A GLUT1 inhibitor-based probe significantly ameliorates the sensitivity of tumor detection and diagnostic imaging

Article abstract of DOI:10.1039/d1cc00343g

We report a non-antibody GLUT1 inhibitor probe NBDQ that is 30 times more sensitive than the traditional GLUT1 transportable tracer for cancer cell imaging and Warburg effect-based tumor detection. NBDQ reveals significant advantages in terms of tumor sel

Full text of DOI:10.1039/d1cc00343g

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A GLUT1 inhibitor-based probe significantly
ameliorates the sensitivity of tumor detection and
Cite this: DOI: 10.1039/d1cc00343g
diagnostic imaging†
Received 20th January 2021,
Accepted 23rd April 2021
a
ab
ac
ad
a
Shengnan Liu, * Weijie Song, Yujun Cui, Ziru Sun, Zhenhua Huang and
a
Qingzhi Gao
*
DOI: 10.1039/d1cc00343g
rsc.li/chemcomm
We report a non-antibody GLUT1 inhibitor probe NBDQ that is 30 structurally compact green fluorophore, NBD (nitrobenzoxadia-
4
times more sensitive than the traditional GLUT1 transportable zole), at the C-2 position of glucosamine (Scheme 1). 2-NBDG
tracer for cancer cell imaging and Warburg effect-based tumor can be transported by GLUT1 and produce short-lived, fluores-
5
detection. NBDQ reveals significant advantages in terms of tumor cent 2-NBDG-6-phosphate in high GLUT1-expressing cells and
selectivity, fluorescence stability and in vivo biocompatibility in has proven to be useful for in vitro glucose uptake monitoring in
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xenograft tumor imaging, including triple-negative breast cancer.
many different types of GLUT1 overexpressing cancer cells.
However, similar to the other transportable glucose tracers, the
The Warburg effect has been consistently recognized in cancers application of 2-NBDG in tumor labeling has been reported to
through increased rates of glucose transport and metabolism. have several downfalls, including high treatment dosage,
Targeting this elevated glycolysis or overexpressed GLUTs has low signal intensity due to the intracellular metabolism
provided a promising therapeutic strategy, which has been a induced fluorescence decay, and low in vivo biocompatibility by
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key catalyst for targeted therapeutic cancer drug development competitive physiological glucose inhibition and impairment.
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and clinical diagnostic tumor imaging.
F-FDG-PET (2-[ F]-
To overcome these challenges, in this work, we propose and
fluoro-2-deoxy-D-glucose based positron emission tomography) demonstrate a novel strategy by employing a fluorescently
is currently the most commonly used clinical modality for labeled GLUT1 inhibitor: NBDQ, as a new class of tumor
2
tumor diagnosis, cancer staging and recurrence monitoring.
diagnostic agent for more efficient detection and monitoring
Although FDG-PET has revolutionized cancer diagnosis, the use of Warburg effect-activated glycolysis. NBDQ was derived from
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of F-FDG still has many disadvantages aside from the technical the natural flavonoid quercetin through a simple regioselective
and logistical problems associated with radioactivity, including Mannich coupling with the fluorophore NBD (Scheme 1).
high cost, very short half-life (B110 min) that needs to be treated
as a perishable substance and is only applicable for immediate
evaluation, and particularly the need of fasting for the plasma
3
glucose control. All these disadvantages led efforts to develop
non-isotopic bioprobes. The most widely used fluorescent GLUT
substrate is 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-
deoxyglucose (2-NBDG), which is designed by installing a
a
Institute of Molecular Plus, Tianjin Key Laboratory for Modern Drug Delivery &
High-Efficiency, School of Pharmaceutical Science and Technology,
Tianjin University, 92 Weijin Rd, Nankai, Tianjin 300072, P. R. China.
