Pre-clinical evaluation of Magnetically labeled Cells for Cellular MRI

通过将常用的硫酸鱼精蛋白(Pro)与超顺磁性葡聚糖包覆的纳米氧化铁(SPIO)铁氧化物(FE)结合，形成了一种可用于对干细胞和其他哺乳动物细胞进行磁性标记的复合体。细胞通过巨噬细胞吞噬来摄取FEPro复合体。通过细胞核磁共振早期检测体内标记细胞需要开发新的脉冲序列或图像处理，以提高对组织中少量氧化铁标记细胞的敏感性。我们已经证明，对胶质瘤抗原致敏的脾细胞的FEPro标记将适用于在大鼠模型中植入C6胶质瘤细胞的过继移植。我们还研究了磁性标记的CD34CD133+造血干细胞在肿瘤血管生成活跃部位的迁移和归巢的影响因素。将PEFro标记的CD133+细胞与大鼠脑胶质瘤细胞或人黑色素瘤细胞混合，接种于裸鼠腹侧。肿瘤包括周围组织行MRI检查。肿瘤切片行普鲁士蓝(PB)、PDGF、HIF-1、SDF-1、基质金属蛋白酶-2、血管内皮生长因子和内皮细胞标志物染色。MRI显示肿瘤周边低信号区，PB+/AC133+细胞内皮细胞标志物阳性。在PB+/CD133+细胞部位，HIF-1和SDF-1均呈强阳性表达，PDGF和MMP-2在肿瘤及其周围组织中有广泛表达。PB+CD133+细胞定位与肿瘤细胞中血管内皮生长因子的表达无明显相关性。免疫印迹显示SDF-1、MMP-2和PDGF在肿瘤周边有较强的表达。HIF-1和HIF-1在肿瘤的周边和中心部位均有表达。本工作表明，磁性标记细胞可以作为磁共振成像和组织学鉴定的探针。我们还解决了干细胞中的外源标记如FEPro或BrdU在直接注射到组织中后会发生什么的问题。将细胞直接植入靶组织可导致80%的细胞因创伤或凋亡而死亡。被激活的巨噬细胞(AM)对标记细胞的旁观者摄取可能会混淆对结果的解释。我们使用Boyden小室炎症模型研究AM摄取BrdU或Spion的频率。将Spion/BrdU标记的骨髓基质细胞(BMSC)或HeLa(HC)、AM与小鼠(MF)或人成纤维细胞(HF)按不同比例混合在Matrigel上室中，培养96h。用化学方法诱导AM迁移到下腔。用抗CD68、抗葡聚糖抗体对上、下室AM进行流式细胞仪分析，96小时后10%~20%的葡聚糖或10%的BrdU呈阳性反应。在标记细胞中铁向AM的转移约占总铁负荷的12%。BrdU和Spion的摄取取决于标记细胞对炎性细胞的密度和微环境条件。将BrdU/Spion标记的细胞直接植入靶组织可导致AM摄取标记，因此应注意通过旁观者细胞标记的染色和与MRI成像的相关性来验证移植细胞的组织学。

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.