Experimental Study Of Mesenchymal Stem Cells Tropism For Malignant Gliomas And Distribution Pattern

Objective (1) To investigate the tropism capacity of rat mesenchymal stem cells (MSCs) for gliomas. (2) To in vitro evaluate the labeling efficiency of (MSCs) with different labeling concentrations of superparamagnetic iron oxide (SPIO) nanoparticels. (3) To delineate the pattern of MSCs distribution in glioma, and to track the migration and incorporation of magnetically labeled MSCs with clinical 1.5T MRI. (4) To investigate whether macrophage chemoattractant protein-1 (MCP-1) and stromal cell-derived factor-1a (SDF-1a) could play important roles in the migration of MSCs toward gliomas.Method (1) MSCs were isolated from Fischer 344 rats, cultured and labeled with enhanced green fluorescence protein (EGFP); The procedure of MSCs tropism to a F98 clone was captured by sequential photograph at 12 hours interval and the tropism capacity of MSCs was quantitatively assayed by Transwell system in vitro. For assessing the distribution of MSCs throughout the brain glioma burdened rats, their brain, heart, lungs, liver, kidneys and spleen examined histologically at 14 days after MSCs transplantation. (2) Various concentrations of SPIO nanoparticles were used to magnetically label cells. Cell viability was evaluated by trypan blue dye exclusion assay in relation to the concentration of SPIO and incubation times. To assess the effects of the particles on cell proliferation, MTT growth curves of each group were obtained at different time points. (3) Fisher344 rats MSCs were co-labeled with superparamagnetic iron oxide nanoparticles (SPIO) and enhanced green fluorescence protein (EGFP). To in vivo track the migration of MSCs, magnetic resonance imaging (MRI) was performed at 7 days and 14 days after systematic administration of labeled MSCs. After scanned, the distribution patterns of MSCs in glioma burdened rats were examined by Prussian blue and fluorescence staining. (4) RT-PCR and flow cytometry analysis were used to detect whether MSCs, in vitro, expressed CCR2 and CXCR4. The effect of MCP-1 and SDF-1a on MSCs migration was studied with migration assay. For neutralization studies, glioma cell conditioned medium was incubated with rabbit anti-rat MCP-1 or rat MSCs were incubated with anti-CXCR4 polyclonal antibody.Result (1) To visualize the migratory properties of MSCs, in vitro studies first assessed the relative migratory capacity of MSCs when mixed with glioma cells. 48 hours later, most of GFP labeled MSCs aggregated around the clone of glioma cells, while merely plated at any other place. To further quantitatively evaluate the migratory pattern of MSCs towards glioma cells, we assay the migration rate through calculating EGFP labeled green MSCs in the lower chamber of Transwell system by fluorescent microscope. Normal brain tissue lysate or saline induced only minimal migration of MSCs and fibrablast cells in vitro. By contrast, lysate from F98 gliomas and cultured F98 cells induced migration of MSCs. Using one-way ANOVA, we found a highly significant difference in the cell migration pattern between the different populations of cells (F=22.34; P<0.001). And there were significant differences between the different stimuli (F=7.65; P=0.002). Importantly, MSCs possessed significantly greater migratory capacity than fibrablast cells (P<0.001), and lysate of F98 glioma / cultured F98 cells showed more capacity to induce migration of cells than other stimuli (P<0.05). For assessing the distribution of MSCs throughout the brain glioma burdened rats, their brain, heart, lungs, liver, kidneys and spleen examined histologically at 14 days after MSCs transplantation. X-gal stained cells were found scarcely distributed in the organs examined, except for brain tumor mass, where blue MSCs were highly concentrated. The number of blue MSCs was significantly higher (MSCs in brain tumor versus MSCs in other tissues respectively, P<0.001) in the brain tumors (97.52±16.13/section) compared to contralateral side of nontumor-bearing brain (7.64±2.31/section) and other organs (heart: 0/section; lungs: 3.76±1.74/section; livers:9.64±1.55/section; kidneys: 19.78±2.63/section; and spleen: 5.74±1.98/section). (2) Prussian blue staining showed numerous blue stained particles in the cytoplasm of the labeled cells. When the iron concentration was 25μg/ml, no significant difference (i.e., labeled cells [12%-15%] vs. unlabeled cells [approximately 15%]) was detected in the percentage of dead cells compared with the unlabeled control MSCs at 24 and 72 hr. However, at the two highest concentrations of iron per milliliter evaluated (i.e., 100 and 250μg/ml), the percentage of viable cells (57%-78%) was significantly (P<0.001) decreased compared with the percentage of viable unlabeled control cells of 85% at 24 and 72hr. There is no difference of cell proliferation viability between SPIO-labeled and unlabeled MSCs by MTT detecting method. (3) At the 7 days after MSCs transplantation, SPIO/EGFP co-labeled MSCs infiltrated the tumor and distributed throughout the tumor. While, at the 14 days, the co-labeled cells no longer scattered through the tumor, but mostly found at the border between tumor and normal parenchyma. This result revealed that incorporation of systemically transplanted MSCs to brain tumors varied with the development of tumor. Moreover, SPIO labeled MSCs could not only be seen to distribute themselves to its invading edge or throughout the tumor, but could be seen to "trail" individual aggressive, dark red, elongated infiltrating tumor cells that migrate away from the main tumor mass. Additionally, EGFP-labeled MSCs were found to incorporate into the vessels at the edge of the tumor, and some labeled MSCs penetrated the vessels and streamed as a chain pattern toward glioma. In vivo MRI did demonstrate a hypointense region with small and well-defined dark features in the tumor at day 7 post transplantation in animals receiving the intravenous injection of SPIO labeled cells, as compared to animals that received unlabeled cells. At 14 days after transplantation, the hypointense areas developed as discontinued amorphous dark curves at the margin of tumor. For this method, we matched the MR images with histological sections as much as possible. The hypointense signals on MR images in panel are correspondent with multiple Prussian blue stained cells in the histological sections. (4) RT-PCR and FACS showed that MSCs express CCR2 and CXCR4, the respective receptors for MCP-1 and SDF-1a. In vitro analysis revealed that MCP-1 and SDF-1a induce the migration of MSCs. Futhermore, addition of the anti-MCP-1 neutralizing antibody or anti-CXCR4-blocking antidoby significantly attenuated the migration of MSCs toward glioma cell conditioned medium.Conclusion (1) MSCs have the ability to migrating toward gliomas. (2) MSCs can be easily and efficiently labeled by SPIO without interference on the cell viability and proliferation. (3) Systemically transplanted MSCs 'home' to glioma with high specificity with a temporal-spatial pattern, which can be tracking by MRI. (4) MCP-1 and SDF-1a mediate the migration of MSCs toward gliomas in vitro.