Enzyme-Activated Dual-Locked Probes for Detecting Nitroreductase and Carboxylesterase.

1
Introduction
Owing to its high incidence
and mortality rates worldwide, lung
cancer remains a significant global health challenge.
,
Approximately 340 people die each day from lung cancernearly
2.5 times more than the number of people who die from colorectal cancer,
which ranks second in cancer deaths. Lung
cancer causes far more deaths each year than do colorectal, breast,
and prostate cancers combined. Surgical
resection represents the primary therapeutic intervention for lung
cancer. For early-stage diseases, postoperative survival rates can
exceed 80%. However, in clinical practice, precise and rapid methodologies
for delineating tumor margins before and after tumor surgery are lacking.
The tumor microenvironment is an important factor for the effective
diagnosis and treatment of lung cancer.
,

Hypoxia
is a hallmark of solid tumors, including lung cancer, and
is known to alter tumor physiology and impact clinical diagnosis and
treatment. Hypoxia can directly lead to
the upregulation of nitroreductase (NTR), which is valuable for distinguishing
tumor tissues from normal tissues. In addition, fatty acids are relevant
to tumor growth; as carboxylesterase (CarE) is a key enzyme that releases
bioactive free fatty acids, CarE is likely involved in cancer progression.
,
Carboxylesterase has been reported to be upregulated in various
cancers, including lung cancer.
,
Hypoxia and increased
fatty acids represent the most distinguishing metabolic features of
lung cancer cells compared to surrounding stromal cells. Thus, both
NTR and CarE can be employed for achieving tumor tissue imaging.
The common clinical methods for tumor diagnosis are X-ray computed
tomography (CT), positron emission tomography
(PET), single-electron emission computed
tomography (SPECT), and magnetic resonance
imaging (MRI). However, the application
of CT, PET, and SPECT is limited due to radiation risk.
−

MRI cannot achieve real-time imaging, and the detection process
is time-consuming. Fluorescent imaging
has the advantages of a fast response time and lower radiation hazard.
−

Recently, several excellent fluorescent probes for NTR or CarE imaging
have been reported (Table S1).
−

However, to the best of our knowledge, no dual-locked fluorescent
probe that can simultaneously detect NTR and CarE is available.
In this work, a series of fluorescent probes were designed and
synthesized. Among these probes, Flu-NitroCa-3 and Flu-NitroCa-4 presented
faster response rates for NTR, and Flu-NitroCa-4 presented 48-fold
fluorescence emission at 515 nm after contact with NTR and CarE. Flu-NitroCa-4
was used for imaging hypoxia in A549 cells and patient-derived lung
cancer organoids. We constructed subcutaneous tumor and orthotopic
murine lung tumor models to validate NTR- and CarE-locked fluorescence
imaging of Flu-NitroCa-4. Moreover, the probe was applied to distinguish
the tumor area from the normal area in human lung cancer and adjacent
lung tissues.

2
Experimental
Section
2.1
Synthesis of Fluorescent Probes
2.1.1
Synthesis of 5- and 6-Nitrofluorescenin
Resorcinol
(2.92 g, 26.5 mmol) and 4-nitrophthalic acid (2.8 g,
13.2 mmol) were mixed and heated to 200 °C overnight. The mixture
was subsequently cooled to 100 °C, and HCl (0.6 M) was added.
The mixture was further cooled to room temperature, filtered, and
washed with water. The resulting solid was dried at 100 °C in
a drying oven.

