Fluorescence Detection of Prostate Cancer by an Activatable Fluorescence Probe for PSMA Carboxypeptidase Activity

Article abstract of DOI:10.1021/jacs.9b04412

Prostate cancer (PCa) is a common malignant tumor among adult males, and convenient intraoperative detection of PCa would reduce the risk of leaving positive surgical margins, especially during nerve-sparing procedures. To achieve rapid, fluorescence-based visualization of PCa, we focused on the glutamate carboxypeptidase (CP) activity of prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein that is attracting attention as a PCa biomarker. Based on our finding that aryl glutamate conjugates with an azoformyl linker are recognized by PSMA and have a sufficiently low LUMO (lowest unoccupied molecular orbital) energy level to quench the fluorophore through photoinduced electron transfer, we designed and synthesized a first-in-class activatable fluorescence probe for CP activity of PSMA. The developed probe allowed us to visualize the CP activity of PSMA in living cells and in clinical specimens from PCa patients and is expected to be useful for rapid intraoperative detection and diagnosis of PCa.

Full text of DOI:10.1021/jacs.9b04412

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Article
Fluorescence detection of prostate cancer by an activatable
fluorescence probe for PSMA carboxypeptidase activity
Minoru Kawatani, Kyoko Yamamoto, Daisuke Yamada, Mako Kamiya, Jimpei Miyakawa,
Yu Miyama, Ryosuke Kojima, Teppei Morikawa, Haruki Kume, and Yasuteru Urano
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b04412 • Publication Date (Web): 07 Jun 2019
Downloaded from http://pubs.acs.org on June 7, 2019
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Fluorescence detection of prostate cancer by an activatable fluores-
cence probe for PSMA carboxypeptidase activity
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1,‡
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1,4,*
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Minoru Kawatani , Kyoko Yamamoto , Daisuke Yamada , Mako Kamiya , Jimpei Miyakawa ,
5
1,4
5
3
1,2,6,*
Yu Miyama , Ryosuke Kojima , Teppei Morikawa , Haruki Kume , Yasuteru Urano
1
2
Graduate School of Medicine, and Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1
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Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Department of Urology, Faculty of Medicine, The University of Tokyo,
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Kawaguchi, Saitama 332-0012, Japan. Department of Pathology, Graduate School of Medicine, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. AMED-CREST, Japan Agency for Medical Research and
4
-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho,
5
6
Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan.
ABSTRACT: Prostate cancer (PCa) is a common malignant tumor among adult males, and convenient intraoperative de-
tection of PCa would reduce the risk of leaving positive surgical margins, especially during nerve-sparing procedures. To
achieve rapid, fluorescence-based visualization of PCa, we focused on the glutamate carboxypeptidase (CP) activity of
prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein that is attracting attention as a PCa
biomarker. Based on our finding that aryl-glutamate conjugates with an azoformyl linker are recognized by PSMA and
have a sufficiently low LUMO (lowest unoccupied molecular orbital) energy level to quench the fluorophore through pho-
to-induced electron transfer, we designed and synthesized a first-in-class activatable fluorescence probe for CP activity of
PSMA. The developed probe allowed us to visualize the CP activity of PSMA in living cells and in clinical specimens from
PCa patients, and is expected to be useful for rapid intraoperative detection and diagnosis of PCa.
Introduction
activated specifically at the tumor site16, 17, 18. But, in spite of
the great advantages of activatable probes, such as a high tu-
mor-to-normal (T/N) signal ratio, only a few examples of
PSMA-targeting activatable fluorescence probe based on
Prostate cancer (PCa) is one of the most prevalent male can-
cers worldwide. Radical prostatectomy, in which the entire
prostate gland is resected and the bladder and urethra are su-
1
9, 20
1, 2
PSMA antibody have been reported so far
, and these do
tured, is the standard operative strategy for PCa . However,
not show an adequate degree of activation and suitable activa-
tion kinetics for intraoperative imaging of PCa.
patients often suffer from postoperative complications such as
erectile dysfunction and urinary incontinence as a result of
nerve damage or resection around the prostate. Nerve-sparing
Here, we newly focused on the carboxypeptidase (CP) activi-
3
21
surgery reduces the risks of such complications , but involves
ty of PSMA, which is elevated in PCa , and we set out to
the risk of recurrence as a result of leaving positive surgical
margins , since it is quite difficult to distinguish cancerous
tissue from normal tissue visually. Therefore, a convenient
method to detect cancerous lesions and to determine their pre-
cise localization in the tissue during operation would be ex-
tremely useful.
