Development of an Activatable Fluorescent Probe ... - ACS Publications

Subscriber access provided by The University of British Columbia Library

Article

Development of an Activatable Fluorescent Probe for Prostate Cancer Imaging Takao Yogo, Keitaro Umezawa, Mako Kamiya, Rumi Hino, and Yasuteru Urano Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00233 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Figure 1 473x283mm (96 x 96 DPI)

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Page 2 of 24

Page 3 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60





1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Page 4 of 24

Page 5 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60





1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Page 6 of 24

Page 7 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

Development of an Activatable Fluorescent Probe for Prostate Cancer Imaging Takao Yogo,† Keitaro Umezawa,‡ Mako Kamiya,†,§ Rumi Hino,‖ and Yasuteru Urano*,†,‡,┴

† Graduate School of Medicine and ‡Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ‖ Department of Sports and Health Science, Daito Bunka University, 560 Iwadono, Higashimathuyama-shi, Saitama 355-8501, Japan. ┴ AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 24

Yogo et al.

Abstract Technology to visualize small prostate cancers is urgently needed because of the difficulty of discriminating prostate cancer from normal tissue with the naked eye, and a fluorescence imaging method would be advantageous. Here we describe the design and synthesis of a fluorogenic probe (Ac-KQLR-HMRG) that is activated by hepsin and matriptase (proteases overexpressed in prostate cancer). Ac-KQLR-HMRG exhibited significant turn-on fluorogenicity in the presence of hepsin (180-fold) and matriptase (80-fold), and allowed specific fluorescence imaging of various prostate cancer cell lines in vitro. In addition, the probe enabled rapid imaging (within 1-10 minutes) of small prostate cancer nodules in mouse models of disseminated peritoneal tumor and orthotopic tumor.

Ac-KQLR-HMRG

HMRG

Colorless, non-fluorescent

Colored, highly fluorescent Tumor

hepsin matriptase

Activated on prostate cancer

White light

Fluorescence image


Page 9 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

Introduction Prostate cancer is one of the most common malignant tumors among adult men, and its incidence has been increasing; indeed more than 1.1 million cases and 307,000 deaths were recorded in 2012.1 Resection of the entire prostate gland, including surrounding tissues and nerves in some cases, is the standard operative strategy2-4 to avoid the risk of leaving tiny cancers that could not be detected with the naked eye. However, this strategy often leads to serious side effects, such as erectile dysfunction. Thus, there is an urgent need for methodology to visualize prostate tumors during surgery, so that cancer tissues can be completely removed without damage to or loss of surrounding uninvolved tissues. Although commonly used imaging modalities such as ultrasound, CT and MRI have advanced considerably in recent decades and are useful for accurate diagnosis of prostate cancer, they have substantial limitations for intraoperative imaging.5,6 We considered that a fluorescence imaging method would be advantageous for this purpose, due to its high sensitivity, real-time capability and high compatibility with image-guided surgery.7 Various biomarkers of prostate cancer have been discovered. For instance, prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) are up-regulated in prostate cancer.8-10 In addition, hepsin and matriptase, which are members of the type 2 transmembrane serine protease (TTSP) family, are overexpressed in prostate cancer.11-23 These biomarkers can be targeted with specific antibodies/ligands, and indeed, agents such as fluorescence-labeled hepsin/matriptase-targeted peptide/antibody11,24,25 have been developed and applied for fluorescence imaging of prostate cancers. However, most of these imaging agents are “always on” (i.e., always fluorescent under irradiation with excitation light), and this feature generally results in a high background signal due to excess probe and to non-specific accumulation of probe molecules in normal tissues; consequently, a period of hours to days may be required for washout/clearance to obtain a sufficient tumor-to-normal (T/N) signal ratio. On the other hand, “turn-on” (activatable) fluorescent probes, which are non-fluorescent in themselves but become fluorescent after incorporation into cancer tissues, are expected to enable high-contrast imaging, and indeed, some activatable fluorophore-antibody conjugates have been reported (e.g. PSMA-targeted antibody26). But, the problem of a relatively long activation time (hours to days) remains, because degradation of the antibody is still required to trigger fluorescence, and this slow response makes them impractical for routine or real-time clinical use. Also, the fluorescence activation ratio is not so high (< 20 folds) due to a relatively inefficient self-quenching mechanism, which should be improved for better tumor imaging. Here, we aimed to develop a new type of turn-on fluorescent probe for prostate cancer that would enable rapid visualization of cancer with a high T/N ratio on a time scale of seconds to minutes without the need for a washout/clearance process. For this purpose, we focused on our previous work, in which we utilized the spirocyclization equilibrium of hydroxymethylrhodamine green (HMRG) to design activatable turn-on fluorescent 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 24