2.1.2
Synthesis of Flu-NitroCa-2
The
isomeric mixture (3.2 g, 8.5 mmol) was refluxed in acetic anhydride
(12.8 g) for 2 h. The reaction mixture was cooled to room temperature,
filtered, and washed with acetic anhydride. The acetic anhydride filtrate
was concentrated to dryness. The solid was subsequently dissolved
in benzene and heated to 60 °C. The resulting crystals were filtered
off and recrystallized with benzene to obtain Flu-NitroCa-2 (yield
= 18.2%). 1H NMR (400 MHz, DMSO-d
6) δ 8.54 (dd, J = 8.4, 1.9 Hz, 1H),
8.41 (d, J = 1.8 Hz, 1H), 8.33 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 2.1 Hz, 2H), 7.02 (d, J = 8.7 Hz, 2H), 6.96 (dd, J = 8.6, 2.4
Hz, 2H), 2.30 (s, 6H). 13C NMR (101 MHz, DMSO-d
6) δ: 169.30, 167.06, 153.20, 153.12, 152.76, 151.48,
131.04, 129.95, 127.54, 126.46, 120.42, 119.01, 115.58, 111.00, 82.27.
ESI-HRMS (m/z): [M-H]− calculated for Flu-NitroCa-1:376.0457, found 376.7348.

2.1.3
Synthesis of Flu-NitroCa-1
Compound 5 (0.48
g, 1.03 mmol) was dissolved in sodium hydroxide solution
and methanol and warmed gently. The precipitate was filtered off and
dissolved in water. The solution was acidified and allowed to stand
overnight in the dark. The resulting solids were filtered off and
washed with water to obtain Flu-NitroCa-1 (yield = 88.1%). 1H NMR (400 MHz, DMSO-d
6) δ 10.19
(s, 2H), 8.50 (dd, J = 8.4, 1.9 Hz, 1H), 8.26 (d, J = 8.3 Hz, 1H), 8.11 (d, J = 1.7 Hz, 1H),
6.62–6.76 (m, 4H), 6.56 (dd, J = 8.7, 2.3
Hz, 2H). 13C NMR (101 MHz, DMSO-d
6) δ: 167.34, 160.24, 153.74, 152.92, 152.43, 131.48,
129.80, 127.20, 126.03, 119.86, 113.16, 108.87, 102.82, 84.30. ESI-HRMS
(m/z): [M-H]− calculated
for Flu-NitroCa-1:376.0457, found 376.7348.

2.1.4
Synthesis
of Flu-NitroCa-4
The
mixture (3.2 g, 8.5 mmol) was refluxed in acetic anhydride (12.8 g)
for 2 h. The reaction mixture was cooled to room temperature, filtered,
and washed with acetic anhydride. The precipitate was subsequently
washed with ethanol and recrystallized in acetic anhydride to obtain
yellowish white crystals of Flu-NitroCa-4 (yield = 17.2%). 1H NMR (400 MHz, DMSO-d
6) δ 8.73
(d, J = 2.3 Hz, 1H), 8.61 (dd, J = 8.4, 2.1 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.33
(d, J = 2.2 Hz, 2H), 7.04 (d, J =
8.7 Hz, 2H), 6.98 (dd, J = 8.7, 2.2 Hz, 2H), 2.30
(s, 6H). 13C NMR (101 MHz, DMSO-d
6) δ: 169.28, 166.87, 157.19, 152.86, 151.28, 149.82,
131.17, 129.94, 127.78, 126.59, 121.18, 119.18, 115.42, 111.09, 82.12,
21.33. ESI-HRMS (m/z): [M-H]− calculated for Flu-NitroCa-2:460.0669, found 460.7673.

2.1.5
Synthesis of Flu-NitroCa-3
Compound
7 (0.86 g, 1.87 mmol) was dissolved in sodium hydroxide solution and
methanol and warmed gently. The precipitate was filtered off and dissolved
in water. The solution was acidified and allowed to stand overnight
in the dark. The resulting solids were filtered off and washed with
water to obtain Flu-NitroCa-3 (yield = 60.5%). 1H NMR (400
MHz, DMSO-d
6) δ 10.22 (s, 2H), 8.66
(d, J = 1.9 Hz, 1H), 8.57 (dd, J = 8.1, 2.1 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 6.65–6.73
(m, 4H), 6.56 (dd, J = 8.7, 2.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d
6) δ: 167.15,
160.41, 157.72, 152.31, 149.55, 130.77, 129.82, 128.28, 126.39, 120.80,
113.28, 108.79, 102.83. ESI-HRMS (m/z): [M-H]− calculated for Flu-NitroCa-3:376.0457,
found 376.7376.