develop an activatable fluorescence probe with suitable prop-
erties for practical application. Although PSMA is known to
hydrolyze the C-terminal glutamate of peptides such as N-
acetyl-L-aspartyl-L-glutamate (NAAG) or poly-γ-glutamated
4
2
2, 23, 24
folate
, most of the currently available methods for de-
tecting CP activity are based on HPLC analysis or coupled
25, 26, 27
assay
. Very recently, our group reported for a first time
Prostate-specific membrane antigen (PSMA), also known as
glutamate carboxypeptidase II (GCPII) or N-acetyl-L-aspartyl-
a rational design strategy for activatable fluorescence probes
2
8
to detect CP activity , in which structural conversion from
aliphatic carboxamide to aliphatic carboxylate due to CP reac-
tion is transformed into fluorescence change by utilizing the
intramolecular spirocyclization of rhodamines. However, un-
fortunately a glutamate-conjugated derivative based on this
strategy was not recognized as a substrate by PSMA, which
presumably has strict substrate specificity. Therefore, we re-
quired a different design strategy. Here, we report a newly
established design strategy for activatable fluorescence probes
to visualize CP activity. We further show that the developed
probe can visualize the CP activity of PSMA in living cultured
cells and in clinical specimens from PCa patients, and we be-
lieve it will be suitable for rapid intra-operative detection and
diagnosis of PCa.
L-glutamate peptidase I (NAALADase I), is a type II trans-
5, 6, 7
membrane glycoprotein that is overexpressed in PCa
, and
its expression level is positively correlated with the stage of
8
, 9
disease and Gleason score . Thus, PSMA has attracted con-
siderable attention recently as a target for imaging and therapy
10
of PCa . So far, various PSMA ligands, including antibodies,
aptamers and low-molecular-weight ligands, have been used to
target imaging agents such as positron-emitting radionuclides
1
1
for positron emission tomography (PET) , a radionuclide for
single-photon-emission computed tomography (SPECT), iron
1
2, 13
oxide for magnetic resonance imaging (MRI)
, and fluoro-
phores for fluorescence imaging14, 15. In contrast to these “al-
ways-on” probes, there is another category of activatable
probes for fluorescence imaging, whose fluorescence can be
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Results and discussion
pared five aryl-glutamate conjugates 1-5 with several linkers
(urea: 1-2, carbamate: 3, azoformyl: 4, amide: 5) and exam-
ined their reactivity with PSMA by means of fluorescent deri-
vatization of the released glutamate with o-phthaldialdehyde
Evaluation of aryl-glutamate conjugates as PSMA sub-
strates. In order to develop an activatable fluorescence probe
for the CP activity of PSMA, we started by searching the liter-
ature for CP substrates in which substrate amino acids or pep-
tides are conjugated to aryl groups via -conjugation. We
found that amino acids conjugated to aryl groups directly (am-
3
4
and -mercaptoethanol (Figure 1a,b, Scheme S1) , using
NAAG peptide as a positive control. We found that, among
the compounds tested, only conjugate 4 with an azoformyl
linker (Ph-AF-Glu) is recognized by PSMA and hydrolyzed to
afford glutamate. Further, this reaction was inhibited in the
29
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ide bond ) or via self-immolative linkers such as urea , car-
2
9, 30
31, 32, 33
bamate
, or azoformyl
, can be recognized by various
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presence
of
a
PSMA
inhibitor,
2-
(Figure
types of CPs, though it is not known whether human PSMA
also recognizes these conjugates. Therefore, we newly pre-
35, 36
(phosphonomethyl)pentanedioic acid (2-PMPA)
1
b, Figure S1). Phenyl- or anisyl-azoformyl compounds have
been developed as CPA (carboxypeptidase A) and CPB
(carboxypeptidase B) substrates, enabling the enzyme
reactions to be monitored by measuring the decrease of
absorption at 350 nm due to loss of the azo chromophore.