Yogo et al.

probes for proteases.27 For instance, we showed that γ-glutamate-conjugated HMRG (gGlu-HMRG) is selectively cleaved by γ-glutamyltransferase (GGT), which is overexpressed on the cell surface of some types of human tumors, and this property can be used for rapid fluorescence imaging of cancer in a mouse model28 and in resected specimens from breast cancer patients.29,30 Here, we adopted a similar strategy to design a HMRG-based fluorescent probe for prostate cancer by targeting hepsin/matriptase. According to the literature, TTSP family members specifically recognize the peptide sequence XX-KQLR-XX and efficiently cleave the peptide bond at the R-X position.31 Indeed, hepsin and matriptase have common substrates, such as hepatocyte growth factor, whose activation

cleavage

site

is

KQLR-VVNG.31

Therefore,

we

designed

a

new

hepsin/matriptase-activatable turn-on fluorescent probe for prostate cancer by conjugating Ac-KQLR and HMRG to produce Ac-KQLR-HMRG. The probe is expected to show fluorescence activation upon cleavage of the peptide unit by hepsin/matriptase (Figure 1a). Herein we report the synthesis and optical properties of Ac-KQLR-HMRG, and its application to imaging of live cells of various prostate cancer cell lines, and imaging of prostate cancers in mouse models of disseminated peritoneal tumor and orthotopic tumor.

Figure 1. (a) Scheme for enzymatic reaction of Ac-KQLR-HMRG in the presence of hepsin/matriptase. (b) pH-dependent absorbance changes of Ac-KQLR-HMRG (black) and HMRG (red). Error bars represent standard deviation (N = 3). Solvent: 200 mM sodium phosphate buffer, including 0.1% DMSO as a cosolvent. Absorption: 501 nm for HMRG; 495 nm for 4 ACS Paragon Plus Environment

Page 11 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

Ac-KQLR-HMRG. (c) Fluorescence spectra of Ac-KQLR-HMRG (10 µM) before (black) and 30 min after (red) reaction with hepsin (10 ng, left) and matriptase (10 ng, right) at pH 7.4 (50 mM tris buffer). Excitation: 500 nm.

Results Photophysical and kinetic properties of Ac-KQLR-HMRG Ac-KQLR-HMRG was synthesized according to scheme S1 in the Supporting Information. First, we evaluated the pH dependency of the optical properties of Ac-KQLR-HMRG. As shown in Figure 1b, Ac-KQLR-HMRG showed a pH-dependent absorption/fluorescence decrease at neutral/alkaline pH, owing

to

pH-dependent

change

in

the

spirocyclization

(spirolactone-rhodamine equilibrium), as discussed previously.

