In vivo tracking of superparamagnetic iron oxide nanoparticle–labeled mesenchymal stem cell tropism to malignant gliomas using magnetic resonance imaging

Laboratory investigation

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Object

Mesenchymal stem cells (MSCs) have been shown to migrate toward tumors, but their distribution pattern in gliomas has not been completely portrayed. The primary purpose of the study was to assay the tropism capacity of MSCs to gliomas, to delineate the pattern of MSC distribution in gliomas after systemic injection, and to track the migration and incorporation of magnetically labeled MSCs using 1.5-T magnetic resonance (MR) imaging.

Methods

The MSCs from Fischer 344 rats were colabeled with superparamagnetic iron oxide nanoparticles (SPIO) and enhanced green fluorescent protein (EGFP). The tropism capacity of MSCs was quantitatively assayed in vitro using the Transwell system. To track the migration of MSCs in vivo, MR imaging was performed both 7 and 14 days after systemic administration of labeled MSCs. After MR imaging, the distribution patterns of MSCs in rats with gliomas were examined using Prussian blue and fluorescence staining.

Results

The in vitro study showed that MSCs possessed significantly greater migratory capacity than fibroblast cells (p < 0.001) and that lysis of F98 glioma cells and cultured F98 cells showed a greater capacity to induce migration of cells than other stimuli (p < 0.05). Seven days after MSC transplantation, the SPIO–EGFP colabeled cells were distributed throughout the tumor, where a well-defined dark hypointense region was represented on gradient echo sequences. After 14 days, most of the colabeled MSCs were found at the border between the tumor and normal parenchyma, which was represented on gradient echo sequences as diluted amorphous dark areas at the edge of the tumors.

Conclusions

This study demonstrated that systemically transplanted MSCs migrate toward gliomas with high specificity in a temporal–spatial pattern, which can be tracked using MR imaging.

Abbreviations used in this paper: DMEM = Dulbecco modified Eagle medium; EDTA = ethylenediaminetetraacetic acid; EGFP = enhanced green fluorescent protein; FBS = fetal bovine serum; MR = magnetic resonance; MSC = mesenchymal stem cell; NSC = neural stem cell; PBS = phosphate-buffered saline; PFA = paraformaldehyde; SDF-1 = stromal cell-derived factor-1; SPIO = super-paramagnetic iron oxide nanoparticles.

Article Information

Address correspondence to: Xing Wu, M.D., Department of Neurosurgery, Huashan Hospital, Shanghai Medical School, Fudan University, 12 Wulumuqizhong Road, Shanghai, 200040, China. email: bttnirvina@gmail.com.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Photomicrographs (A and B) and electromicrographs (C and D) of MSCs in culture after labeling with iron oxide nanoparticles (Prussian blue staining). A cluster of iron nanoparticles is shown surrounded by a cell membrane in the close vicinity of the Golgi apparatus (C and D, arrows), confirming the presence of iron inside the cell. The boxed area in C is shown at higher magnification in D. Original magnification × 200 (A), × 400 (B), × 4000 (C), and × 21,000 (D).

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    Photomicrographs of the EGFP-labeled MSCs cultured with F98 cells for 48 hours. The MSCs migrated toward F98 clones and clustered around the clones, whereas no green MSCs could be seen in any other place (A). The process of MSC tropism to an F98 clone was captured by sequential photographs at 12-hour intervals (B–D). Only 1 green MSC was found in the F98 clone 12 hours after coculturing MSCs and F98 cells (B), whereas 24 hours later at least 5 green MSCs clustered around the F98 clone (C), and 36 hours later the green MSCs occupied almost half of the F98 clones (D). Original magnification × 100 (A) and × 200 (B–D).

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    Bar graph showing the migratory capacity of MSCs and fibroblast cells. The F98 cells and lysis of F98 glioma cells significantly stimulated the directional migration of the MSCs compared with saline or normal brain tissue extract (*p < 0.01 for lysis of F98 glioma cells and *p < 0.05 for F98 cells, t-test). As expected, glioma cell lines (F98 cells) significantly stimulated MSC migration (up to 3-fold compared with the control) but interestingly, lysis of F98 glioma cells induced the highest chemotactic response (up to 5-fold). Normal brain tissue extract or saline did not change the basal migration rate. In addition, the response of fibroblast cells in migration capacity was not significantly different under the stimulus. Compared with fibroblast cells, F98 cells and lysis of glioma cells all promoted migration of MSCs significantly (#p < 0.001, t-test). Values shown are means ± standard error from 3 independent experiments.

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    Photomicrographs of SPIO–EGFP colabeled MSCs after transplantation. A–C: Images obtained 7 days after transplantation showing that the SPIO–EGFP colabeled MSCs infiltrated and were distributed throughout the tumor. Prussian blue staining (A), EGFP staining (B), and combination of A and B (C). D–F: After 14 days, most colabeled MSCs were found at the border between the tumor and normal parenchyma, and only a few of the MSCs had infiltrated the tumor bed. Prussian blue staining (D), EGFP staining (E), and combination of D and E (F). G and H: The blue MSCs appeared to “follow” the invading tumor cell into surrounding tissue (G). This “trailing” of individual glioblastoma cells migrating away from the main tumor bed is examined in greater detail in H. The blue-stained MSCs are in direct juxtaposition to a single migrating and invading neutral red spindle-shaped tumor cell; the MSC “rides” the glioma cell in a “piggyback” fashion. The sections were costained with Prussian blue (allowing the SPIO-labeled MSCs to stain blue) and neutral red (allowing the elongated glioblastoma cells to stain dark red). I and J: Immunostaining showed EGFP-labeled green MSCs incorporated into the vessels at the edge of the tumor (Tu). The boxed area in I is shown at higher magnification in J. Some of the EGFP-labeled MSCs penetrated the vessels and then migrated in a chain pattern toward the glioma bed after 14 days (I). Original magnification × 200 (A–G and I) and × 400 (H and J).

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    Bar graph showing the distribution pattern of systemically injected MSCs in different organs. At 14 days after MSC transplantation, Prussian blue–stained cells were found to scarcely distribute in the organs examined, except for within the brain tumor mass (glioma), where blue MSCs were highly concentrated. The number of blue MSCs was significantly higher in the brain tumors compared with the contralateral side of the nontumor-bearing part of the brain and other organs. *p < 0.001.

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    Magnetic resonance images of SPIO–EGFP colabeled MSCs. These MSCs were examined at 7-day intervals for 2 weeks after MSC transplantation with T2-weighted spin echo and T2*-weighted gradient echo pulse sequence MR imaging. At 7 days the glioma showed a slightly high-signal intensity on the spin echo sequence (A, white arrow), whereas a well-defined dark hypointense region was shown on gradient echo sequence (B). Fourteen days after MSC transplantation, the tumor grew larger as demonstrated by a hyperintense edema region surrounding a significantly hyperintense necrosis core on the spin echo sequence (D), whereas the hypointense areas developed as 2 diluted amorphous curves at the tumor's edge on the gradient echo sequence (E). The hypointense signals on MR images in panels B and E correspond to multiple Prussian blue–stained cells in the histological sections in panels A and D of Fig. 4. As the 3D reconstructions (C and F) show, hypointense regions—which represent SPIO-labeled MSCs—in the MR slices are indicated by the yellow structures with the yellow arrows. The 3D images of SPIO-labeled MSCs in C and F are fitted with the histological sections (insets) and MR images in B and E, respectively. The tumors cannot be reconstructed in 3D because they cannot be shown in gradient echo sequences.

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