However, these azoformyl substrates are not suitable for live-
cell imaging of CP activity due to the limited sensitivity of
absorption spectrophotometry. Considering that enzyme-
catalyzed hydrolysis of the amino acid of a phenylazoformyl
conjugate is followed by self-decomposition to afford molecu-
lar carbon dioxide, nitrogen, and a phenyl group, together with
the strong electron-withdrawing ability of the azoformyl group,
we thought that CP-mediated hydrolysis would induce a sig-
nificant change in the electron density of the aryl group, which
could be translated into significant fluorescence activation via
a photo-induced electron transfer (PeT) process. Specifically,
we aimed to develop an activatable CP probe whose fluores-
cence would be quenched through a donor-excited PeT (d-
38
PeT) process , i.e., electron transfer from the fluorophore to
the phenyl group, but which would be converted to a highly
fluorescent molecule after hydrolysis by the enzyme owing to
a drastic change in the lowest unoccupied molecular orbital
(
LUMO) energy level of the phenyl group (Figure 1c).
A first-in-class activatable fluorescence probe for PSMA.
In order to examine whether the phenylazoformyl (Ph-AF)
group works as an electron acceptor from the fluorophore, we
calculated the LUMO energy levels of some Ph-AF deriva-
tives and compared the values with those of compounds previ-
ously reported to quench the fluorescence of fluorescein
through d-PeT. As the electron donor, we chose a xanthene
moiety, the fluorophore of fluorescein derivatives. We found
that the LUMO energy levels of the Ph-AF derivatives were
lower than that of trimellitic acid trimethyl ester (Figure S2,
Table S1). Since the fluorescein derivative bearing trimellitic
acid trimethyl ester as the benzene moiety has the low fluores-
Figure 1. Evaluation of aryl-glutamate conjugates as
PSMA substrates. (a) Chemical structures of aryl-glutamate
conjugates 1-5, and NAAG (endogenous substrate of PSMA).
3
8
cence quantum yield (QY) of 0.001 , we expected that a fluo-
rescein derivative bearing Ph-AF as the benzene moiety would
show suppressed fluorescence. Encouraged by this result, we
prepared two fluorescein derivatives, 4GluAF-Fl and
(b) In vitro reaction of conjugates 1-5 with recombinant hu-
man PSMA, followed by the fluorescent derivatization of
released glutamate with o-phthalaldehyde with -
mercaptoethanol. A 50 M solution of conjugates 1-5 or
NAAG was incubated with recombinant human PSMA (1.45
5
GluAF-Fl, in which glutamate was conjugated to a benzene
moiety through an AF linker at the 4 and 5 position, respec-
tively (Figure 2a, Scheme S2). As expected, both 4GluAF-Fl
and 5GluAF-Fl are well quenched, and the fluorescence QY
values were calculated to be 0.003 and 0.002, respectively

g/mL) for 1 hr at 37 °C in the absence or presence of 2-
PMPA (1M), a specific inhibitor of PSMA. *P < 0.0001,
*P < 0.001 (Student’s t-test, n = 3). (c) Molecular design of
*
(
5
Table 1). When incubated with recombinant human PSMA,
GluAF-Fl showed a large fluorescence increase (> 200 fold),
activatable fluorescence probes for CP activity of PSMA
based on the d-PeT process. The probe fluorescence is
quenched by d-PeT before reaction with PSMA, but PSMA-
catalyzed hydrolysis of the glutamate moiety, followed by the
release of molecular carbon dioxide and nitrogen, affords a
highly fluorescent molecule.
while 4GluAF-Fl did not show fluorescence activation (Fig-
ure 2b). LC-MS analysis confirmed that 5GluAF-Fl was
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mainly converted to highly fluorescent fluorescein, together
ity towards cancer cells expressing PSMA, we incubated
5GluAF-Fl with lysates of two PCa cell lines, LNCaP cells
(PSMA-positive) and PC3 cells (PSMA-negative). 5GluAF-Fl
showed a large fluorescence increase when incubated with
LNCaP cell lysate, but not when incubated with PC3 cell ly-
sate or in the presence of 2-PMPA (Figure 2c). Further, when
5GluAF-Fl was applied to living LNCaP and PC3 cells seed-
ed in 96-well plates and the fluorescence change from each
well was recorded on a plate reader, fluorescence activation
was observed only in the presence of LNCaP cells, suggesting
that the CP activity of PSMA in living cells can be successful-
ly monitored with 5GluAF-Fl (Figure S5). However, when we
performed live-cell imaging using confocal fluorescence mi-
croscopy, we did not observe a marked fluorescence increase
inside LNCaP cells (Figure 2d, Figure S6). Considering that
with small quantity of by-products, while 4GluAF-Fl was not
hydrolyzed (Figure S3). It has been reported that compounds
with structural similarities to PSMA substrates are not always
2
5, 39
recognized by PSMA
; thus, the difference in reactivity
between 4GluAF-Fl and 5GluAf-Fl presumably reflects strict
substrate specificity of PSMA. The PSMA-catalyzed hydroly-
sis of 5GluAF-Fl was inhibited in the presence of 2-PMPA
(
Figure 2b, Figure S4a).
