Bell Biosystems, San Francisco, CA, USA.
Pediatrics-Neonatology and Molecular Imaging Program (MIPS), Stanford University, Palo Alto, CA, USA.
Mol Imaging Biol. 2018 Feb;20(1):55-64. doi: 10.1007/s11307-017-1094-6.
The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.
The ME's surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing.
The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ∼30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ∼350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking.
MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.
本研究的目的是描述磁共生体（ME）对哺乳动物细胞的标记，并确定这些活对比剂的亚细胞命运。ME 是新型磁共振成像（MRI）对比剂，用于细胞追踪研究。了解 ME 在宿主细胞中的命运对于设计体内细胞追踪实验非常有价值。
使用免疫细胞化学（ICC）、铁和 MRI 对比测量以及定量聚合酶链反应（qPCR）分别研究 ME 的表面表位、产生对比的顺磁磁粒体铁和基因组。这些测定方法与其他常见测定方法相结合，验证了 ME 细胞的标记，并剖析了 ME 的亚细胞处理过程。
上述测定方法提供了细胞标记的定性和定量评估、ME 的亚细胞定位和命运。使用 ME 特异性抗体的 ICC 结果定性显示 ME 均匀标记。铁嗪测定法显示 ME 平均含有 7 fg Fe/ME，其中约 30% 有助于 MRI 对比，ME 标记的 MDA-MB-231（MDA-231）细胞一般含有 2.4 pg Fe/细胞，这意味着每细胞约有 350 个 ME。调整 ME 生长培养基中的铁浓度会降低非 MRI 产生对比的铁浓度。来自 qPCR 测定的结果，该测定定量了标记细胞中的 ME 基因组，表明 ME 的处理在 MDA-231 细胞中在 24 小时内开始。ICC 结果表明，这些 ME 的细胞内消化通过溶酶体降解途径发生。用李斯特菌溶素 O（LLO）包被的 ME 能够逃避初级吞噬体，但随后与 LC3 共定位，LC3 是一种与自噬相关的分子，并被加工消化。在自噬暂时受到抑制的胚胎中，ME 显示出更高的生存能力，甚至复制能力。最后，对 ME 标记的 MDA-231 细胞的透射电子显微镜（TEM）证实，磁粒体（产生 MRI 对比的颗粒）保持完整，能够进行体内细胞追踪。
ME 用于标记哺乳动物细胞，以便在体内进行 MRI 细胞追踪。本文所述的各种测定方法（ICC、铁嗪和 qPCR）允许对标记效率进行定性和定量评估，并提供对 ME 亚细胞处理的详细了解。在某些细胞类型中，ME 被消化，但产生 MRI 的颗粒仍然存在。LLO 涂层允许 ME 逃避初级吞噬体，略微增强保留，并证实 ME 最终通过自噬进行处理。许多细胞内细菌和所有共生衍生的细胞器都进化出了分子机制来避免细胞内清除，确定 ME 清除涉及的特定过程为开发在哺乳动物细胞中具有增强保留能力的 ME 提供了框架。
