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The ability of mesenchymal stem cells (MSCs) to specifically home to tumors has suggested their potential use as a delivery vehicle for cancer therapeutics. MSC integration into tumors has been demonstrated in animal models using histopathological 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 demonstrated that as few as 1000 MSCs carrying iron oxide nanoparticles can be detected by MRI one month after their coinjection with breast cancer cells which formed sub-cutaneous tumors. Subsequently we show that intravenously 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 utilize MRI to track MSCs to lung metastases in vivo. This technique has the potential to demonstrate MSC integration into human tumors, allowing early phase clinical studies examining MSC homing in patients with metastatic tumors.
The poor survival of both lung cancer patients and those with other forms of pulmonary metastatic disease relates partly to the inability to deliver locally targeted therapeutic agents. A recent body of work has utilized exogenous mesenchymal stem cells (MSCs) from the bone marrow compartment to attenuate several carcinoma models (1-6). In some of these studies the MSCs carrying anti-tumor therapies have been delivered locally (2), while in others they have been delivered systemically and migrate to the site of the tumor where they contribute to tumor reduction (1, 3-6).
We have shown previously in murine cancer studies that human MSCs expressing TRAIL can provide targeted delivery of this proapoptotic agent to breast cancer metastases (1). Similarly, MSCs transduced to express IFNβ or the immunostimulatory chemokine CX3CL1 have also been shown to reduce tumor burden in murine glioma (3), breast (5), melanoma (4), and colorectal models (6), with an improvement in survival.
The ability of bone marrow-derived stem cells to migrate to areas of injury in a range of pathological conditions suggests that they may be ideal vectors for therapeutic delivery. MSCs possess a number of properties that make them suitable candidates. They are easily obtained from a simple bone marrow aspirate and are readily expanded in culture without losing their multi-lineage potential. They are readily transducible, allowing for simple ex vivo modification (7). Finally, they appear to be relatively non-immunogenic (8) due to their expression of major histocompatibility complex 1 (MHC1), and lack of MHC2 and co-stimulatory molecules CD80, CD86 and CD40 (9). This may allow the delivery of genetically dissimilar MSCs without the need for immunomodulation or subsequent immunosuppressive therapy for the recipient. Because of these properties, MSCs have considerable therapeutic potential in tumour therapy.
To deliver cell therapy for cancer in the clinical setting, there is a need for imaging confirmation of targeted delivery. Novel imaging contrast agents have emerged that open up the possibility of visualizing stem cell transplants in vivo using magnetic resonance imaging (MRI). Superparamagnetic iron oxide (Fe3O4) nanoparticles have been used for tracking engrafted cells in a variety of tissues (10), as well as targeted cell delivery (11). The nanoparticles generate a local magnetic field pertubation, which leads to a marked shortening of the MRI parameter T2*. This is exhibited as hypointensity on MR images, leading to the possibility of imaging the localization of these particles (10,12). We have exploited this phenomenon, using MRI to track the fate of MSCs labeled with magnetic nanoparticles in a metastatic lung cancer model.
Here we have introduced bio-compatible iron oxide nanoparticles into MSCs to enable localized cellular-level sensing while retaining full viability. Using a combination of cancer cells and iron-nanoparticle containing MSCs, we have shown in a subcutaneous tumor model that the MSCs can be imaged down to very low numbers. Finally we demonstrate in a metastatic cancer model, that systemically delivered cells can be tracked to pulmonary metastases, which is subsequently confirmed histopathologically.
Tissue culture reagents were purchased from Invitrogen (Paisley, UK) unless otherwise stated. MDAMB231 breast cancer cells were obtained from Cancer Research UK, London Research Institute (CRUK, London, UK) and were cultured in DMEM and 10% fetal bovine serum (FBS). Human adult mesenchymal stem cells were purchased from Tulane University (New Orleans, USA) and cultured in αMEM with 16% FBS. Both cell lines were obtained directly from cell banks that perform cell line characterizations (DNA fingerprinting and short tandem repeats (MDAMB231), flow cytometry and differentiation analysis (MSC)), and passaged for less than 6 months. FluidMAG iron nanoparticles (NC-D, Chemicell GmbH, Berlin, Germany) with a hydrodynamic diameter of 200nm and a magnetite core were coated by the manufacturer with starch.
