Ongoing Imaging Project

Imaging Glucose Uptake into Cells by Fluorescent Glucose Analogues

Background
 Most living things take up and metabolize naturally occurring D-glucose as essential fuel. By contrast, little is known about use of its mirror image isomer L-glucose. Mammalian cells take up D-glucose through glucose transporters such as GLUTs. A green fluorescence-emitting D-glucose analogue 2-NBDG1,2) can visualize this process at the single cell level (Fig. 1A).3-6) For obtaining a precise control substrate for 2-NBDG, we developed 2-NBDLG, a fluorescent analogue of L-glucose (fLG) (Fig. 1B).7-9)

   Fig. 1  2-NBDG and 2-NBDLG, fluorescent D-, and L-glucose analogue, respectively.


Research Results
 Beyond our expectation, when 2-NBDLG was administered to three-dimensionally accumulated insulinoma spheroids, remarkable uptake was detected in cells showing nuclear heterogeneity, one of major cytological criteria in clinical diagnosis for tumor cells suspected of high grade of malignancy.10) Moreover, a combined use of 2-NBDLG with a membrane-impermeable, Texas Red-bearing L-glucose analogue 2-TRLG11) provided a unique method for visualizing cellular state of individual tumor cells in multiple colors.10,12) Furthermore, topical application of fLGs in vivo to a hamster model of bile duct cancer successfully demonstrated good correlation between fLG images and pathological diagnoses for corresponding lesions.13)
 Clinical studies using fLGs are currently underway in Hirosaki University for discriminating cancerous/pre-cancerous cells/tissues from those with no such anomaly. Compared to conventional D-glucose analogues, lower uptake of 2-NBDLG into non-cancerous/normal cells resulted in higher signal-to-background ratio in tumor imaging.12)

   Fig. 2  Confocal microscopic images of 12 DIV MIN6 spheroids.

     (from Sasaki, A. et al., Human Cell 29: 37-45, 2016 with permission).

Future Prospects
 This method will facilitate accurate diagnosis and necessary and sufficient removal of lesions that have potential of developing cancer while sparing normal functions as much as possible. Use of the method as a drug delivery system is a potential as well. We would like to proceed with students and colleagues who believe that “Freedom to pursuit things what ‘you’ think important” may open a new door towards future.

 These researches are mainly funded by JST (1996-2000), JST (2008-2011), JST-AMED (2011-2016; 2011-2017), Hirosaki University Institutional Research (2012-2015, 2016-2019).

References
1) 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose
2) Yoshioka, K. et al., Biochim. Biophys. Acta 1289: 5-9 (1996).
3) Yamada, K. et al., J. Biol. Chem. 275: 22278-22283 (2000).
4) Ohtsubo, K. et al., Cell 123: 1307-1321 (2005).
5) Yamada, K. et al., Nature Protocols 2: 753-762 (2007).
6) Rouach, N. et al., Science 322: 1551-1555 (2008).
7) 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose
8) Yamamoto, T. et al., Tetrahedron Lett. 49: 6876-6878 (2008).
9) Yamada, K. et al., EP2325327B1, US8986656B2, JP5682881.
10) Sasaki, A. et al., Human Cell 29: 37-45 (2016).
11) Yamamoto, T. et al., Bioorg. Med. Chem. Lett. 21: 4088-4096 (2011).
12) Yamada, K. et al., WO2012/133688, JP6019500, ZL201280015126.5.
13) Yokoyama, H. et al., Human Cell 29: 111-121 (2016).