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Next, in order to examine whether 5GluAF-Fl shows reactiv-
4
0
the active site of PSMA is located in the extracellular domain
and the hydrolysis product, fluorescein, is highly hydrophilic,
it seems likely that 5GluAF-Fl reacted with PSMA on the cell
surface to produce cell-impermeable fluorescein, resulting in a
fluorescence increase exclusively in the extracellular space.
Thus, we need to improve the cell permeability of the reaction
product to visualize PSMA-positive cells.
Live-cell imaging of the CP activity of PSMA. For improv-
ing the cell permeability of the reaction product, we modified
5
GluAF-Fl by changing the 2’-substituent from carboxylic
acid to methoxycarbonyl or methyl, affording 5GluAF-FM or
GluAF-2MeTG, which would produce a more hydrophobic
hydrolysis product: fluorescein methyl ester (FM) or 2’Me
5
4
1
TokyoGreen (2’MeTG) , respectively (Figure 3a, Scheme S3,
S4). The photophysical properties indicated that the fluores-
cence of these derivatives was well quenched, and the fluores-
cence QYs were as low as that of 5GluAF-Fl (Table 1). We
confirmed that both probes can be hydrolyzed by recombinant
Table 1. Photophysical properties of the developed fluo-
rescence probes for CP activity of PSMA, and the putative
hydrolysis products.
Compounds
Absorbance
maximum
Emission
maximum
(nm)
Fluorescence
quantum
yield
Figure 2. A first-in-class activatable fluorescence probe
for PSMA. (a) Chemical structures of 4GluAF-Fl and
(
nm)
5
GluAF-Fl, and the enzymatic reaction with PSMA. (b) Fluo-
4
GluAF-Fla)
494
494
496
495
521
526
521
516
0.003
0.002
0.004
0.002
rescence spectra of 4GluAF-Fl (left) and 5GluAF-Fl (right)
before and after reaction with recombinant human PSMA. A
GluAF-Fla)
5
10 M probe solution in tris-buffered saline, pH 7.4, was
incubated with recombinant human PSMA (0.44 g) for 10
hr at 37 °C in the absence or presence of 2-PMPA (10 M),
and then diluted in tris-buffered saline, pH 7.4, to give a
final probe concentration of 0.67M for fluorescence meas-
urement. (c) Time course of fluorescence activation of
5GluAF-FMa)
5
2
GluAF-
MeTG
a)
5
GluAF-Fl upon addition of lysate from LNCaP cells (PSMA-
Fluoresceinb)
492
494
491
511
518
510
0.85
0.76
positive) or PC3 cells (PSMA-negative). A 10 M probe solu-
tion was incubated with cell lysate (0.36 mg/mL) for 10 hr at
37 °C in the absence or presence of 2-PMPA (10 M). Error
bars represent standard deviation (SD) from a single exper-
iment in triplicate. (d) Live-cell imaging of LNCaP cells or
PC3 cells treated with 5GluAF-Fl by confocal microscopy.
Cells were incubated with 10 M 5GluAF-Fl for 5 hr at 37°C,
and bright-field (BF) and fluorescence images were cap-
tured. Ex/em: 490/500-600 nm. Scale bars, 50 μm.
FMc)
2MeTGb)
0.85
a)
b)
Measured in 0.2 M sodium phosphate buffer, pH 7.4. Ref-
4
1
c)
38
erence . Reference .