27

equilibrium

of

the

probe

The equilibrium constant of

Ac-KQLR-HMRG (pKcycl: the pH value at which the extent of spirocyclization is sufficient to reduce the absorbance of the compound to one-half of the maximum absorbance) was calculated to be 4.8 (Table S1). Thus, the compound showed almost no absorption or fluorescence in buffer at pH 7.4, where it is predominantly in the spirocyclized form. On the other hand, HMRG has a pKcycl value of 8.1, and shows intense color and high fluorescence at pH 7.4 (Figure 1b). These results suggest that drastic fluorescence enhancement would be observed in response to the enzymatic cleavage reaction. Next, we investigated the reactivity of Ac-KQLR-HMRG with hepsin and matriptase by monitoring changes in fluorescence intensity after addition of these enzymes. Addition of hepsin and matriptase to the probe solution resulted in a dramatic and rapid fluorescence increase (80-fold with matriptase, and 180-fold with hepsin after 30 min reaction, respectively) (Figure 1c, Table S2); we also confirmed that this fluorescence enhancement was strongly suppressed by the addition of an enzyme inhibitor (Figure S1). These results demonstrate that Ac-KQLR-HMRG is available as an activatable fluorescent probe targeting hepsin/matriptase, as expected.

Fluorescence imaging of cultured cell lines Next, we performed live-cell fluorescence imaging of cultured human prostate cancer cells of several lines (LNCaP, PC3, DU145, which were established from metastases in lymph nodes, lumbar bones, and the central nervous system, respectively32) and a human prostate normal cell line (PrEC) with Ac-KQLR-HMRG. As shown in Figure 2, intense fluorescence signals were observed in all prostate cancer cell lines after incubation for 5 min with Ac-KQLR-HMRG, whereas only weak signal was observed in PrEC cells. In order to confirm whether this fluorescence increase was specific for hepsin/matriptase activities, we co-incubated the cells with Ac-KQLR-HMRG and hexamidine, an inhibitor of matriptase/hepsin33. As shown in Figure 2, the fluorescence increase in LNCaP, PC3 and DU145 cells was significantly suppressed by hexamidine. These results indicate that Ac-KQLR-HMRG was selectively activated by hepsin and/or matriptase expressed on the cell 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 24

Yogo et al.

membrane to yield fluorescent HMRG, which subsequently accumulated in the cancer cells. Co-staining of Ac-KQLR-HMRG and Lysotracker Red (Invitrogen) revealed that HMRG localizes in lysosomes (Figure S2). Furthermore, almost no fluorescence signal was detected in any of the cell lines tested when we used gGlu-HMRG instead28 (Figure 3). This clearly demonstrates the importance of selecting appropriate targets depending on the tumor of interest, since each tumor type has a distinct enzymatic profile. Considering that hepsin is known to be overexpressed only in LNCaP cells, but not in PC3 and DU145 cells,11,12 while up-regulation of matriptase is reported in all three cell lines,12 Ac-KQLR-HMRG may mainly target matriptase, since all of the three typical prostate cancer cell lines (LNCaP, PC3, DU145) were visualized. Thus, Ac-KQLR-HMRG should be widely applicable for visualizing not only less aggressive cancer cell lines such as LNCaP, but also more aggressive lines such as PC3 and DU145.

Figure 2. (a) Fluorescence images of three cultured cell lines derived from human prostate cancer cells (LNCaP, PC3, DU145) and normal prostate cells (PrEC) using Ac-KQLR-HMRG in the absence or presence of hexamidine (20 μM). Cells were incubated with 1 μM probe solution for 5 min and then images were captured with a Leica TCS SP5X confocal microscopy system (excitation: 488 nm, emission: 500 – 560 nm). Scale bar, 50 μm. (b) Averaged fluorescence in the regions of interest for the four cell lines. Error bars represent standard deviation (n = 10).

***

p < 0.005

(Student’s t-test).


Page 13 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

Figure 3. (a) Fluorescence imaging of three cultured cell lines derived from human prostate cancer cells (LNCaP, PC3, DU145) using Ac-KQLR-HMRG and gGlu-HMRG. Cells were incubated with 1 μM probe solution for 5 min and then images were captured with a Leica TCS SP5X confocal microscopy system (excitation: 488 nm, emission: 500 – 560 nm). Scale bar, 50 μm. b) Averaged fluorescence in the regions of interest for the three cell lines. Error bars represent standard deviation (n = 10). *** p < 0.005 (Student’s t-test).

