Background

[PubMed]

Optical fluorescence imaging is increasingly used to monitor biological functions of specific targets in small animals (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–650 nm) are used. Near-infrared (NIR) fluorescence (650–900 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a noninvasive complement to radionuclide imaging in small animals or with probes in close proximity to the target in humans (4). Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment (5-8). It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumor cells and endoscopic imaging of the pancreas and colon.

The phosphorylation of glucose, an initial and important step in cellular metabolism, is catalyzed by hexokinases (HKs) (9). There are four HKs in mammalian tissues (HKI–HKIV). HKI, HKII, and HKIII have molecular weights of ~100,000 each; HKI is found mainly in the brain, and HKII is insulin-sensitive and is found in adipose and muscle cells. HKIV, also known as glucokinase, has a molecular weight of ~50,000 and is specific to the liver and pancreas. Most brain HK is bound to mitochondria, enabling coordination between glucose consumption and oxidation. Tumor cells are known to be highly glycolytic because of increased expression of glycolytic enzymes and HK activity (10), which was detected in tumors from patients with lung, gastrointestinal, and breast cancers. The HKs, by converting glucose to glucose-6-phosphate, help maintain the downhill gradient that results in the transport of glucose into cells through the facilitative glucose transporters (GLUT1–13) (11). GLUT1 is considered to be the main transporter of glucose uptake. GLUT4 and HKII are the major transporter and HK isoform in skeletal muscle, heart, and adipose tissue, wherein insulin promotes glucose utilization. HKIV is associated with GLUT2 in liver and pancreatic β cells.

2-Deoxy-d-glucose (2DG) was first developed to inhibit glucose utilization by cancer cells (12). HKs phosphorylate 2DG to 2DG-6-phosphate, which inhibits phosphorylation of glucose. 2-[¹⁸F]Fluoro-2-deoxy-d-glucose ([¹⁸F]FDG) was later developed for molecular imaging studies (13). FDG is moved into cells by glucose transporters, where it is phosphorylated by HK to FDG-6-phosphate. FDG-6-phosphate cannot be metabolized further in the glycolytic pathway and remains in the cells. Tumor cells do not contain a sufficient amount of glucose-6-phosphatase to reverse the phosphorylation. The elevated rates of glycolysis and glucose transport in many types of tumor cells and activated cells enhance the uptake of FDG in these cells relative to other normal cells. Positron emission tomography (PET) with [¹⁸F]FDG has been used to assess alterations in glucose metabolism in brain, cancer, cardiovascular diseases, Alzheimer’s disease and other central nervous system disorders, and infectious, autoimmune, and inflammatory diseases (14-19). Various NIR dyes (such as cypate, Cy5.5, and IRDye800CW) were conjugated to 2DG (20-22) as optical imaging agents for in vivo imaging of glucose utilization in tumors in mice. Cypate is a reactive carbocyanine dye, which is derived from indocyanine green (ICG) (23). Cypate exhibits an absorbance maximum at 778 nm and an emission maximum at 805 nm, with a high extinction coefficient of 224,000 M−¹cm−¹. Cypate is a lipophilic dye and contains two carboxyl functional groups for covalent conjugation to the amino group of biomolecules. Guo et al. (24) evaluated cypate-2DG for in vivo NIR optical imaging in tumor-bearing mice.

Synthesis

[PubMed]

Cypate-N-hydroxysuccinimide ester (0.0128 mmol) was reacted with 2-amino-2-DG (0.064 mmol) for 18 h at room temperature in sodium phosphate buffer (pH 9) (24). Cypate-2DG was purified with high-performance liquid chromatography and verified with mass spectroscopy. There were two 2DG molecules per cypate-2DG. The yield of cypate-2DG was ~35%, with 90% purity. Cypate-2DG displayed similar spectral properties as cypate, with maximum absorption at 782 nm and maximum emission at 821 nm.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro uptake studies of rhodamine-2DG (RhB-2DG) were performed with MCF-7/estradiol, MDA-MB-435, U87MG, and MCF-7 tumor cells in culture (24). These studies showed that high, medium, and low fluorescence intensity values correlated with the tumors' GLUT1 expression levels, respectively. Fluorescence microscopy showed that RhB-2DG accumulated in the cytoplasm of the tumor cells. Excess 2DG was able to block the NIR fluorescence signal in the cytoplasm.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

References

1.

Achilefu S. Lighting up tumors with receptor-specific optical molecular probes. Technol Cancer Res Treat. 2004;3(4):393–409. [PubMed: 15270591]

2.

Becker A., Hessenius C., Licha K., Ebert B., Sukowski U., Semmler W., Wiedenmann B., Grotzinger C. Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nat Biotechnol. 2001;19(4):327–31. [PubMed: 11283589]

3.

Ntziachristos V., Bremer C., Weissleder R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol. 2003;13(1):195–208. [PubMed: 12541130]

4.