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Figure 3. Live-cell imaging of CP activity of PSMA. (a) Chemical structures of 5GluAF-FM and 5GluAF-2MeTG, and the
enzymatic reaction with PSMA. (b) Fluorescence spectra of 5GluAF-FM (left) and 5GluAF-2MeTG (right) before and after reac-
tion with recombinant human PSMA. A 10M probe solution in tris-buffered saline, pH 7.4, was incubated with recombinant
human PSMA (0.44 g) for 10 hr at 37 °C in the absence or presence of 2-PMPA (10 M), and then diluted in tris-buffered sa-
line, pH 7.4, to make a final probe concentration of 0.67 M for fluorescence measurement. (c) Time courses of fluorescence
activation of 5GluAF-FM (upper) and 5GluAF-2MeTG (bottom) upon addition of lysate from LNCaP cells (PSMA-positive) or
PC3 cells (PSMA-negative). A 10M probe solution was incubated with cell lysate (0.36 mg/mL) for 10 hours at 37 °C in the
absence or presence of 2-PMPA (10 M). Error bars represent standard deviation (SD) from a single experiment conducted in
triplicate. (d) Live cell imaging of LNCaP cells and PC3 cells treated with 5GluAF-FM (upper) and 5GluAF-2MeTG (bottom) by
confocal microscopy. Cells were incubated with 10 M 5GluAF-FM or 5GluAF-2MeTG for 5 hr at 37 °C, and bright-field (BF)
and fluorescence images were captured. Ex/em: 490/500-600 nm. Scale bars, 50 μm.
PSMA and PSMA in cell lysate to produce 2Me-TG and FM;
the hydrolysis kinetics were in the order 5GluAF-2MeTG >
5GluAF-FM > 5GluAF-Fl (Figure 3b,c, Figure S3b,c, Figure
S7, Table S2). Further, when we performed live-cell imaging
of LNCaP cells, 5GluAF-2MeTG showed a significant fluo-
rescence increase in the cells, while 5GluAF-FM did not
activation was observed when the probe was applied to PC3
cells (Figure 3d, Figure S8). These results demonstrated that
5AFGlu-2MeTG is a first-in-class activatable fluorescence
probe for the CP activity of PSMA, and is capable of visualiz-
ing PSMA-expressing living cells.
(
Figure 3d, Figure S8). LC-MS analysis of the extracellular
Fluorescence detection of PCa in resected clinical speci-
mens. We next examined whether 5AFGlu-2MeTG could
visualize PCa in clinical tissue specimens. Resected specimens
from prostate cancer patients who had not received any hor-
mone therapy, chemotherapy or radiotherapy were collected
during radical prostatectomy. The specimens were incubated
with a 50 μM solution of 5GluAF-2MeTG on a heating plate
solution revealed that 2Me-TG was mainly produced during
incubation of 5GluAF-2MeTG, while in the case of 5GluAF-
FM (m/z = 548), we observed a mass peak whose molecular
weight is higher by 2 units (m/z = 550), suggesting that
5
(
be accounted for by the difference in the LUMO energy level
of the azoformyl benzene moiety (Figure S2). The observed
fluorescence activation of 5GluAF-2MeTG in LNCaP cells
was inhibited in the presence of 2-PMPA (Figure S10), and no
GluAF-FM tends to be reduced in the cellular environment
Figure S9). The difference in susceptibility to reduction might
(40 °C) for 30 minutes, which would be an acceptable duration
for incorporating this imaging into actual surgical procedures,
and the fluorescence images were captured with a Maestro
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Figure 4. Ex vivo fluorescence imaging of PCa in surgically resected clinical specimen from prostate cancer patients.
(a) White-light and fluorescence images of resected tissue after incubation with 5GluAF-2MeTG for 0 min and 30 min. The
specimen was incubated with a 50M solution of 5GluAF-2MeTG in tris-buffered saline, pH 7.4, for 30 min on a heating plate
(
(
40 °C). Fluorescence images were captured with a Maestro In-Vivo imaging system (PerkinElmer), using the blue-filter setting
excitation, 465/30 nm; emission, 515 nm long-pass). The fluorescence image at 540 nm was extracted and presented. The spec-
imen corresponds to Case 4 in figure (e). Scale bars, 1 cm. (b) Time course of fluorescence activation during incubation for 30
minutes with 5GluAF-2MeTG at the ROIs indicated in (a). (c) Table of fluorescence increase, cancer existence, Gleason score
and PSMA expression level at each ROI. (d) Representative images of hematoxylin and eosin (HE) staining and immunohisto-
chemistry (IHC) of PSMA. Images at ROI #7 and ROI #1 are presented as a cancer region with high PSMA expression and a
normal region with low PSMA expression, respectively. Asterisks (*) indicate stromal cells, in which PSMA expression was not
observed. Scale bars, 100 m. (e) Fluorescence increase at ROIs in 4 clinical specimens incubated with 5GluAF-2MeTG were
analyzed and ordered by activity. Each case was from a different prostate cancer patient (Case 1-Case 4), and # represents the
ROI number in each specimen. Red bars show cancer-containing regions, and blue bars show non-cancerous regions.