Fluorescence imaging of intraperitoneally disseminated prostate cancer cells in a mouse model In order to investigate whether Ac-KQLR-HMRG would also work for in vivo imaging, we performed fluorescence imaging of intraperitoneally disseminated prostate cancer nodules in mice.28 PC3 (3.0 × 106 or 8.5 × 106 cells in 150 µl PBS) and DU145 (3.8 × 106 cells in 150 µl PBS) were each disseminated by intraperitoneal injection into male mice, and after 10–14 days, fluorescence imaging of DU145 and PC3 cancer nodules was conducted by intraperitoneal injection of the probe. As shown in Figure 4 and 5a, small disseminated cancer nodules (ca. 0.5 - 2.0 mm size), which can hardly be detected with the naked eye, could be clearly visualized just 5 minutes post injection of the probe. In contrast, a much weaker fluorescence signal was observed from normal tissue and from the mesentery of control mice (without tumor implantation), demonstrating that the probe is selectively activated by tumor-associated proteases in vivo, and the resulting fluorescent product is rapidly incorporated and accumulated in cancer cells. The results of hematoxylin-eosin staining of the sections including fluorescence-positive and negative regions confirm that the fluorescence is well colocalized with cancer tissue (Figure 5b and c). It is well known that the pH in cancer tissues is lower (around 6 to 7) than that in normal tissues,34 but since non-reacted Ac-KQLR-HMRG is predominantly in the closed, non-fluorescent state at this pH (Figure 1b), the above fluorescence 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 24

Yogo et al.

enhancement is considered to be due to activation of the probe by hepsin/matriptase. Consequently, we conclude that rapid and highly sensitive fluorescence imaging of small prostate cancer nodules can be achieved with Ac-KQLR-HMRG.

Figure 4. Ex vivo imaging of the mesentery of mouse models of disseminated peritoneal tumor (PC3, DU145, and controls) 5 min after intraperitoneal injection of Ac-KQLR-HMRG (0 or 50 µM, 300 µl in PBS). Scale bar: 1 cm. Top: white-light image. Middle: fluorescence images (465/30 nm band-pass filter for emission and a 515 nm long-pass filter for emission). Bottom: Unmixed spectral image of the mesentery. (green; HMRG signal, red; autofluorescence signal derived from digested pelleted feed). N = 4 (for PC3, DU145 and control with 50 µM probe), N = 3 (for control without probe).


Page 15 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

Figure 5. a) Ex vivo imaging of the pancreas of a mouse model of disseminated peritoneal tumor (PC3) 5 min after intraperitoneal injection of Ac-KQLR-HMRG (50 µM, 300 µl in PBS). Scale bar: 1 cm. Left: color image taken by a digital camera. Middle: fluorescence images (465/30 nm band-pass filter for emission and a 515 nm long-pass filter for emission). Right: Unmixed spectral image of the pancreas. (green; HMRG signal, red; autofluorescence signal derived from the pancreas). b, c) Sections containing fluorescence-positive and negative regions was cut along the corresponding dashed yellow lines in Figure 5a right, and stained with hematoxylin and eosin. Scale bar: 1 mm. i-iii) Enlarged images of the orange squares in Figure 5b and c. Scale bars: 100 µm. T: tumor, PA: pancreas.

Fluorescence imaging of orthotopic tumor-bearing mouse model We also examined fluorescence imaging in an orthotopic tumor-bearing mouse model. PC3 cell suspension (1.6 × 106 cells in 40 μl PBS) was injected into the prostate gland. After 14 days, when the resulting prostate tumor had reached approximately 5 – 7 mm in size, 20 µl Ac-KQLR-HMRG (50 µM in PBS) was directly injected to the prostate gland. Fluorescence enhancement in the prostate cancer was observed from just 1 minute after the injection (Figure 6a and b), and was sufficient to allow the tumor to be clearly distinguished from the surrounding tissues, such as intestine, urinary bladder, vessels, and peritoneum. Further, the fluorescence signal of the orthotopic tumor was significantly higher than that of the prostate of control sham-operated mice (without tumor implantation), and the T/N ratio reached 3.3 after 10 min (Figure 6c). Fluorescence images of isolated prostate gland including cancer tissue at 30 min after in vivo injection of the probe revealed 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 24