2.2
Fluorescence Measurement
in Solution
All fluorescence measurements were conducted
on a Hitachi F2700 fluorescence
spectrophotometer at room temperature. The probes, including Flu-NitroCa-1,
Flu-NitroCa-2, Flu-NitroCa-3, and Flu-NitroCa-4, were dissolved in
DMSO. The probe solution was diluted to 10 μM with PBS to further
investigate the detection performance for NTR and CarE.

2.3
Cell Culture and Fluorescence Imaging
A549 and LLC
cells were purchased from the Cell Bank of the Chinese
Academy of Sciences. As previously described,
−

Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum was used to culture A549 and LLC cells. A549 cells were
incubated in a CO2 incubator at 37 °C under normoxia
and in a triple gas incubator at 37 °C with 2% O2 under
hypoxia. Hypoxic A549 cells were pretreated with or without dicoumarin
(an NTR inhibitor) or bis­(4-nitrophenyl) phosphate (BNPP, a CarE inhibitor).
These cells were subsequently incubated with 10 μM Flu-NitroCa-4
for 30 min. The cells were subsequently rinsed three times with PBS
to remove excess Flu-NitroCa-4. Fluorescence imaging was performed
via confocal microscopy (Zeiss, LSM900).

2.4
Animal
Administration and Fluorescence Imaging
All experiments involving
4–6-week-old male C57BL/6 mice
and BALB/c nude mice were approved by the Ethics Committee of Naval
Military Medical University. All of the mice were purchased from the
Model Organisms Center (Shanghai) and housed under temperature-controlled,
specific pathogen-free (SPF) conditions. As previously described,
−

for the subcutaneous tumor model, A549 cells (5 × 106 cells) were injected subcutaneously into the left axillary region
of BALB/c nude mice. Tumor volume was calculated via the formula:
0.5 × (larger diameter) × (smaller diameter)2. For the orthotopic model of lung cancer, LLC cells (2 × 105 cells) were injected slowly into the upper lobe (avoiding
major vessels) of the lung. Three weeks later, the mice were anesthetized,
and the probe was delivered via intratracheal intubation for pulmonary
distribution. The remaining mice were euthanized, and their lung tissues
were harvested for staining and subsequent experiments. Mouse lung
tissue samples were quickly frozen and cut into consecutive sections
via a freezing sectioning machine (Leica, CM1950). Probes were diluted
in a 1:100 ratio and applied to stain the sections. After 30 min,
images were acquired via a fluorescence microscope.

2.5
Patients and Specimens
Lung cancer
tissue (approximately 1–3 cm3) was obtained from
surgically resected lung specimens at the Thoracic Surgery Department
of Changhai Hospital, Naval Military Medical University, and patients
provided informed consent. LC diagnosis was based on histology. The
samples were collected in accordance with the relevant regulations
on the management of human genetic resources in China. A total of
three patients with lung cancer were included in this study between
April 3, 2024 and May 3, 2024 (Table S2). The research protocol was approved by the Ethics Committee of
Changhai Hospital, Naval Military Medical University.

2.6
Human Tissue Preparation and Fluorescence
Imaging
Human tissue samples were immersed in cold Hank’s
balanced salt solution (HBSS) supplemented with 2% antibiotics and
transported to the laboratory within 1 h postcollection. The samples
were washed three times with ice-cold PBS containing 2% antibiotics.
The tissues were minced into approximately 1–2 mm3 fragments via sterile surgical scissors and forceps. One fragment
was fixed for Flu-NitroCa-4 incubation, hematoxylin and eosin (H&E)
staining, and immunohistochemical (IHC) validation. Another fragment
was used for establishing patient-derived organoid (PDO) models. Fresh
surgically resected lung cancer patient samples were quickly frozen
and cut into consecutive sections via a freezing sectioning machine
(Leica, CM1950). Flu-NitroCa-4 (20 μM) was then incubated with
the tissues. After 30 min, images were acquired via a fluorescence
microscope.

