放射发光显微镜单细胞成像揭示 [18F]HFB 的意外结合靶标。
Single-Cell Imaging Using Radioluminescence Microscopy Reveals Unexpected Binding Target for [18F]HFB.
机构信息
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA, USA.
Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA.
出版信息
Mol Imaging Biol. 2018 Jun;20(3):378-387. doi: 10.1007/s11307-017-1144-0.
PURPOSE
Cell-based therapies are showing great promise for a variety of diseases, but remain hindered by the limited information available regarding the biological fate, migration routes and differentiation patterns of infused cells in trials. Previous studies have demonstrated the feasibility of using positron emission tomography (PET) to track single cells utilising an approach known as positron emission particle tracking (PEPT). The radiolabel hexadecyl-4-[F]fluorobenzoate ([F]HFB) was identified as a promising candidate for PEPT, due to its efficient and long-lasting labelling capabilities. The purpose of this work was to characterise the labelling efficiency of [F]HFB in vitro at the single-cell level prior to in vivo studies.
PROCEDURES
The binding efficiency of [F]HFB to MDA-MB-231 and Jurkat cells was verified in vitro using bulk gamma counting. The measurements were subsequently repeated in single cells using a new method known as radioluminescence microscopy (RLM) and binding of the radiolabel to the single cells was correlated with various fluorescent dyes.
RESULTS
Similar to previous reports, bulk cell labelling was significantly higher with [F]HFB (18.75 ± 2.47 dpm/cell, n = 6) than 2-deoxy-2-[F]fluoro-D-glucose ([F]FDG) (7.59 ± 0.73 dpm/cell, n = 7; p ≤ 0.01). However, single-cell imaging using RLM revealed that [F]HFB accumulation in live cells (8.35 ± 1.48 cpm/cell, n = 9) was not significantly higher than background levels (4.83 ± 0.52 cpm/cell, n = 12; p > 0.05) and was 1.7-fold lower than [F]FDG uptake in the same cell line (14.09 ± 1.90 cpm/cell, n = 13; p < 0.01). Instead, [F]HFB was found to bind significantly to fragmented membranes associated with dead cell nuclei, suggesting an alternative binding target for [F]HFB.
CONCLUSION
This study demonstrates that bulk analysis alone does not always accurately portray the labelling efficiency, therefore highlighting the need for more routine screening of radiolabels using RLM to identify heterogeneity at the single-cell level.
目的
细胞疗法在多种疾病的治疗中显示出巨大的潜力，但由于临床试验中关于输注细胞的生物学命运、迁移途径和分化模式的信息有限，仍然受到限制。先前的研究已经证明，利用正电子发射断层扫描（PET）追踪单个细胞是可行的，这种方法称为正电子发射粒子追踪（PEPT）。由于其高效且持久的标记能力，十六烷基-4-[F]氟苯甲酸酯（[F]HFB）被确定为 PEPT 的有前途的候选物。本研究的目的是在体内研究之前，在体外对[F]HFB 的单细胞水平的标记效率进行特征描述。
方法
使用批量伽马计数在体外验证[F]HFB 与 MDA-MB-231 和 Jurkat 细胞的结合效率。随后，使用一种称为放射光显微镜（RLM）的新方法在单个细胞中重复进行测量，并将放射性标记物与各种荧光染料结合与单个细胞的结合进行关联。
结果
与先前的报告类似，[F]HFB（18.75±2.47 dpm/细胞，n=6）的细胞批量标记明显高于 2-脱氧-2-[F]氟-D-葡萄糖（[F]FDG）（7.59±0.73 dpm/细胞，n=7；p≤0.01）。然而，使用 RLM 进行的单细胞成像显示，[F]HFB 在活细胞中的积累（8.35±1.48 cpm/细胞，n=9）并不明显高于背景水平（4.83±0.52 cpm/细胞，n=12；p>0.05），并且比同一细胞系中[F]FDG 的摄取低 1.7 倍（14.09±1.90 cpm/细胞，n=13；p<0.01）。相反，发现[F]HFB 与与死细胞核相关的碎片化膜结合明显，表明[F]HFB 的替代结合靶标。
结论
本研究表明，仅进行批量分析并不总是能准确描述标记效率，因此需要使用 RLM 对放射性标记物进行更常规的筛选，以识别单细胞水平的异质性。
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