Real-Time Bioluminescence Imaging of Nitroreductase in Mouse Model

Article abstract of DOI:10.1021/acs.analchem.6b01160

Nitroreductase (NTR) is an endogenous reductase overexpressed in hypoxic tumors; however, its precise detection in living cells and animals remains a considerable challenge. Herein, we developed three reaction-based probes and a related bioluminescence assay for the real-time NTR detection. The high sensitivity and selectivity of probe 3, combined with its remarkable potential of bioluminescence imaging, affords a valuable approach for in vivo imaging of NTR in a tumor model mouse.

Full text of DOI:10.1021/acs.analchem.6b01160

Letter
pubs.acs.org/ac
Real-Time Bioluminescence Imaging of Nitroreductase in Mouse
Model
Ping Feng,†,‡ Huateng Zhang,§,‡ Quankun Deng,¶ Wei Liu,¶ Linghui Yang,¶ Guobo Li,¶ Guo Chen,¶
Lupei Du,§ Bowen Ke,*,¶ and Minyong Li*,§
†Institute of Clinical Trials, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
¶The Laboratory of Anesthesiology and Critical Care Medicine, Translational Neuroscience Center, West China Hospital, Sichuan
University, Chengdu, Sichuan 610041, China
§Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan,
Shandong 250012, China
S
* Supporting Information
ABSTRACT: Nitroreductase (NTR) is an endogenous
reductase overexpressed in hypoxic tumors; however, its
precise detection in living cells and animals remains a
considerable challenge. Herein, we developed three reaction-
based probes and a related bioluminescence assay for the real-
time NTR detection. The high sensitivity and selectivity of
probe 3, combined with its remarkable potential of bio-
luminescence imaging, affords a valuable approach for in vivo
imaging of NTR in a tumor model mouse.
ypoxia, defined as a condition of oxygen deprivation in
been widely employed as an activating enzyme in developing
Hthe tissue level to induce cell death if severe or and screening anticancer nitroaromatic prodrug.8,9 Given the
importance of NTR to diagnosis and treatment of a tumor, so
far, great efforts have been undertaken to develop a variety of
optical NTR probes, among which specific fluorescent and
chemiluminescent NTR probes were fashioned to operate as a
valuable toolkit in a biological application.10−19 However, the
intrinsic photobleaching and autofluorescence of fluorescent
probe, as non-negligible flaws of fluorescence modality, restrict
its further applications.
It is most desirable that an ideal probe for in vivo imaging
should possess indispensable properties, such as high
sensitivity, deep tissue penetration, rapid molecular diffusion,
and excellent biocompatibility. In order to construct a
promising approach for localizing NTR and visualizing its
dynamic changes in a living animal, we sought to employ firefly
bioluminescence imaging (BLI) as a noninvasive alternative
owing to its ability to afford high-sensitivity in vivo imaging.20
Without a detectable light signal from mammalian cells or
tissues, bioluminescence imaging during the transformation of
D-luciferin into oxyluciferin in the presence of luciferase and
other cofactors such as ATP, O2, and Mg2+ features low
background and high signal-to-noise contrast, which obviates
the intrinsic limitations of fluorescence and provides a sensitive
prolonged, occurs during the onset and development of diverse
diseases, including cancer, vascular ischemia,1,2 and inflamma-
tory disease.3 As a pathophysiological characteristic resulting
from poor perfusion and anemia within the malignant solid
tumor, low oxygen tension causes a transcription program so as
to promote an aggressive tumor phenotype in association with
increased metastasis, poor prognosis, and resistance to radiation
therapy.4 As such, an accurate and reliable hypoxia detection
would be beneficial to discern tumor status and identify
pathological variables, as well as predict anticancer treatment
efficacy. Though direct evidence of hypoxia in human cancers
was shown by using oxygen-sensitive electrodes, an invasive
nature of electrode measurement may cause inconsistent results
in a spatial distribution of hypoxia, because of regional
heterogeneity in the tumor.5 Accordingly, hypoxia detection
strategies have been moving away from direct oxygen
measurement to the indirect evaluation of a related biological
state or condition, such as bioreductive metabolism and
hypoxia-inducible factor.
As a biomarker involved in the transcriptional response to
low oxygen tension, nitroreductase (NTR) is directly correlated
to the degree of hypoxia in solid tumor.6 NTR can catalyze the
reduction of nitro groups on aromatic compounds in the
presence of a cofactor, such as nicotinamide adenine
dinucleotide (NADH) or nicotinamide adenine dinucleotide
phosphate (NADPH).7 Using this reductive activity, NTR has
Received: March 23, 2016
Accepted: May 20, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.analchem.6b01160
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Analytical Chemistry
Letter
optical approach to monitoring molecular and cellular events in
living animals. By using the luciferase/luciferin system, highly
responsive bioluminescent probes have been well established
for specific biomolecules of interest, such as caspase,21 β-
galactosidase,22 β-lactamase,23 aminopeptidase N,24 hydrogen
peroxide,24,25 fluoride,26 and hydrogen sulfide.27,28 Recently, an
NTR bioluminescence sensor (probe 2) was reported with
striking features including high selectivity and a robust turn-on
for the whole cell and lysate samples.29 In the current study, we
presented a highly selective and sensitive bioluminescent probe
with improvements for the detection of nitroreductase in living
cells and animals.