Yogo et al.

selective fluorescence imaging of cancer tissues, which were clearly distinguished from healthy prostate and bladder (Figure 6d). This was further confirmed by histopathological analysis (Figure 6e). Consequently, our newly developed fluorescent probe, Ac-KQLR-HMRG, should be suitable for rapid and selective imaging of prostate cancer tissues in vivo.

Figure 6. In situ fluorescence imaging of orthotopic tumor in a mouse model. 540-nm fluorescence images were exported by Maestro software. a) Representative white-light and fluorescence images (Em: 540 nm) at 1, 2, 3, 5 and 10 min after injection of Ac-KQLR-HMRG (50 µM, 20 µl). Scale bar: 1 cm. b) Enlarged images of the yellow square in Figure 6a. Scale bar: 0.5 cm. T: tumor, BL: bladder, PR: prostate gland. c) Changes in tumor fluorescence signals in prostate tissue in orthotopic tumor mice model (red) and sham-operated mice (black). Data are mean fluorescence intensities of tumor at different time points, and error bars represent standard deviation (N = 3).

***

p < 0.005

(Student’s t-test). d) White-light (left), fluorescence (middle) and merged (right) images of isolated prostate gland and bladder. The tissue was cut and separated to expose two surfaces of the same cancer tissue (top and bottom). Scale bar: 500 µm. e) A section containing fluorescence-positive and negative regions was cut along the dashed yellow line in Figure 6d (right), and stained with hematoxylin and eosin. Scale bar: 1 mm. i-iii) Enlarged images of the orange squares in Figure 6e left. Scale bars: 200 µm.

Discussion We have developed the first turn-on fluorescent probe targeting hepsin/matriptase, which are overexpressed on the surface of prostate cancer, and we showed that this probe can visualize minimally aggressive LNCaP cells, moderately aggressive DU145, and highly aggressive PC3 cell 10 ACS Paragon Plus Environment

Page 17 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

lines. In addition, small cancer nodules in mouse models of disseminated peritoneal tumor and orthotopic tumor were visualized within 1 – 10 min (much faster than can be achieved with antibody-based activatable fluorescent probes26), with sufficiently high T/N ratio for easy detection with the naked eye. These results demonstrate the potential utility of our probe to visualize prostate cancer in the clinical situation.28 Although peritoneal dissemination of prostate cancer is rare,35 our results in the mouse model of disseminated peritoneal tumor clearly show the potential of this probe for detecting small peritoneal cancer metastases (< 1 mm) from prostate cancer during surgery, whereas such submillimeter-scale metastases are difficult to detect with preoperative imaging examinations such as CT and MRI.36,37 The orthotopic model is much closer to the usual clinical situation, and thus the results in the orthotopic tumor-bearing mice directly suggest practical utility of our probe. At present, radical prostatectomy is still the standard operative method for patients with localized prostate cancer. However, resection of the entire prostate gland, including surrounding tissues, involves the risk of side effects such as erectile dysfunction and urinary incontinence. Nerve-sparing radical prostatectomy can be performed only when invasion of cancer cells into the neurovascular bundles can be ruled out. According to a multi-institutional study, better recovery of sexual function was found among patients who underwent nerve-sparing prostatectomy than among those who did not.38 Therefore, maintenance of erectile function after radical prostatectomy depends strongly on whether or not autonomic cavernous nerves in the neurovascular bundles can be preserved during surgery. Further, a multi-institutional study of 603 patients who had undergone radical prostatectomy indicated that 52% of patients experienced urine leakage more than once a day for two months after surgery.38 It is important to preserve normal anatomical and functional structure in the pelvis, such as bladder neck, neurovascular bundle, puboprostatic ligament, pubovesical complex and urethral length,39 to improve postoperative urinary incontinence. Although robot-assisted radical prostatectomy (RARP) has been shown to improve postoperative continence rates compared with retropubic and laparoscopic radical prostatectomy,40 further improvements in the accuracy of intraoperative techniques are required to minimize side effects after radical prostatectomy. So far, surgeons have to decide whether to perform tissue-preserving operation based on preoperative visual inspection and palpation of the gland and its relationship to the neurovascular bundles, together with the results of preoperative diagnostics such as ultrasound, CT and MRI. However, it is extremely difficult to detect all cancerous tissue, especially tiny cancerous nodules, with these standard diagnostic methods. Further, intraoperative assessment of the margin status is known to have a high false-negative rate, and is not reliable to determine which cases require excision of the neurovascular bundle.41 In this situation, the ability of our newly developed probe to visualize small prostate cancer nodules within a few minutes might improve the accuracy of intraoperative cancer diagnosis, 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 24

