Nozaki Satoshi, Nakatani Yuka, Mawatari Aya, Hume William Ewan, Doi Hisashi, Watanabe Yasuyoshi
Laboratory for Pathophysiological and Health Science, RIKEN Center for Biosystems Dynamics Research, 6-7-3 Minatojima-Minamimachiinamimachi, Chuo-Ku, Kobe, Hyogo, 650-0047, Japan.
Novel PET Diagnostics Laboratory, RIKEN Innovation Center, Hyogo, Japan.
EJNMMI Res. 2023 Apr 28;13(1):36. doi: 10.1186/s13550-023-00988-1.
(S)-2-amino-3-[3-(2-F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid (F-FIMP) as a promising PET probe for imaging the tumor-specific L-type amino acid transporter (LAT) 1. Our previous study revealed that F-FIMP had a higher affinity for LAT1 than for LAT2 abundantly expressed even in normal cells. F-FIMP showed high accumulation in LAT1-positive tumor tissues and low accumulation in inflamed lesions in tumor-bearing mice. However, the affinity of F-FIMP for other amino acid transporters was not determined yet. Here, we aimed to determine whether F-FIMP has affinity for other tumor-related amino acid transporters, such as sodium- and chloride-dependent neutral and basic amino acid transporter B(0 +) (ATB), alanine serine cysteine transporter 2 (ASCT2), and cystine/glutamate transporter (xCT).
Cells overexpressing LAT1, ATB, ASCT2, or xCT were established by the transfection of expression vectors for LAT1, ATB, ASCT2, or xCT. Protein expression levels were determined by western blot and immunofluorescent analyses. Transport function was evaluated by a cell-based uptake assay using F-FIMP and C-labeled amino acids as substrates.
Intense signals were observed only for expression vector-transfected cells on western blot and immunofluorescent analyses. These signals were strongly reduced by gene-specific small interfering ribonucleic acid treatment. The uptake values for each C-labeled substrate were significantly higher in the transfected cells than in the mock-transfected cells and were significantly inhibited by the corresponding specific inhibitors. The F-FIMP uptake values were significantly higher in the LAT1- and ATB-overexpressing cells than in the corresponding mock cells, but no such increase was seen in the ASCT2- or xCT-overexpressing cells. These F-FIMP uptake values were significantly decreased by the specific inhibitors for LAT1- and ATB.
We demonstrated that F-FIMP has affinity not only for LAT1, but also for ATB. Our results may be helpful for understanding the mechanisms of the whole-body distribution and tumor accumulation of F-FIMP.
（S）-2-氨基-3-[3-（2-氟乙氧基）-4-碘苯基]-2-甲基丙酸（F-FIMP）是一种有前景的正电子发射断层扫描（PET）探针，用于成像肿瘤特异性L型氨基酸转运体（LAT）1。我们之前的研究表明，F-FIMP对LAT1的亲和力高于对即使在正常细胞中也大量表达的LAT2的亲和力。F-FIMP在荷瘤小鼠的LAT1阳性肿瘤组织中显示出高摄取，而在炎症病变中显示出低摄取。然而，F-FIMP对其他氨基酸转运体的亲和力尚未确定。在此，我们旨在确定F-FIMP是否对其他与肿瘤相关的氨基酸转运体具有亲和力，例如钠和氯依赖性中性和碱性氨基酸转运体B(0 +)（ATB）、丙氨酸丝氨酸半胱氨酸转运体2（ASCT2）和胱氨酸/谷氨酸转运体（xCT）。
通过转染LAT1、ATB、ASCT2或xCT的表达载体建立过表达LAT1、ATB、ASCT2或xCT的细胞。通过蛋白质印迹和免疫荧光分析确定蛋白质表达水平。使用F-FIMP和碳标记的氨基酸作为底物，通过基于细胞的摄取试验评估转运功能。
在蛋白质印迹和免疫荧光分析中，仅在表达载体转染的细胞中观察到强信号。这些信号通过基因特异性小干扰核糖核酸处理而显著降低。在转染细胞中，每种碳标记底物的摄取值显著高于mock转染细胞，并且被相应的特异性抑制剂显著抑制。F-FIMP摄取值在过表达LAT1和ATB的细胞中显著高于相应的mock细胞，但在过表达ASCT2或xCT的细胞中未观察到这种增加。这些F-FIMP摄取值被LAT1和ATB的特异性抑制剂显著降低。
我们证明F-FIMP不仅对LAT1具有亲和力，而且对ATB也具有亲和力。我们的结果可能有助于理解F-FIMP的全身分布和肿瘤摄取机制。