fluorescence imaging system. Although there was a substantial
fluorescence signal at 0 min due to the combination of tissue
auto-fluorescence and background fluorescence of the probe,
we observed heterogeneous fluorescence activation in the
specimens during 30 min (Figure 4a, Figure S11). In order to
examine the correlation between the fluorescence activation
and the results of pathological/immunohistochemical analysis,
several representative ROIs (regions of interest) were selected,
cut out, and pathologically examined for the existence of PCa
chemical analysis. We found that most of the strongly fluores-
cent regions contained cancer strongly expressing PSMA,
while weakly fluorescent regions were mostly normal prostate
tissue or cancer-containing regions with weak PSMA expres-
sion (Figure 4b,c,d, Figure S11). We also confirmed the ab-
sence of PSMA expression in stromal cells (Figure 4d). Figure
4e summarizes the results of applying 5GluAF-2MeTG to 4
clinical specimens, arranged in descending order of fluores-
cence increase. Although there are some false-negatives and
false-positives, probably due to limited PSMA expression in
cancer tissue and substantial PSMA expression in normal
4
2
and the histological grade (Gleason score) . In addition, the
PSMA expression level was determined by immunohisto-
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prostate tissue, the fluorescence activation of 5GluAF-
rescence probes for other carboxypeptidase activities by re-
placing the substrate moiety, as well as red-shifted analogues
by changing the fluorophore.
2MeTG after incubation for 30 minutes was mostly well cor-
related with the presence of cancer, reflecting the expression
level of PSMA in tissues. It is particularly noteworthy that tiny
millimeter-sized cancer regions such as ROI #7 in Figure 4 or
ROI #6 in Figure S11a can be clearly visualized as fluorescent
spots; such small cancer regions would be difficult to detect by
means of conventional preoperative diagnostic methods such
as CT and MRI. These results demonstrate that 5GluAF-
Experimental section.
Synthesis. For synthetic protocols, see the supplementary
information.
Cell lines and culture. LNCaP and PC3 cells were obtained
from RIKEN Cell Bank. Both cell lines were cultured in
RPMI 1640 medium (Wako) supplemented with 10% (v/v)
FBS and 1% (v/v) penicillin streptomycin (Invitrogen) in an
incubator at 37°C under 5% CO2 in air.
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MeTG can detect the CP activity of PSMA even in small
cancer regions of clinical specimens, and therefore should be
useful as an intraoperative diagnostic tool for PCa.
Live-cell confocal imaging. An 8-well chamber (μ-slide, 8-
well; Ibidi) was coated with 30 μg/mL fibronectin solution
Conclusion.
In conclusion, we have designed and synthesized 5GluAF-
(063-05591, Wako) in DPBS (-) at 37°C for 2 hours. Then,
2
MeTG as a first-in-class activatable fluorescence probe for
LNCaP cells or PC3 cells were seeded at 2.5 × 104 cells per
well and cultured overnight at 37°C under 5% CO2 in air. The
medium of each well was replaced with 150 μL of HBSS (+),
then 50 μL of a 40 μM probe solution in HBSS (+) was added
to each well, and incubation was continued under the same
conditions. Fluorescence images were captured for 5 hours
with a confocal fluorescence microscope (TCS SP 8 STED,
Leica) equipped with a CO2 incubator. Excitation and emis-
sion wavelengths were 490 nm and 500-600 nm, respectively.
HC PL APO CS2 40x/1.30 Oil was used as the objective lens.
For inhibitor assay, the cells were incubated with 10 μM probe
solution in HBSS (+) in the presence of 10 μM 2-PMPA at
37°C under 5% CO2 in air for 15 hours. Fluorescence images
were captured with a confocal fluorescence microscope (TCS
SP 8 STED, Leica). After imaging, the extracellular solution
was collected and centrifuged at 15000 rpm at 4 °C, and the
supernatant was stored at -80°C for LC-MS analysis.