2.7
Culture of Lung Cancer
Organoids
To establish lung cancer organoids (LCOs), the
remaining samples
were minced and then incubated at 37 °C in 5 mL of collagenase
B (5 mg/mL; Roche, #11088815001) and DNase I (100 μg/mL; MilliporeSigma,
#6918230) for 2 h with intermittent agitation. After incubation, the
suspension was filtered through a 70 μm cell strainer (BD Falcon,
# 352350). The filtered cell pellet was centrifuged at 350 ×
g for 5 min, resuspended in Matrigel solution (Corning, #354234),
and seeded into a confocal microplate (Absin, #abs-7020). After the
Matrigel solidified, 250 μL of culture medium (Table S1) was added to each well. Patient-derived LCOs were
cultured in a humidified incubator at 37 °C with 5% CO2, and the medium was changed twice weekly. LCOs were stained with
a probe for 30 min, and then imaging was performed via confocal microscopy
(Zeiss, LSM800).

2.8
Histology and Imaging
Tissues were
first fixed in 4% paraformaldehyde (PFA), followed by dehydration,
paraffin embedding, sectioning, and standard H&E staining. For
IHC staining, the samples were incubated with the following primary
antibodies: anti-Napsin A (1:200 dilution, Novocastra, #NCL-L-Napsin
A), anti-TTF-1 (1:200 dilution, Novocastra, #NCL-L-TTF-1), and anti-Ki67
(1:400 dilution, Dako, #M7018). The samples were subsequently incubated
with secondary antibodies (Absin, #abs996) at a 1:5000 dilution. Detection
was performed via the UltraView Universal DAB Detection Kit (Ventana
Medical Systems, Arizona, USA). Nuclei were counterstained with Harris
hematoxylin. Images were acquired via a Leica Eclipse E600 microscope.

3
Results and Discussion
3.1
Designed
and Synthesized Fluorescent Probes
Although many fluorophores
have been developed, few fluorophores
have been approved for clinical use as contrast dyes, including fluorescein,
ICG, and methylene blue (Figure
A). Furthermore, to develop
dual-locked fluorescent probes, the two recognition 0 groups of NTR
and CarE should be combined into one molecule. Among these clinically
used fluorophores, only fluorescein has at least two fluorescence
modification sites. Thus, fluorescein
was selected as the fluorophore in this study. Based on the activity
of NTR and CarE and the structural characteristics of fluorescein,
we designed four fluorescent probes: Flu-NitroCa-1, Flu-NitroCa-2,
Flu-NitroCa-3, and Flu-NitroCa-4. We then synthesized four fluorescent
probes in a few steps (Figure
B). All the fluorescent probes were characterized by 1H NMR, 13C NMR, and HRMS (Figures S13–S24).

3.2
Spectroscopic Properties
of Probes with NTR
and CarE
After these probes were obtained, we further tested
the detection performance of NTR and CarE. NTR caused a 33.2-fold
increase in the fluorescence of Flu-NitroCa-3 at 515 nm and a 3.6-fold
increase in the fluorescence of Flu-NitroCa-1 at 517 nm. For Flu-NitroCa-2
and Flu-NitroCa-4, neither NTR nor CarE caused a similar fluorescence
enhancement (Figures
A and S1). However, Flu-NitroCa-4 presented
a 47.9-fold increase in fluorescence at 515 nm after contact with
NTR (90 μg/mL) and CarE (10 U/mL), whereas Flu-NitroCa-2 presented
a 4.7-fold increase in fluorescence at 517 nm (Figure
A). We then observed the fluorescence emission
spectra of the probes upon the gradual addition of NTR, with or without
CarE (Figure
A). Figure
D,E shows that the
fluorescence intensity of all four probes increased with increasing
amounts of NTR or CarE, and there was an excellent linear correlation
between the fluorescence intensity and various concentrations of NTR,
with or without CarE. According to the LOD = 3σ/κ, the
LODs of Flu-NitroCa-1, Flu-NitroCa-2, Flu-NitroCa-3, and Flu-NitroCa-4
for NTR were 4.97, 1.96, 0.35, and 0.18 μg/mL in PBS, respectively,
whereas Flu-NitroCa-2 and Flu-NitroCa-4 displayed low LODs of 0.41
and 0.03 U/mL for CarE in PBS, respectively. The fluorescence intensity
of these probes gradually increased and reached a maximum within 15
min for Flu-NitroCa-1 and Flu-NitroCa-3 or 30 min for Flu-NitroCa-2
and Flu-NitroCa-4 (Figures
F and S4). In a solution containing
10% fetal bovine serum, the LOD of Flu-NitroCa-4 toward NTR and CarE
was 0.325 μg/mL and 0.042 U/mL, respectively (Figure S2). The main advantage of Flu-NitroCa-4 lies in its
requirement for activation by both NTR and CarE, which helps reduce
false-positive signals in lung cancer tissue identification. However,
in terms of detection sensitivity, Flu-NitroCa-4 shows some limitations
compared to reported highly sensitive probes for NTR, such as XN3
and Cy5-NTR, as well as the probe for CarE, TCFCl-CES.
,,
These results suggested that
fluorescein with 5-nitro groups is much more sensitive than fluorescein
with 6-nitro groups but has similar response rates for NTR and CarE.
In addition, Flu-NitroCa-4 presented a low LOD and a fast response
rate for NTR and CarE.