Labeling of MSCs with iron nanoparticles was performed by overnight incubation with 0.5mg/ml nanoparticles in cell culture medium. The cells were vigorously washed with PBS 8 times to remove any free particles before use.
Adipogenic and osteogenic differentiation of MSCs was performed as previously described (13, 14). Cell viability was performed using an MTS NAD(P)H-dependent assay (15) according to the manufacturer's guidelines (Promega, Southampton UK). Cell apoptosis was analyzed using an Annexin-V-FITC/Propidium Iodide (A-V/PI) assay (ApoTarget™, Invitrogen), 72 hours after labeling. Ten samples were analyzed using a flow cytometer (FACSCalibur, Becton Dickenson, Oxford, UK), and 6×103 - 8×103 cells were scored per analysis (CellQuestPro, Becton Dickenson). Annexin V−/PI− cells were judged to be viable, Annexin V+/PI− cells were considered to be undergoing apoptosis, and Annexin V+/PI+ cells were considered late apoptotic or necrotic, and recorded as dead (1).
Cell migration was performed as previously described (1). Briefly, 1.5×105 MDAMB231 cells were plated in 800μl medium on the bottom well of a transwell plate (Becton Dickenson), with 4×104 MSCs in 300μl plated in the upper well. The MSCs were allowed to migrate across the 8μm pore membrane for 24 hours at 37°C. The cells attached to the upper side of the membrane were removed with a cotton bud, and the cells on the lower side that had migrated through the membrane were fixed, stained (Rapid Romanowsky, Raymond Lamb, Eastbourne, UK), and counted (5 fields/well, triplicate wells) at ×10 magnification (Olympus BX40, Watford, UK).
Prussian blue staining (1.2% potassium ferrocyanide with 1.8% hydrochloric acid) was performed on fixed cells (4% paraformaldehyde) 96 hours after labeling. Confocal microscopy was performed on a Leica TCS SP2 microscope (Leica Microsystems Ltd., Bucks., UK). Reflectance was used to visualize iron as previously described (12), and images were processed using Image J. For electron microscopy, cells were fixed with 2% paraformaldehyde, 1.5% glutaraldehyde in 0.1M phosphate buffer pH 7.3. They were then osmicated in 1% OSO4/ 0.1M phosphate buffer, dehydrated in a graded ethanol-water series, cleared in propylene oxide and infiltrated with Araldite resin. Ultra thin sections were cut using a diamond knife, collected on 300 mesh grids, then stained with uranyl acetate and lead citrate. These were viewed in a Jeol 1010 transmission electron microscope (Jeol, Herts., UK) and the images were recorded using a Gatan Orius CDD camera (Gatan, Abingdon, UK).
We used a superconducting quantum interference device (SQUID) (16) to measure the amount of Fe3O4 in the cells. The samples were saturated in a field of 2 Tesla, which was subsequently removed to leave the superparamagnetic iron oxide (SPIO) particles in a magnetized state. Comparison of this remnant signal with a sample of known Fe3O4 concentration allowed quantification of Fe3O4 per cell.
All animal studies were performed in accordance with British Home Office procedural and ethical guidelines. Six-week old NOD/SCID mice (Harlan, Bicester, UK) were kept in filter cages.
Subcutaneous tumors were obtained by the injection of 2 × 106 MDAMB231 cells in 200μl PBS, subcutaneously into the left flank with a 29G needle (17). Metastatic lung tumors were produced by the intravenous delivery of two million MDAMB231 in 200μl PBS into the lateral tail vein (1).