CP activity of PSMA and succeeded in applying it for fluores-
cence visualization of PCa in cultured cells and in clinical
specimens. We first established that Ph-AF-Glu is a substrate
of PSMA, and then developed 5GluAF-Fl, whose fluores-
cence was controlled by d-PeT. But, although 5GluAF-Fl
showed significant fluorescence activation upon reaction with
PSMA, it failed to visualize PSMA-positive cells, apparently
due to poor cellular permeability of the hydrolysis product.
Therefore, we modified the chemical structure to afford
5
GluAF-2MeTG, which produces cell-permeable 2MeTG as
its PSMA-catalyzed hydrolysis product, making it possible to
visualize PSMA-positive cells. To our knowledge, this is the
first example of an activatable fluorescence probe detecting
the CP activity of PSMA in living cells. When 5GluAF-
2
MeTG was applied to resected specimens from PCa patients,
we observed significant fluorescence activation, which corre-
sponded well with the pathological localization of PCa.
Prostate cancer patients. This study was conducted with the
approval of the Research Ethics Committee of the University
of Tokyo (registration number: 11623). All experiments were
performed in accordance with guidelines and regulations ap-
proved by the Research Ethics Committee of the University of
Tokyo. Informed consent was obtained from all patients. Pros-
tate cancer patients examined or treated at the University of
Tokyo Hospital in Tokyo, Japan, were prospectively included
in this study.
These results demonstrate the potential utility of our probe to
visualize PCa in clinical situations. Nerve-sparing surgery is
an important treatment option to preserve nerve function and
urinary function postoperatively, in cases where invasion of
cancer into the neurovascular bundles can be ruled out based
on preoperative diagnostics such as ultrasound, CT, MRI, and
needle biopsy. However, it is difficult to detect all cancerous
tissue preoperatively, especially tiny regions, and so there has
been concern about the risk of increased rates of recurrence if
positive surgical margins are left during nerve-sparing radical
prostatectomy. In this context, our newly developed probe
might be useful to improve the accuracy of intraoperative can-
cer detection, enabling efficient nerve-sparing by clearly dis-
tinguishing cancerous regions near the neurovascular bundle.
Thus, we believe that our probe might enable more confident
minimally invasive prostate resection, without increased risk
of complications such as erectile dysfunction and urinary in-
continence.
Ex vivo fluorescence imaging of freshly resected prostate
specimens. Surgical specimens were rinsed with tris-buffered
saline, pH 7.4, and a 50 μM solution of 5GluAF-2MeTG in
tris-buffered saline, pH 7.4, was added to cover the entire
specimen, which was then incubated on a heating plate (40 °C).
Fluorescence images were captured with a Maestro In-Vivo
imaging system (Perkin Elmer) for 30 minutes, using the blue-
filter setting (excitation, 465/30 nm; emission, 515 nm long-
pass).
Histological analysis and immunohistochemistry. Resect-
ed specimens were evaluated pathologically. Hematoxylin and
eosin (HE) staining was used to confirm the presence of can-
Furthermore, it was recently reported that PSMA is highly
expressed in the neovasculature of other cancers, such as
4
3
44
45
breast cancer , lung cancer , gastric cancer , colorectal can-
42
4
5
46
cer and the histological grade (Gleason score) , together with
cer and bladder cancers , and it is also expressed in a variety
of neurological diseases . Thus, we think that 5GluAF-
MeTG could also be useful as a tool to study the roles of
4
7
immunohistochemistry (IHC) to determine the PSMA expres-
sion level. IHC staining was performed by an automated IHC
slide-staining instrument (Ventana BenchMark XT, Roche),
using primary antibody against PSMA (mouse monoclonal
antibody, clone 3E6, Dako M3620) at 1:200 dilution and an I-
2
PSMA in the pathophysiology of these diseases, as well as in
the development of therapeutics. Our newly established design
strategy should also be applicable to develop activatable fluo-
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VIEW DAB universal kit (760-041, Roche). 3,3’-
margin during radical prostatectomy. Urol. Oncol.: Seminars and
Original Investigations 2015, 33, 18.e1-18.e6.