3.3
Molecular Docking of Probes to NTR and CarE
After the initial validation of the detection performance of the
probes, docking calculations were introduced to illustrate the binding
relationships between the probes and both CarE (PDB ID: 5A7H) and NTR (PDB ID: 4DN2). Flu-NitroCa-1,
Flu-NitroCa-2, Flu-NitroCa-3, and Flu-NitroCa-4 were docked to NTR
(Figures
A and S7B,C). Among all of the probes, the Flu-NitroCa-4
probe presented the lowest binding free energy during optimal docking
with NTR (−7.9 kcal/mol), suggesting a greater affinity between
Flu-NitroCa-4 and NTR. The results from Discovery Studio revealed
that Flu-NitroCa-4 formed a single hydrogen bond with ARG11 of NTR
(Figure
A). Additionally,
van der Waals interactions were observed between Flu-NitroCa-4 and
13 surrounding amino acid residues, including ARG10, VAL13, LEU128,
and GLY129. π–π interactions were also identified
with PRO162 and PRO166 (Figure
A). Furthermore, a nonclassical hydrogen bond involving the
C–H bond as the hydrogen bond donor was noted with GLY165,
although this interaction was significantly weaker than that of conventional
hydrogen bonds (Figure
A). The binding relationships between the probes and CarE were also
determined. As shown in Figures
B and S7A, the acetyl groups
of Flu-NitroCa-2 and Flu-NitroCa-4 fit well into the binding site
of CarE. Flu-NitroCa-2 and Flu-NitroCa-4 formed hydrogen bonds and
hydrophobic interactions with residues of CarE. These results were
supported by the detection performance of Flu-NitroCa-1, Flu-NitroCa-2,
Flu-NitroCa-3, and Flu-NitroCa-4 for NTR.

3.4
Selectivity of Flu-NitroCa-4 toward NTR and
CarE
The specificity of Flu-NitroCa-4 for NTR and CarE was
subsequently investigated. We observed that the presence of NTR (10
μg/mL), CarE (20 U/mL), and NADH (500 μM) caused an approximately
50-fold increase in the fluorescence intensity at 515 nm (Figure
C). Then, Flu-NitroCa-4
was examined for common biological analytes or other enzymes, including
amino acids (Cys, Asn, Phe, and Lys), metal ions (CaCl2, ZnCl2, and CuSO4), and enzymes (NTR alone,
CarE alone, AChE, BuChE, ALP, TYR, and PPE). As shown in Figure
C, the fluorescence
response of Flu-NitroCa-4 was insensitive to the different types of
influencing factors in the organism. We also attempted to add complex
biological samples such as human plasma to the probe solution and
found that a significant increase in fluorescence was only detected
when both CarE and NTR were added simultaneously (Figure S5). These data suggested that the probe can be used
for the selective detection of NTR and CarE.