In the subcutaneous model, varying numbers of MSCs labeled with CM-DiI (Invitrogen, as per manufacturer's instructions), and iron nanoparticles were delivered concurrently with the cancer cells. In metastatic models, 7.5×105 MSCs were suspended in 200μl PBS and injected into the lateral tail vein at day 35 after the cancer models had been set up. As controls, MSCs not bearing nanoparticles, 100ng of free iron, or iron nanoparticle-labeled MSCs which were killed in 70% ethanol (cell death confirmed with trypan blue staining) were delivered with the cancer cells.
Images were acquired on a 9.4T horizontal bore Varian (VNMRS) system using a 39mm RF coil (RAPID Biomedical GmbH). Lung in vivo images were obtained before, one hour and 24 hours after MSC injection, at day 35 after the metastatic model had been initiated (n=4 mice). They were acquired using a fast spin-echo sequence with cardiac and respiratory gating (TR~1s, effective TE=5ms, 100um in-plane resolution, 1mm slice thickness, NSA=4). Subcutaneous tumor images were obtained 28 days after subcutaneous injection of MDAMB231 cells and MSCs and acquired ex vivo using the same sequence and similar parameters (TR=1.5s, effective TE=5ms, 100um in-plane resolution, 1mm slice thickness, NSA=4) (n=14 mice; 2 per group). Signal-to-noise ratios were obtained from three consecutive coronal slices for 4 lung areas (right and left, upper and lower), using the average signal intensity (SI) of each area, the SI of shoulder muscle and the standard deviation of the noise, within each slice.
Mice were sacrificed by CO2 asphyxiation followed by exsanguination following the MRI at day 28 in the subcutaneous tumour experiment and post final MRI (1 hour or 24 hours following MSC delivery) in the metastatic experiment. Subcutaneous tumors were removed and fixed in 4% paraformaldehyde for histology. The lungs were excised and inflated with a fixed 20 cm pressure of 4% paraformaldehyde and then bathed in 4% paraformaldehyde for histology.
Fixed specimens were embedded in paraffin and cut into 3μm sections for Haematoxylin and Eosin (H&E) staining. Prussian Blue staining was used to detect iron and fluorescent microscopy was used to detect DiI positive cells with DAPI counterstain. Macrophages were stained with a monoclonal rat anti-mouse Mac-2 primary antibody (1/10000 dilution, Cedarlane, Ontario, Canada) for immunohistochemistry and a monoclonal rat anti-mouse F4/80 primary antibody (1/50 dilution, Ebiosciences, Herts., UK) for immunofluorescence. Microscopy was performed using light (Olympus BX40) or fluorescent (Carl Zeiss Ltd., Axioskop 2, Welwyn Garden City, UK) microscopes.
Statistical analysis was performed using GraphPad Prism v4 (GraphPad Software, California, USA). Multiple groups were analysed by Anova. Single group data was assessed using Student's t-test or Mann-Whitney test. Results were considered to be statistically significant for p<0.05.
The MSCs readily internalized the iron nanoparticles. This was confirmed by Prussian blue staining, electron microscopy and confocal microscopy (Figure 1Ai-iv). Cells contained up to 30pg of iron oxide per MSC, quantified using SQUID magnetometry. The labeled cells retained their MSC characteristics, with the ability to differentiate into stromal tissues, including bone and fat (Figure 1Av-vi). Furthermore, the iron nanoparticle-labeled and unlabeled MSCs demonstrated equivalent in vitro tumor homing (104.4 ± 5.6 vs. 113.1 ± 16.1 cells/field) in transwell migration studies (non-significant (ns), t-test) (Figure 1B). There was also no effect of iron nanoparticles on MSC proliferation (ns, 2-way Anova), as demonstrated by the MTS proliferation assay (Figure 1C), or cell viability as demonstrated by Annexin V flow cytometry apoptosis assay (33.0 ± 4.2 % cells dead or apoptotic cells with no iron nanoparticles, compared to 29.4 ± 2.1% with iron nanoparticles, ns, Mann-Whitney) (Figure 1D).
