injection and MRI. i) Prussian blue and ii) DiI-staining (red) on contiguous sections from mice, demonstrating the MSCs migrate to and incorporate into lung metastases after intravenous delivery (scale bar 20μm). iii) Macrophage immunohistochemistry (brown) stains different cells to SPIO-loaded cells (blue stain), iv) Macrophage immunofluorescence (green) stains different cells to DiI-labeled (red) cells (scale bar 5μm).