By combining commonly used Protamine Sulfate (Pro) with superparamagnetic dextran coated iron oxide nanoparticle (SPIO) Ferumoxides (FE), a complex is formed that can be used to magnetically label stem cells and other mammalian cells. Cells take up FEPro complexes by macropinocytosis. Early detection of labeled cells in vivo by cellular MRI requires the development of novel pulse sequences or image processing to improve the sensitivity to low numbers of iron oxide labeled cells in tissues. We have shown that FEPro labeling of sensitized splenocytes to glioma antigens will home to following adoptive transplantation to implanted C6 glioma cells in a rat model. We also investigated the factors responsible for migration and homing of magnetically labeled CD 34 CD 133+ hematopoietic stem cells at the sites of active angiogenesis in tumor. CD133+ cells labeled with PEFro were mixed with either rat glioma or human melanoma cells and implanted in flank of nude mice. MRI of the tumors including surrounding tissues was performed. Tumor sections were stained for Prussian blue (PB), PDGF, HIF-1α, SDF-1, MMP-2, VEGF and endothelial markers. MRIs demonstrated hypointense regions at the periphery of the tumors where the PB+/AC133+ cells were positive for endothelial cells markers. At sites of PB+/CD133+ cells, both HIF-1α and SDF-1 were strongly positive and PDGF and MMP-2 showed generalized expression in the tumor and surrounding tissues. There was no significant association of PB+CD133+ cells localization and VEGF expression in tumor cells. Western blot demonstrated strong expression of the SDF-1, MMP-2 and PDGF at the peripheral parts of the tumors. HIF-1α was expressed at both the periphery and central parts of the tumor. This work demonstrated that magnetically labeled cells maybe used as probes for MRI and histological identification administered cells. We also addressed the issue of what happens to exogenous label such as FEPro or bromodeoxyuridine (BrdU) in stem cells following direct injection into tissues. Direct implantation of cells into target tissue can result in >80% cell death due to trauma or apoptosis. Bystander uptake of labeled cells by activated macrophages (AM) can confound the interpretation of results. We investigated the frequency of BrdU or SPION uptake by AM using Boyden chamber model of inflammation. SPION/BrdU labeled bone marrow stromal cells (BMSC) or HeLa (HC), AM and mouse (MF) or human fibroblasts (HF) were mixed in various ratios in Matrigel, in upper chamber and incubated for up to 96 hours. The AM were chemotactically induced to migrate to the lower chamber. FACS analysis of AM from both, lower and upper chamber, in the presence of either MF or HF using anti-CD68, anti-dextran antibodies revealed 10 to 20% dextran or 10% BrdU positive AM after 96 hours of incubation. Transfer of iron to AM was about 12% of the total iron load in labeled cells. The uptake of BrdU and SPION was dependent on the density of labeled cells to inflammatory cells and microenvironmental conditions. Direct implantation of BrdU/SPION labeled cells into target tissue can result in uptake of label by AM, therefore care should be taken to histologically validate implanted cells by staining for bystander cell markers and correlation with MRI imaging.
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