To determine the sensitivity of MRI in visualizing MSCs carrying iron nanoparticles, we used subcutaneous tumors, rather than lung tissue, in combination with our lung imaging MRI sequence to assess the dose response of iron labeled cells, as the air spaces in the lung could confound this assessment. We grew subcutaneous MDAMB231 tumors (2×106 cells) in NOD/SCID mice with increasing numbers of DiI-labeled human MSCs carrying nanoparticles (100, 1×103, 1×104, and 1×105) for 28 days (n=2 per group). Using a 9.4T MRI system we were able to visualize as few as 1000 MSCs labeled with nanoparticles in tumours 28 days after injection of the MDAMB231 cells (Figure 2Ai-iii). Signal voids were not visible at 28 days when non-iron-labeled MSCs, dead iron-labeled MSCs (Figure 2Av), or free iron (Figure 2Avi) were coadministered with the tumour cells. Histopathological examination confirmed that iron was present only in the tumors injected with live nanoparticle-labeled MSCs. This was demonstrated by co-localization of the Prussian blue staining of iron and DiI fluorescence with the MSCs (Figure 2B).
In the following experiments, 2×106 MDAB231 cells were injected into the tail vein. This model reproducibly forms pulmonary metastases throughout all lung lobes. We were able to detect lung metastases using MRI, visualized as diffuse hyperintensities in all five lobes 38 days after tumour cell injection (Figure 3A). As we have shown previously, MSCs show tropism to pulmonary tumors (1). Therefore 35 days post intra-venous delivery of MDAMB231 cells, MSCs double-labeled with DiI and iron nanoparticles were injected intravenously. We used MRI to confirm the fate of the intravenously injected, iron-labeled MSCs within metastases in vivo, by acquiring MR images pre-, one hour, and 24 hours post-injection. MRI images post MSC injection showed a decrease in signal intensity in areas of metastatic deposits detected in pre-MSC delivery images, which correlated with the iron-labeled MSCs integrating or lodging into these tumors (n=4) (Figure 3A). To examine MSC engraftment throughout the lung, the signal intensity across the lung was examined before and after MSC injection in three consecutive slices. This was compared to the standard deviation of the signal noise of each slice, giving a within-slice signal-to-noise ratio (SNR) for each examined area, which was averaged across the three slices. There was a significant reduction in the SNR following the MSC injection, which was consistent in all lung areas (p=0.005, 2-way Anova, n=3) (Figure 3B). There were no differences in the SNR decrease between the lung areas (ns, 2-way Anova). Immunohistochemistry confirmed our previous findings that DiI/Fe staining cells were found within or adjacent to tumors (Figure 3Ci,ii). As previous studies have suggested that iron-labeled cells may represent macrophages, we performed immunohistochemistry and immunofluorescence for macrophages and iron or DiI (18, 19). There was no colocalisation of the macrophage marker with the iron nanoparticles or DiI-positive cells with either technique (Figure 3Ciii-iv).
MSCs have enormous potential as vehicles for directed cancer delivery. The mechanism responsible for the homing of mesenchymal stem cells to tumors is likely to involve chemokine ligands and receptors in a similar fashion to the recruitment of leukocytes to areas of inflammation. However, unlike with leukocytes, the specific chemokines responsible for homing and migration of mesenchymal stem cells are less well characterised (20). Nevertheless, homing to tumors has been confirmed by many studies using the labeling of MSCs and subsequent immunohistochemistry (21). Identification of the MSCs in these previous studies has necessitated histological tissue and animal sacrifice. In this preliminary study we have demonstrated the ability to detect and visualize homing of iron-labeled MSCs in real time, in vivo.
For clinical applications, the ability to track MSC homing to primary tumors and metastases using a simple non-invasive scan would be of great benefit. Although murine models have shown a lot of promise for transduced MSCs in cancer therapy, many uncertainties still remain. The ability to systematically visualize the therapy and the response of the tumor will allow for more informed decisions as to the optimum timing of MSC therapy, as well as the number of treatments.