In designing an NTR bioluminescent reporter, we first
envisaged chemoselective probes based on a luciferin/luciferase
system as a responsive platform with different caged groups and
linkers, which may afford variances on reactivity between probe
and analyte. Utilizing the reductive characteristic of the enzyme,
turn-on probes with the introduction of nitro group may
possess high selectivity and reactivity toward NTR. Upon a
selective reduction in which the nitro group was transformed
into the amino group and the subsequent cleavage reaction,
small molecule probes could be unmasked to release free firefly
luciferin for activating the catalytic reaction with luciferase and
subsequently producing a photon. To demonstrate our initial
hypothesis, bioluminescent probes 1−3 with appropriate NTR-
responsive groups were well designed as depicted in Figure 1.
dependent manner. Without NTR, all probes were expected to
be stable and have a weak bioluminescent response in Tris-HCl
buffer in the presence of luciferase and ATP. After being
incubated with a series of concentrations of NTR at 37 °C
within 60 min, probes were triggered to release free firefly
luciferin (or aminoluciferin) and subsequently to provide a
robust turn-on response in bioluminescence intensity (Figure
S1). Among these molecules, probe 3 exhibited distinctive
response capability and high sensitivity to nitroreductase. In
response to NTR (5 μg/mL), probe 3 presented a 552-fold
bioluminescent enhancement. Even when the concentration of
NTR was as low as 0.001 μg/mL, a considerable growth (20%)
of bioluminescence intensity still can explicitly be observed
(Figure S1). Moreover, varying from 0 to 2.5 μg/mL of NTR,
the relative bioluminescence intensity could reflect a real
“linear” growth (R2 = 0.99), which could be used to quantify
the concentration of NTR. These results fully indicated that
probes are capable of interacting with NTR at various
concentrations in aqueous systems.
To further assess the selectivity of probes toward NTR in
aqueous systems, a range of pertinent species were tested in a
bioluminescent assay, including D-cysteine (Cys), sodium
ascorbate (VcNa), sodium hydrosulfide (NaSH), β-nicotin-
amide adenine dinucleotide (NADH), and sodium hyposulfite-
(Na2S2O4). As shown in Figure 2, our bioluminescent probes
Figure 1. Scheme of bioluminescent probes for selective detection of
nitroreductase.
Figure 2. (A) Bioluminescence imaging of selectivity of the probes 1−
3 with various relevant reductants (NADH, NTR, Cys, VcNa, NaSH,
Na2S2O4) after adding luciferase and ATP in a 96-well plate; (B)
quantification of the relative bioluminescent intensity of probes for
each condition.
To design and prepare probe 1, aminoluciferin scaffold was
substituted with a nitro group, which could be easily reduced to
an amino group by nitroreductase. Probe 2 was well designed
and synthesized by using a nitro-substituted benzylic linker to
acylate the 6′-NH2 group of aminoluciferin. As an immolvative
linker, this caging group could be triggered by the reduction of
nitro group and release aminoluciferin to produce light. We also
designed and prepared bioluminescent probes 3 via introducing
a self-immolative linker into luciferin scaffold at the 6′-position.
The detailed synthesis and structural characterization are
provided in the Supporting Information.
(60 μM) showed no to little bioluminescent response to these
reductants, even if their concentrations were high up to 10 mM.
However, after adding NADH and NTR simultaneously, the
remarkable enhancements to a different extent in bio-
luminescence intensity were measured (2.24-, 20-, and 450-
fold increase for probes 1, 2, and 3, respectively). As a result,
probe 3 with high sensitivity and selectivity, combined with
favorable characteristics of the bioluminescence technique, can
afford potential utility for cellular and in vivo imaging.
Initial experiments focused on the evaluation of NTR
detection efficiency of these probes in an aqueous environment
at physiological pH. We first evaluated their responsiveness and
related sensitivity of these probes to NTR in a concentration-
Following the preceding characterization of probes, we
determined probe 3 as a versatile molecule for further study in
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DOI: 10.1021/acs.analchem.6b01160
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bioimaging, due to its unique features compared to other
probes. To test the performance of probe 3 for detecting
endogenous NTR in living-cell assays, cobalt chloride was
utilized to stimulate luciferase-transfected 4T1 (4T1-luc) cells
to elevate the intracellular NTR level.19,30 As a result, upon
gradual growth of concentrations (0−62.5 μM) of CoCl2,
sustained increases in bioluminescence intensity were detected
before the attainment of the maximum value (186% relative
increase) at 62.5 μM CoCl2, as shown in Figure 3. It was
Figure 4. Bioluminescence imaging of endogenous nitroreductase
activities in 4T1 tumor mouse model by using probe 3. (a) Time-
dependent bioluminescence imaging of mouse tumor model with or
without injection of dicoumarol; (b) quantification of the total flux of
(a); (c) representative bioluminescence imaging at 40 min with (right)
or without (left) injection of dicoumarol; (d) quantification of the
total flux of (c).