3.5
Proposed
Mechanism of Flu-NitroCa-4 Binding
to NTR and CarE
Considering the spectral evidence presented
above, the potential reaction mechanisms of Flu-NitroCa-4 with NTR
and CarE are depicted in Figure
A. Either nitro groups or acetyl groups can inhibit
the fluorescence emission of fluorescein. CarE-triggered cleavage
of the acetyl group on Flu-NitroCa-4 induced the formation of Compound 6. Compound 6 could further react with NTR in
the presence of NADH, leading to the reduction of the nitro group
to an amino group. The final products of Flu-NitroCa-4 with NTR and
CarE were separated and characterized by 1H NMR, 13C NMR, and HRMS (Figures S25–S27) as well as UV–vis absorption spectra (Figure S1), which revealed that the reactions of Flu-NitroCa-4
with NTR and CarE proceeded as shown in Figure
A.

3.6
Theoretical Calculation of Flu-NitroCa-4 for
NTR and CarE
The off-on mechanism of Flu-NitroCa-4 was inferred
from time-dependent density functional theory. As shown in Figure
B, the electrons
of Flu-NitroCa-4, Compound 8, and Compound 12 were concentrated mainly
on the fluorophores in the HOMO orbit. The electrons of Flu-NitroCa-4
and Compound 8 were strongly transferred and almost centered at one
end of the fluorophores in the LUMO orbit. Strong electron transfer
destroyed the original conjugated system for Flu-NitroCa-4 and Compound
8, and intramolecular fluorescence self-quenching occurred. Although
the electrons of Compound 10 did not transfer greatly, the electrons
remained concentrated at one end of the fluorophores in the HOMO and
LUMO orbits. As the product of the reaction between Flu-NitroCa-4
and NTR and CarE, Compound 12 formed a large π-conjugated system
with strong fluorescence. These results indicated that Flu-NitroCa-4
and some reaction intermediates have a PET effect, and when the nitro
and acetyl groups are transferred or released, the product of Flu-NitroCa-4
can cause strong fluorescence.

3.7
Confocal
Imaging of NTR and CarE in Living
Cells under Hypoxia
We further tested Flu-NitroCa-4 as a
fluorescent probe for NTR and CarE in A549 cells. First, the cytotoxicity
of Flu-NitroCa-4 was measured via a CCK-8 assay. As shown in Figure S8, the adverse effect of Flu-NitroCa-4
on cell viability was minimal. Then, Flu-NitroCa-4 was employed to
image NTR and CarE in living A549 cells under hypoxia. The time-dependent
fluorescence response was imaged during the first 60 min. Figure
A shows two enzyme-induced
increases in emission intensity, with a plateau appearing at approximately
40 min. Compared with normoxic (20% O2) conditions, either
hypoxic (2% O2) conditions for 8 h or CoCl2 (100
μM) treatment for 12 h significantly increased the green fluorescence
in Flu-NitroCa-4-loaded A549 cells. A clear reduction in green fluorescence
in probe-loaded A549 cells was recorded after treatment with dicoumarin
(an NTR inhibitor) or bis­(4-nitrophenyl) phosphate (BNPP, a CarE inhibitor; Figure
B). These results
suggested that Flu-NitroCa-4 is capable of detecting NTR and CarE
in living cells.