We have previously described the use of MSCs in delivering the proapoptotic protein TRAIL (1). In these experiments, the expression of TRAIL was sensitively controlled by doxycycline via an inducible lentivirus. The ability to detect the proximity of the transduced MSCs to the tumors with MRI could be utilized in such a model, expressing the anti-tumor agent only when the MSCs are in their optimal position. Although the spatial proximity of the MSC homing to the tumors and the benefit of MR monitoring for TRAIL therapy was not investigated as part of this study, we envisage that MSC tracking will help define the optimal time window by detection of MSCs in the lung and any regression of the metastases.
The non-invasive tracking of MSCs has previously been studied with the use of bioluminescence (22) and whole body micro PET (23) with MSCs labeled with firefly luciferase or transduced to express HSV1-TK respectively. The use of superparamagnetic iron oxide particles has the advantage of labeling MSCs without transduction, but with the use of agents and facilities which are now frequently used in medical practice, thus providing direct clinical applicability. In our study we found that iron particles had no effect on MSC differentiation, migration, survival and proliferation capacity.
Previous groups have studied the use of nanoparticles for detecting MSCs in vivo with direct injection into a cardiac scar (18) and direct injection into the brain (24). MSCs have also been tracked after intravenous injection in a Kaposi's sarcoma model (25). This is the first study, to our knowledge, that assesses the applicability of a systemic delivery of SPIO nanoparticle-labeled MSCs to metastatic disease.
As a non-invasive imaging modality that uses non-ionizing radiation, MRI and in general the use of iron nanoparticles, may have an important future in human applications. The approach described here could augment tracking of cells in other cancer models, and will be crucial in the monitoring of cell localization prior to clinical gene therapy studies.
One of the limitations of this approach is that the detection of iron nanoparticles by MRI does not ensure that these cells are the labeled MSCs. Indeed, other studies have suggested that some of the MRI signal may be the result of either the release of free iron, or the uptake of iron by macrophages after labeled-MSC death (18, 19). In our experiments, there was no MRI signal with the use of free iron or dead iron-labeled MSCs, suggesting that the iron is cleared in both these situations, and that the MRI signal is generated exclusively by viable, labeled cells, as also demonstrated in other studies (26, 27). The MSCs containing the iron nanoparticles were also labeled with the fluorescent dye DiI and histochemistry for iron of both subcutaneous and metastatic tumors demonstrated the persistent colocalisation of the iron with the fluorescent marker DiI. DiI labelling has been validated as a technique for tracking human cells transplanted into mice in a study whereby DiI-stained cells were characterised as human origin by in situ hybridisation (28), and we have previously demonstrated that the transplanted DiI cells retain characteristics of MSCs with the use ex vivo vimentin staining, in the same in vivo models as this study (1). This present study has also demonstrated that there was no colocalisation of the iron with macrophage markers, again suggestive that the iron signal represents MSCs.
In conclusion, targeted technologies, utilizing pro-apoptotic methods such as TRAIL in conjunction with non-invasive MR monitoring, may become an important adjuvant to the use of ionizing radiation and chemotherapeutic agents, opening up a variety of possibilities for the future of cancer treatment.
MRL is a MRC Clinical Training Fellow. SMJ was an MRC Clinician Scientist. This work was partly undertaken at UCLH/UCL who received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres funding scheme (SMJ), the Institute of Child Health, Child Health Research Appeal Trust, the A.S. Onassis Public Benefit Foundation (PGK), the Biotechnology and Biological Sciences Research Council (AP, ML), the British Heart Foundation (ML), an EPSRC Nanotechnology Grand Challenge Grant (QP) and King's College London and UCL Comprehensive Cancer Imaging Centre, CR-UK & EPSRC, in association with the MRC and DoH (England) (ML).
Conflicts of Interest
No conflicts of interest to declare.