E-mail: shengnan_liu@tju.edu.cn, qingzhi@tju.edu.cn
b
Tianjin Medical University Cancer Institute and Hospital, National Clinical
Research Center for Cancer, West Huanhu Road, Hexi, Tianjin 300060, P. R. China
Transplantation Center, Tianjin First Central Hospital, 24 Fukang Road, Nankai, Tianjin
3
00192, P. R. China
Scheme 1 Design concept of the Warburg effect targeted GLUT1 inhibitor
bioprobe. 2-NBDG: the well-known GLUT1 transportable fluorescent tracer
for glucose uptake assessment; NBDQ: the newly designed GLUT1 inhibitor
bioprobe based on the natural flavonoid quercetin in this study.
d
Department of Biology, Gudui BioPharma Technology Inc., 5 Lanyuan Road, Huayuan
Industrial Park, Tianjin 300384, P. R. China
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/d1cc00343g
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We revealed that NBDQ deserves a particular merit for
more effective Warburg effect based cancer cell detection and
significantly improves the in vivo tumor diagnostic imaging.
Our study reveals the first inhibitor-based approach on
non-antibody mediated molecular imaging of GLUT1
overexpression.
Several classes of GLUT inhibitors were identified and
synthesized by scientists during their approaches for either
diabetes management studies or cancer drug research. Namely,
the natural flavonoids, including quercetin, phloretin,
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9
phlorizin, the Greek cytos relaxation molecule cytochalasin B,
and the natural compound forskolin which is isolated from the
plant Coleus forskohlii, were frequently used as SGLTs (sodium
1
0
dependent glucose transporters) and GLUT inhibitors. In
addition to the natural compounds, artificially synthesized
GLUT1 inhibitor molecules and GLUT targeting nanoparticles
have also been extensively pursued for potential targeted drug
Fig. 1 (A) Fluorescence excitation and emission spectra of NBDQ (50 mM
of NBDQ in PBS). (B) The expression level of GLUT1 in MCF10A, MDA-MB-
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68, BEAS-2B, and A549 cells. (C) The sensitivity in live-cell imaging of
NBDQ (0.3–50 mM) and 2-NBDG (3–500 mM) in A549 cells under physio-
analog that can be a potent GLUT1 inhibitor, we chose the logical conditions for 1 h. (D) The fluorescence microscopic imaging of
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1
delivery and biomedical diagnosis. In order to make an optical
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0 mM of NBDQ labeled A549 cells at 3 h (see Fig. S7B, ESI† for 2-NBDG).
natural flavonoid quercetin, which is abundant in onions, green
tea, and other flavonoids and polyphenol containing plants, as a
template for fluorescent derivatization. Quercetin has been
found to strongly inhibit glucose transport and intracellular 2-NBDG (up to 500 mM) was needed for achieving an acceptable
uptake in several cancer cells. The inhibitory effect of quercetin signal-to-noise ratio under physiological cell culture conditions.
on glucose uptake was established by impairing the Warburg In the same cell line, the inhibitor probe NBDQ shows a
effect, and most importantly, the glucose inhibition of quercetin significant improvement for live-cell imaging. Under normal cell
1
2
was confirmed to be independent of extracellular glucose.
culture conditions, 10 mM of NBDQ can achieve the same labeling
Based on these observations, NBDQ was designed and effect as 300 mM of 2-NBDG (Fig. 1C). The metabolic deterioration
synthesized by combining quercetin with the fluorophore of of both probes was also remarkably different. For instance, a rapid
NBD to target the tumor specific Warburg effect.
fluorescence quenching within 3 h was observed in the 2-NBDG
The preparation details and analytical data of NBDQ are (500 mM) treated A549 cells (Fig. S1D, ESI†), whereas the
described in the ESI† (Scheme S1 and Fig. S1–S4). In PBS, fluorescence of the NBDQ (50 mM) treated cells were found to
NBDQ exhibits a minor excitation peak at 343 nm and a major be stable (Fig. 1D). Thus, with the rationally designed NBDQ,
excitation peak at 478 nm, with a single emission at 561 nm 10 times lower concentration of the probe can realize a 3-fold
(Fig. 1A). The two excitation peaks presumably reflect two higher fluorescence intensity and the fluorescence lifetime in
distinct states: protonated and unprotonated NBD fluoro- living cells was greatly improved. This result demonstrates that
phores. This unique excitation characteristic makes the probe NBDQ enables significant improvement in the cancer cell labeling
reach an extremely large Stokes shift (up to 218 nm) and more sensitivity and bio- and physiological compatibility over 2-NBDG.