3.8
Fluorescence Imaging of Different Tumor-Bearing
Mouse Models
The ability of Flu-NitroCa-4 to image cancer
in living mice was next examined, and the toxicity of Flu-NitroCa-4
in mice was investigated. As shown in Figures
A and S9, the
Flu-NitroCa-4-treated group presented no obvious changes in histopathological
parameters, which suggested that Flu-NitroCa-4 has low acute toxicity.
To further explore the potential of Flu-NitroCa-4 for detecting cancer
in vivo, a subcutaneous tumor model was first constructed. One hundred
microliter of Flu-NitroCa-4 (1 mM) was injected into A549 tumor-bearing
nude mice, while the same volume of PBS was injected as a control.
Real-time intravital imaging revealed that the control group presented
no fluorescence at 40 min, whereas the Flu-NitroCa-4-treated tumor-bearing
nude mice presented strong green fluorescence at 40 min (Figure
B). Furthermore,
the strong green fluorescence of A549 tumor-bearing nude mice was
almost completely inhibited by DIC pretreatment as well as BNPP. Encouraged
by the above results, we also constructed an orthotopic lung cancer
model, which was confirmed by H&E and IHC staining of CK5/6, Ki67,
and p63, as shown in Figure
E. The mice with lung carcinoma in situ were anaesthetized
and treated with Flu-NitroCa-4 via endotracheal administration (Figure
C). As shown in Figure
D, the fluorescence
intensities between normal lung tissue and lung cancer tissue markedly
differed at 30 min. Additionally, we performed serial cryosections
of lung tissue from tumor orthotopic mice and coincubated the sections
with Flu-NitroCa-4 for 30 min. A distinct boundary was observed between
cancerous and adjacent paracancerous tissues. The carcinoma regions
were confirmed by IHC (CK5/6, Ki67, and p63) and H&E staining
(Figure
E). These
data indicated that the hypoxic tumor site caused more NTR, resulting
in a reaction with Flu-NitroCa-4 to distinguish tumors from normal
tissues. Thus, Flu-NitroCa-4 demonstrates a superior capability for
precise boundary delineation in orthotopic lung cancer imaging.

3.9
Fluorescence Imaging of Patient-Derived Lung
Tumor Organoids and Clinical Patient Samples
Inspired by
the performance of Flu-NitroCa-4 in mice, we further validated the
capability of Flu-NitroCa-4 for real-time intraoperative delineation
of lung cancer boundaries. Consequently, we examined the performance
of Flu-NitroCa-4 in patient-derived lung cancer organoids and clinical
patient samples. CT scanning demonstrated definitive evidence of lung
carcinoma (Figure
A). Fresh human lung cancer specimens and adjacent tissues were resected
via surgery to generate human lung cancer organoids. Human lung cancer
organoids were subsequently used to test the detection performance
of Flu-NitroCa-4 (Figure
B). As shown in Figure
C, human lung cancer organoids presented weak green fluorescence
under normoxia (20% O2). Under hypoxic (2% O2) conditions, the green fluorescence became more apparent in human
lung cancer organoids. Furthermore, the fluorescence in the organoids
was significantly inhibited by dicoumarin (100 μM) or BNPP (200
μM).
Encouraged by the above results, fresh surgically
resected lung
cancer patient samples were quickly frozen, cut, and then incubated
with Flu-NitroCa-4 (20 μM) for 30 min. According to the fluorescence
images and quantitative data, the fluorescence signal intensity of
the lung cancer group was significantly greater than that of the adjacent
tissues (Figures
D
and S10–S12). These results demonstrated
that the probe Flu-NitroCa-4 can accurately distinguish tumor tissue
from normal tissue in the lung by imaging NTR and CarE in the tumor
hypoxic center. The intraoperative imaging data confirmed the ability
of Flu-NitroCa-4 to accurately demarcate tumor margins in real time.
These results demonstrate that Flu-NitroCa-4 enables robust discrimination
between tumor and normal tissues in human subjects, with clear delineation
of tumor boundaries.

4
Conclusions
We developed
Flu-NitroCa-4,
the first dual-locked fluorescent probe
designed for the simultaneous detection of NTR and CarE in lung cancer.
This fluorescein-based probe exhibited excellent performance, including
a 48-fold fluorescence turn-on at 515 nm upon activation by NTR and
CarE, rapid response kinetics (<30 min), high specificity, and
low detection limits. In A549 cells and patient-derived lung cancer
organoids, Flu-NitroCa-4 reliably visualized cellular hypoxia in an
NTR/CarE-dependent manner. This approach enabled clear tumor delineation
within A549 and LLC mouse tumor models. Critically, this method accurately
discriminated human lung cancer tissue from adjacent normal tissue
in clinical samples, demonstrating its direct potential for intraoperative
margin mapping and clinical diagnosis. This work provides a powerful,
clinically translatable tool for precision oncology that targets biomarkers
within the tumor microenvironment.

Supplementary Material