Yogo et al.

enabling efficient detection and resection of tiny nodules close to essential neurovascular tissue and the urinary sphincter during surgery. Although there have been concerns about the risk of increased rates of positive surgical margins during nerve-sparing operation, our probe might reduce the risk of overlooking positive surgical margins and improve the likelihood of achieving minimally invasive prostate resection without complications such as impotence and urinary incontinence. Consequently, we believe our fluorogenic probe for hepsin/matriptase is a promising candidate for application as a tool to enable intraoperative detection of even small prostate cancer nodules.

Experimental procedures Organic Synthesis Ac-KQLR-HMRG (Figure 1a) was synthesized according scheme S1 in the Supporting Information, wherein the synthetic protocols are also described. The purified probe was prepared as a 10 mM stock solution in DMSO and stored at -20 °C.

Spectroscopic measurements Absorption spectra were recorded on a UV-1650PC UV/Vis spectrometer (Shimadzu), and fluorescence spectra were recorded on a F4500 spectrometer (Hitachi) equipped with a R2658P photomultiplier tube (Hamamatsu Photonics) as a fluorescence detector. Construction of pH-dependent fitting curves and calculation of parameters including pKcycl were done with KaleidaGraph software (ver. 4.1).

Kinetics parameters of Ac-KQLR-HMRG Various concentrations of Ac-KQLR-HMRG were dissolved in 150 µl (total volume) of 50 mM tris buffer (pH 7.4). Hepsin and matriptase were added to the probe solution at 37°C, and the change in absorbance at 495 nm was measured. Initial reaction velocity was calculated, plotted against probe concentration, and fitted to a Michaelis-Menten curve. The kinetic parameters were calculated by use of the Michaelis-Menten equation: 𝑉=

𝑉max [S] 𝐾m + [S]

V, [S] and Km denote initial velocity, substrate concentration and the Michaelis constant, respectively.

Inhibitory effect of hexamidine for hepsin and matriptase Solutions of 10 μM Ac-KQLR-HMRG in 50 mM tris buffer at pH 7.4 with and without 20 µM hexamidine (inhibitor) were prepared. Hepsin or matriptase (10 ng) was added and the fluorescence increase during 30 min was evaluated. Fluorescence enhancement was calculated as follows: F30 12 ACS Paragon Plus Environment

Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

min/F0 min.

Cell culture Three established human prostate cancer cell lines (LNCaP, PC3, DU145) and one normal prostate cell line (PrEC) were used for fluorescence imaging. Cells were grown in culture medium at 37°C, in an atmosphere of 5% CO2 in air. LNCaP, PC3 and DU145 cells were grown in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum, 0.03% L-glutamine and PS (100 U/ml penicillin and 100 µg/ml streptomycin). PrEC was grown in PrEGM (Lonza) containing BPE 0.1%, hydrocortisone 0.1%, hEGF 0.1%, epinephrine 0.1%, transferrin 0.1%, insulin 0.1%, retinoic acid 0.1%, triiodothyronine 0.1%, and GA-1000 0.1%.