Figure 3. Imaging the endogenous nitroreductase activities in 4T1-luc
cells by using probe 3. (a) Bioluminescence imaging of living cells
simulated with diverse concentrations (0, 7.8, 15.6, 31.25, 62.5, 125,
250, 500, 1000, 2000 μM) of cobalt chloride; (b) quantification of
bioluminescent intensity of probe 3 for each condition.
bioluminescence was reached in 40 min with ca. a 10-fold
increasing signal in the tumor region relative to the control
animal. These results in the enzyme inhibition experiment not
only confirmed the long-term responsiveness of probe 3 toward
endogenous NTR in living animal but also verified that the
performance of probe 3 was carried out through the reductive
mechanism of NTR.
revealed that not only CoCl2 can effectively stimulate cell
cultures to promote NTR production but also probe 3 is
capable of monitoring dynamic changes in NTR level at cellular
concentrations. The biocompatibility of all probes was
examined by standard MTT assays. It was demonstrated that
all probes exhibited very low cytotoxicity to the cultured cells
(Figure S2). With rising concentrations of CoCl2 incubated
with living cells, the bioluminescent signal was substantially
attenuated in this live-cell assay. Therefore, we proposed that
the cell death resulting from the toxicity of CoCl2 at high
concentrations was a reasonable cause (Figure S3).
Having confirmed that probe 3 can offer a highly selective
and sensitive approach for monitoring oscillations of
endogenous NTR in living cells, we moved on to investigate
whether this capability of probe 3 in cell cultures translated to
the whole animal. Considering the critical roles of NTR in
cancer progression, a breast cancer mouse model was
established, in which 1 × 106 4T1-luc cell lines were
orthotopically implanted by s.c. injection into nude mice to
build 4T1 breast tumor model. After 10 days, a tumor-bearing
mouse model with a 1.6 cm diameter was formed. In the
present study, dicoumarol as a reductase inhibitor was utilized
to decrease NTR activity in the control group.8,19 Different
groups were injected with probe 3 into the tumor, and time-
dependent signal changes were collected every 5 min within 70
min as shown in Figure 4. Due to the enzymatic inhibition,
there is little to no bioluminescence growth observed in the
control group, in which hypoxia tumor was pretreated with
dicoumarol. In contrast, a progressive increase in bio-
luminescence intensity was immediately detected within the
first few minutes after probe injection, and the highest
In an effort to expand the mission of probe 3 to visualize the
degree of hypoxia in the process of tumor growth in living
animal, we assessed the possibility of bioluminescent tracking of
fluctuations in endogenous NTR level in the tumor model by
using this unique chemical tool. For this purpose, different sized
A549 tumors (1.0 and 1.3 cm) in living mice were respectively
injected with probe 3 and the subsequent change of
bioluminescence intensity was kinetically recorded. As shown
in Figure 5, the distinct enhancements in emission signal from
the tumor regions were observed within the first few minutes
after injection of probe 3. The increased bioluminescence
intensity from whole tumors measurably lasted for more than
30 min, and the maximum values were reached within 5.5 min
after injection. The average flux value of the 1.3 cm-diameter
tumor was 5-fold higher than that of the 1.0 cm-diameter tumor
at 5.5 min. These results clearly not only established a clear
correlation between dynamic NTR degrees and different sized
tumors but also suggested that probe 3 is capable of providing a
valuable tool for monitoring alterations of endogenous NTR in
tumor development.
In brief, herein we developed a bioluminescent probe for the
chemoselective visualization of endogenous NTR in hypoxia
tumor model. Even in complex biological systems, probe 3
exhibited high selectivity, sensitivity, and other favorable
properties, which presents a powerful tool in bioimaging and
provides a viable approach for visualizing NTR overexpression
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ACKNOWLEDGMENTS
■
We are grateful for financial support from the National Natural
Science Foundation of China (Nos. 21402130 and 81273471),
the National Program on Key Basic Research Project (No.
2013CB734000), the Program for Changjiang Scholars and
Innovative Research Team in University (No. IRT13028), the
Major Project of Science and Technology of Shandong
Province (No. 2015ZDJS04001), the Shandong Key Research
& Development Project (No. 2015GSF118166), and the
Fundamental Research Funds of Shandong University (No.
2014JC008).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
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chem.6b01160.
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Full experimental procedure, NMR, MS, fluorescence,
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AUTHOR INFORMATION
Corresponding Authors
*Tel./Fax: +86-28-8518-8632. E-mail: bowenke80@hotmail.
com (B.K.).
■
*Tel./Fax: +86-531-8838-2076. E-mail: mli@sdu.edu.cn
(M.L.).
Author Contributions
‡P.F. and H.Z. contributed equally.
Notes
The authors declare no competing financial interest.
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