Diaminobenzidine (DAB) was used as a chromogen and he-
matoxylin was applied for counterstaining. PSMA expression
level in epithelial cells (either normal or cancerous) was
scored from 1 (weak) to 4 (strong) by a pathologist who was
blinded to the results of fluorescence imaging.
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ASSOCIATED CONTENT
Supporting Information. This material is available free of
charge via the Internet at http://pubs.acs.org. Synthesis, ex-
perimental details, and photophysical properties of the com-
pounds
2256-2261.
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7) Su, S. L.; Huang, I.-P.; Fair, W. R.; Powell, C. T.; Heston, W. D.
W., Alternatively spliced variants of prostate-specific membrane
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AUTHOR INFORMATION
Corresponding Author
(8) Bravaccini, S.; Puccetti, M.; Bocchini, M.; Ravaioli, S.; Celli,
M.; Scarpi, E.; De Giorgi, U.; Tumedei, M. M.; Raulli, G.; Cardi-
nale, L.; Paganelli, G., PSMA expression: a potential ally for the
pathologist in prostate cancer diagnosis. Sci. Rep. 2018, 8, 4254.
*
mkamiya@m.u-tokyo.ac.jp, uranokun@m.u-tokyo.ac.jp
Author Contributions
These authors contributed equally.
(
9) Kawakami, M.; Nakayama, J., Enhanced expression of pros-
tate-specific membrane antigen gene in prostate cancer as re-
‡
vealed by in situ hybridization. Cancer Res. 1997, 57, 2321-2324.
(
10) Evans, J. C.; Malhotra, M.; Cryan, J. F.; O'Driscoll, C. M., The
therapeutic and diagnostic potential of the prostate specific
membrane antigen/glutamate carboxypeptidase II
PSMA/GCPII) in cancer and neurological disease. Br. J.
Pharmacol. 2016, 173, 3041-3079.
11) Afshar-Oromieh, A.; Avtzi, E.; Giesel, F. L.; Holland-Letz, T.;
Notes
The authors declare no competing financial interest.
(
ACKNOWLEDGMENT
(
This research was supported in part by AMED/P-CREATE
under grant number JP18cm0106409 (to Y.U.), by
JST/PRESTO grant JPMJPR14F8 (to M. Kamiya), by
MEXT/JSPS KAKENHI grants JP16H02606, JP26111012 (to
Y.U.) and JP15H05951 “Resonance Bio” and JP19H02826 (to M.
Kamiya), by JSPS Core-to-Core Program, A. Advanced Re-
search Networks, by Astellas Foundation for Research on
Metabolic Disorders (to M. Kamiya), and Japan Foundation
for Applied Enzymology (to M. Kamiya), as well as a stipend
from Graduate Program for Leaders in Life Innovation
Linhart, H. G.; Eder, M.; Eisenhut, M.; Boxler, S.; Hadaschik, B.
A.; Kratochwil, C.; Weichert, W.; Kopka, K.; Debus, J.;
Haberkorn, U., The diagnostic value of PET/CT imaging with the
68Ga-labelled PSMA ligand HBED-CC in the diagnosis of recur-
rent prostate cancer. Eur. J. Nucl. Med. Mol. Imaging 2015, 42,
197-209.
(
12) Tse, B. W.-C.; Cowin, G. J.; Soekmadji, C.; Jovanovic, L.; Va-
sireddy, R. S.; Ling, M.-T.; Khatri, A.; Liu, T.; Thierry, B.; Russell,
P. J., PSMA-targeting iron oxide magnetic nanoparticles enhance
MRI of preclinical prostate cancer. Nanomedicine 2015, 10, 375-
386.
(
GPLLI) (to M. Kawatani) and a JSPS stipend (to K.Y.). The
(13) Zhu, Y.; Sun, Y.; Chen, Y.; Liu, W.; Jiang, J.; Guan, W.; Zhang,
Z.; Duan, Y., In vivo molecular MRI imaging of prostate cancer
by targeting PSMA with polypeptide-labeled superparamagnetic
iron oxide nanoparticles. Int. J. Mol. Sci. 2015, 16, 9573.
authors thank Dr. Yusuke Saito, Dr. Taketo Kawai, Dr. Masa-
ki Nakamura, Dr. Motofumi Suzuki, and Dr. Akihiko Matsu-
moto for their supports to collect clinical specimens.
(
14) Chen, Y.; Dhara, S.; Banerjee, S. R.; Byun, Y.; Pullambhatla,
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