Tung C.H. Fluorescent peptide probes for in vivo diagnostic imaging. Biopolymers. 2004;76(5):391–403. [PubMed: 15389488]

5.

Yannuzzi, L.A., Indocyanine green angiography: a perspective on use in the clinical setting. Am J Ophthalmol, 2011151(5): p. 745-751 e1. [PubMed: 21501704]

6.

Yamamoto M., Sasaguri S., Sato T. Assessing intraoperative blood flow in cardiovascular surgery. Surg Today. 2011;41(11):1467–74. [PubMed: 21969147]

7.

Schaafsma B.E., Mieog J.S., Hutteman M., van der Vorst J.R., Kuppen P.J., Lowik C.W., Frangioni J.V., van de Velde C.J., Vahrmeijer A.L. The clinical use of indocyanine green as a near-infrared fluorescent contrast agent for image-guided oncologic surgery. J Surg Oncol. 2011;104(3):323–32. [PMC free article: PMC3144993] [PubMed: 21495033]

8.

Manizate F., Hiotis S.P., Labow D., Roayaie S., Schwartz M. Liver functional reserve estimation: state of the art and relevance for local treatments: the Western perspective. J Hepatobiliary Pancreat Sci. 2010;17(4):385–8. [PubMed: 19936599]

9.

Smith T.A. Mammalian hexokinases and their abnormal expression in cancer. Br J Biomed Sci. 2000;57(2):170–8. [PubMed: 10912295]

10.

Suolinna E.M., Haaparanta M., Paul R., Harkonen P., Solin O., Sipila H. Metabolism of 2-[18F]fluoro-2-deoxyglucose in tumor-bearing rats: chromatographic and enzymatic studies. Int J Rad Appl Instrum B. 1986;13(5):577–81. [PubMed: 3818323]

11.

Wood I.S., Trayhurn P. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br J Nutr. 2003;89(1):3–9. [PubMed: 12568659]

12.

Laszlo J., Humphreys S.R., Goldin A. Effects of glucose analogues (2-deoxy-D-glucose, 2-deoxy-D-galactose) on experimental tumors. J Natl Cancer Inst. 1960;24:267–81. [PubMed: 14414406]

13.

Fowler J.S., Ido T. Initial and subsequent approach for the synthesis of 18FDG. Semin Nucl Med. 2002;32(1):6–12. [PubMed: 11839070]

14.

Phelps M.E. PET: the merging of biology and imaging into molecular imaging. J Nucl Med. 2000;41(4):661–81. [PubMed: 10768568]

15.

Phelps M.E., Mazziotta J.C. Positron emission tomography: human brain function and biochemistry. Science. 1985;228(4701):799–809. [PubMed: 2860723]

16.

Phelps M.E., Mazziotta J.C., Huang S.C. Study of cerebral function with positron computed tomography. J Cereb Blood Flow Metab. 1982;2(2):113–62. [PubMed: 6210701]

17.

Rohren E.M., Turkington T.G., Coleman R.E. Clinical applications of PET in oncology. Radiology. 2004;231(2):305–32. [PubMed: 15044750]

18.

Sokoloff L. Basic principles in imaging of regional cerebral metabolic rates. Res Publ Assoc Res Nerv Ment Dis. 1985;63:21–49. [PubMed: 2992057]

19.

Spence A.M., Mankoff D.A., Muzi M. Positron emission tomography imaging of brain tumors. Neuroimaging Clin N Am. 2003;13(4):717–39. [PubMed: 15024957]

20.

Kovar J.L., Volcheck W., Sevick-Muraca E., Simpson M.A., Olive D.M. Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models. Anal Biochem. 2009;384(2):254–62. [PMC free article: PMC2720560] [PubMed: 18938129]

21.

Cheng Z., Levi J., Xiong Z., Gheysens O., Keren S., Chen X., Gambhir S.S. Near-infrared fluorescent deoxyglucose analogue for tumor optical imaging in cell culture and living mice. Bioconjug Chem. 2006;17(3):662–9. [PMC free article: PMC3191878] [PubMed: 16704203]

22.

Chen Y., Zheng G., Zhang Z.H., Blessington D., Zhang M., Li H., Liu Q., Zhou L., Intes X., Achilefu S., Chance B. Metabolism-enhanced tumor localization by fluorescence imaging: in vivo animal studies. Opt Lett. 2003;28(21):2070–2. [PubMed: 14587818]

23.

Ye Y., Bloch S., Kao J., Achilefu S. Multivalent carbocyanine molecular probes: synthesis and applications. Bioconjug Chem. 2005;16(1):51–61. [PubMed: 15656575]

24.

Guo J., Du C., Shan L., Zhu H., Xue B., Qian Z., Achilefu S., Gu Y. Comparison of near-infrared fluorescent deoxyglucose probes with different dyes for tumor diagnosis in vivo. Contrast Media Mol Imaging. 2012;7(3):289–301. [PubMed: 22539399]