Nakamura Yuko, Mochida Ai, Nagaya Tadanobu, Okuyama Shuhei, Ogata Fusa, Choyke Peter L, Kobayashi Hisataka
Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, United States National Institutes of Health, Bethesda, Maryland 20892, USA.
Oncotarget. 2017 Jun 13;8(24):39512-39521. doi: 10.18632/oncotarget.17080.
SPiDER-βGal is a newly-developed probe that is activated by β-galactosidase and is then retained within cells by anchoring to intracellular proteins. Previous work has focused on gGlu-HMRG, a probe activated by γ-glutamyltranspeptidase, which demonstrated high sensitivity for the detection of peritoneal ovarian cancer metastases in an animal model. However, its fluorescence, after activation by γ-glutamyltranspeptidase, rapidly declines over time, limiting the actual imaging window and the ability to define the border of lesions. The purpose of this study is to compare the fluorescence signal kinetics of SPiDER-βGal with that of gGlu-HMRG using ovarian cancer cell lines in vitro and ex vivo tissue imaging. In vitro removal of gGlu-HMRG resulted in a rapid decrease of fluorescence intensity followed by a more gradual decrease up to 60 min while there was a gradual increase in fluorescence up to 60 min after removal of SPiDER-βGal. This is most likely due to internalization and retention of the dye within cells. This was also confirmed ex vivo tissue imaging using a red fluorescence protein (RFP)-labeled tumor model in which the intensity of fluorescence increased gradually after activation of SPiDER-βGal. Additionally, SPiDER-βGal resulted in intense enhancement within the tumor due to the high target-to-background ratio, which extended up to 60 min after activation. In contrast, gGlu-HMRG fluorescence resulted in decreasing fluorescence over time in extracted tumors. Thus, SPiDER-βGal has the advantages of higher signal with more signal retention, resulting in improved contrast of the tumor margin and suggesting it may be an alternative to existing activatable probes.
SPiDER-βGal是一种新开发的探针，它由β-半乳糖苷酶激活，然后通过锚定细胞内蛋白质而保留在细胞内。先前的研究集中在γ-谷氨酰转肽酶激活的探针gGlu-HMRG上，该探针在动物模型中对检测腹膜卵巢癌转移表现出高灵敏度。然而，其在被γ-谷氨酰转肽酶激活后的荧光会随着时间迅速下降，限制了实际成像窗口以及界定病变边界的能力。本研究的目的是在体外使用卵巢癌细胞系以及在离体组织成像中比较SPiDER-βGal与gGlu-HMRG的荧光信号动力学。体外去除gGlu-HMRG后，荧光强度迅速下降，随后在长达60分钟的时间里逐渐下降，而去除SPiDER-βGal后，荧光在长达60分钟的时间里逐渐增加。这很可能是由于染料在细胞内的内化和保留。在使用红色荧光蛋白（RFP）标记的肿瘤模型进行的离体组织成像中也证实了这一点，其中SPiDER-βGal激活后荧光强度逐渐增加。此外，由于高靶标与背景比，SPiDER-βGal在肿瘤内导致强烈增强，激活后长达60分钟。相比之下，在提取的肿瘤中，gGlu-HMRG荧光随时间导致荧光下降。因此，SPiDER-βGal具有信号更高且信号保留更多的优点，从而改善了肿瘤边缘的对比度，表明它可能是现有可激活探针的一种替代物。