potentially useful in different biological environments as one
The relationship between GLUT1 expression and cytotoxicity
may alternate excitation wavelength to avoid potential induced by the probe was confirmed using two pairs of normal
incompatible cross disturbance with other fluorescent labeling and neoplastic cell lines, namely, the normal bronchial epithelial
(e.g. GFP excitation at B480 nm). The emission spectrum of BEAS-2B cells and the corresponding human non-small-cell lung
NBDQ at 561 nm also gives this probe the potential to perform cancer A549 cells and non-tumorigenic human breast MCF10A
biological studies regarding the single-molecule fluorescence cells and the corresponding human triple-negative breast cancer
resonance energy transfer (FRET) mediated spatial resolution. (TNBC) MDA-MB-468 cells. NBDQ showed low toxicity against the
The fluorophores that can be excited by the emission light of normal BEAS-2B and MCF-10A cells but more toxicity against
NBDQ in terms of a single molecule, as studied by FRET the GLUT1 high expressing A549 and TNBC cells (Fig. S8, ESI†).
analysis, include Cy3 (excitation at B530 nm), dsRed1 This result can be explained by the fact that the specific inhibition
(
excitation at B560 nm) and rhodamine (excitation at of GLUT1 may cause more decreased glucose supply and
B543 nm) (Fig. 1A; for more information including excitation disturbance to the glycolytic influx in both A549 and TNBC cells.
and UV–vis spectra, see Fig. S5 and S6, ESI†). The GLUT1 specificity and inhibitory effect were confirmed
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As reported previously, for the uptake of glucose tracer by the 2-DG uptake assay using human red blood cells as an
-NBDG, increased extracellular concentrations of the probe established system because human erythrocytes express GLUT1
2
1
4
can lead to increased intracellular probe transport in the as their sole glucose transporter. The known GLUT1 inhibitors,
GLUT1 overexpressing A549 cancer cells (Fig. 1B, C and quercetin, phloretin, and CB (Fig. S9A, ESI†), were used to
Fig. S7A, ESI†). However, a very high concentration of determine the effectiveness and magnitude of NBDQ on the
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to phloretin by interacting with GLUT1 at a specific site close to
the exofacial surface of the transporter. The inhibitor binding site
and molecular binding mode of NBDQ with GLUT1 were further
confirmed by a 150 ns molecular dynamics simulation study with
the YASARA program (Fig. S11–S14 and Tables S1–S3, ESI†).
To demonstrate the specificity of NBDQ towards the GLUT1
overexpressing cancer cells, we conducted a simple flow
cytometry-based detection and quantification test by using
GLUT1 low- and high-expressing BEAS-2B and A549 cells. As
an optimal condition, 50 mM of NBDQ with 30 min of incubation
was confirmed to allow specific identification of A549 cells
according to the GLUT1 expression magnitude (Fig. S15A–C,
ESI†). Furthermore, in a spiked mixture, the NBDQ labeling was
Fig. 2 (A) 2-DG uptake inhibition curves in human erythrocytes with validated to be able to differentiate cancer cells (A549) from
different GLUT1 inhibitors. (B) The confocal fluorescence microscopy
imaging of human erythrocytes treated with 50 nM of NBDQ at 37 1C
incubated for 30 min. (C) The competitive effect of cytochalasin B (1–20 mM)
on NBDQ cellular binding in MDA-MB-468 cells. (D) The competitive effect of
normal cells (BEAS-2B) with a very high sensitivity and excellent
accuracy (Fig. S15D and E, ESI†).
To validate the robustness of the probe for in vivo diagnostic
imaging, we performed an application study of NBDQ in both
human TNBC and A549-derived xenograft tumor detection.
TNBC diagnostic imaging remains an unmet need due to the
lack of representing biomarkers (i.e. no estrogen receptor,
phloretin (1–200 mM) on NBDQ cellular binding in MDA-MB-468 cells.
Data are presented as mean Æ S.E.M.
GLUT1 inhibition. According to the half maximal inhibitory progesterone receptor and human epidermal growth factor
16
concentrations (IC50, NBDQ: 7.6 Æ 1.3 mM; CB: 10.9 Æ 2.1 mM; receptor-2). However, a high tumoral glucose uptake regulated
quercetin: 112.0 Æ 8.1 mM; phloretin: 4200 mM) (Fig. 2A), the by the Warburg effect has been fundamentally recognized as a
GLUT1 inhibition effect of NBDQ is much stronger than that of hallmark and promising target of TNBC for diagnostic imaging and
17
quercetin and phloretin but similar to the GLUT1 specific even prognostic staging. Equimolar concentrations of both NBDQ
inhibitor CB, which reduces the 2-DG uptake in erythrocytes to and 2-NBDG were administered by tail vein injection and the mice
5
2.0 Æ 2.3% under 10 mM.
The confocal-laser scanning analysis with 30 min of incubation in vivo imaging system. All animal experiments were approved by
in human erythrocytes using NBDQ (50 nM) and 2-NBDG the Institutional Animal Ethics Committee and complied with the
500 nM) has further clarified the differences between these two current guidelines for treating lab animals of China.
probes (Fig. 2B and Fig. S9B, ESI†). Under the testing conditions, As shown in Fig. 3A, the fluorescence signals of NBDQ were
were imaged after certain time intervals using the Xenogen IVIS
(
the intracellularly transported 2-NBDG and surface binding of clearly differentiated from the surrounding tissues 6 h post a
NBDQ were clearly visualized. To decipher the inhibition single dose injection. The fluorescence intensity of NBDQ
mechanism, the competitive inhibitor binding test was carried gradually increased and accumulated at the tumor site, indicat-
out by combining CB and phloretin with NBDQ, respectively. ing that NBDQ could be preferentially recognized and recruited
In the GLUT1 highly overexpressing MDA-MB-468 cells, the by elevated glycolysis of the TNBC cells (Fig. 3B). The comparison
pretreatment of 20 mM of CB did not cause fluorescence intensity results between NBDQ and equimolar concentration of 2-NBDG
changes of NBDQ, whereas an increased amount of phloretin revealed that the fluorescence signal of NBDQ considerably
significantly attenuated NBDQ cellular binding (Fig. 2C and D). outlasted up to 72 h, while the detectable fluorescence intensity
These results suggest that the GLUT1 inhibition mechanism of for 2-NBDG can only persist for about 12 h post treatment
NBDQ is different from that of CB, whereas phloretin may (Fig. 3A vs. C). The ex vivo optical images and the corresponding
share similar binding determinants with NBDQ. The competitive probe activities of the main organs from animals sacrificed at
inhibition results on the 2-DG uptake using human erythrocytes 72 h for NBDQ and at 24 h for 2-NBDG are shown in Fig. 3A, C
revealed the same findings: the pretreatment of a high concen- and D. As summarized in Table S4 (ESI†), the highest imaging
tration of phloretin (200 mM) results in no further inhibition of contrast indicated as tumor-to-background ratio (Tm/Bkg) for
NBDQ on the intracellular uptake of 2-DG but CB (10 mM) treated NBDQ approaches 12.37 : 1, which is significantly higher than
erythrocytes showed a synergistic inhibitory effect with NBDQ the GLUT1 substrate tracer 2-NBDG (5.44 : 1, Table S4, ESI†).
1
8
(
Fig. S10, ESI†). The kinetic effects of both cytochalasin B and The clinically proved F-FDG/PET imaging has been reported to
phloretin on glucose transport mediated by GLUT1 are well be utilized in the same MDA-MB-468 xenograft mice, but under
known. Cytochalasin B acts as a competitive inhibitor of glucose fasted conditions, and the Tm/Bkg ratio was recorded as 3.21: 1,
1
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transport out of cells by molecular binding to a site accessible which is much lower than NBDQ. The tumor-to-organ ratio
from the endofacial surface of the transporter, whereas phloretin (Tm/Torg) of NBDQ for groups of mice was recorded as 8.60: 1
acts as a competitive inhibitor of glucose uptake into cells by versus 3.48: 1 for 2-NBDG (n = 3), which is also significantly
9,15
binding with a glucose competitive external site.
These results improved (Fig. 3D and Table S4; see Fig. S16, ESI† and Table S5
suggest that NBDQ inhibits glucose transport in a similar fashion for the A549 xenograft imaging study).
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xenograft mice. Up to 72 h following administration of 0.2 mmol mouse
À1
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indicates the location of the tumor. (B) The quantitative analysis of mean
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