Confocal fluorescence imaging Confocal fluorescence images and bright-field images were taken with a TCS SP-5X (Leica) instrument equipped with a white-light laser and an objective lens (HCX PL APO CS 40x/1.25; Leica). The microscope was controlled by LAS AF software. Cells were seeded in each partition of an 8-well chamber (μ-Slide 8 well; Ibidi) and incubated with Ac-KQLR-HMRG (final concentration: 1 μM) or gGlu-HMRG (final concentration: 1 µM) for 5 min. For inhibition experiment, hexamidine was used at 20 μM final concentration. After preincubation with hexamidine for 1 min, the cultured cells were incubated for 5 min with Ac-KQLR-HMRG and images were captured.

Co-staining of LNCaP cells with Ac-KQLR-HMRG and Lysotracker Dual-color fluorescence imaging of LNCaP cells was conducted. Images were captured 15 min after incubation with Ac-KQRL-HMRG (final concentration: 1 µM) and Lysotracker Red (50 nM), respectively.

Preparation of intraperitoneal tumor-bearing model All experimental protocols were in accordance with the policies of the Animal Ethics Committee of The University of Tokyo. Human prostate cancer cell of the PC3 (3.0 x 106 or 8.5 x 106 cells in 150 µl PBS) and DU145 (3.8 x 106 cells in 150 µl PBS) lines were implanted by intraperitoneal injection into male mice (BALB/cA Jcl-nu/nu, Clea Japan Inc.). Experiments with tumor-bearing mice were performed after 10 – 14 days, when disseminated peritoneal nodules had reached about 0.5 – 2 mm in size.

Preparation of orthotopic tumor-bearing model All experimental protocols were in accordance with the policies of the Animal Ethics Committee of The University of Tokyo. Male 7 week old mice (BALB/cA Jcl-nu/nu, Clea Japan Inc.) were used. A 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 24

Yogo et al.

low midline abdominal incision of around 10 mm was made and human prostate cancer cell suspension (PC3 1.6 x 106 cells in 40 μl PBS) was injected into the prostate gland. Fluorescence imaging was performed after 14 days, when the tumor size had reached to approximately 5 - 7 mm.

Fluorescence imaging of prostate cancer in mice In the case of the intraperitoneal tumor-bearing mice, a solution of Ac-KQLR-HMRG in PBS (0 or 50 µM, 300 µl) was injected intraperitoneally into the abdominal cavity. After 5 min, the mice were sacrificed with CO2 and the abdominal cavity was exposed (Figure 4 and 5). Orthotopic tumor-bearing mice were also sacrificed with CO2 and the abdominal cavity was exposed. A 50 µM solution of Ac-KQLR-HMRG in 20 µl PBS was injected directly into the prostate gland and time-lapse imaging was performed (Figure 6a - c). In both cases, fluorescence images were obtained with the Maestro In-Vivo imaging system (PerkinElmer Inc.) equipped with a 465/30 nm band-pass filter for excitation. The tunable filter was automatically stepped in 10 nm increments from 500 to 800 nm, and images at each wavelength corresponding to each pseudo color were integrated to obtain pseudo real color images. Fluorescence images consisting of the spectra of HMRG and autofluorescence derived from digested pelleted feed or pancreas were also obtained as pseudo real color images. Fluorescence imaging of isolated prostate gland (Figure 6d) was done according to the above-described procedure with a different mouse from the one shown in Figure 6a, and the prostate gland including cancer tissues was isolated at 30 min after probe injection. Preparation of the paraffin-embedded slides and hematoxylin-eosin staining were done at Tokyo Central Pathology Laboratory Corporation (Tokyo, Japan).

Associated content Supporting Information: Additional experimental procedures, characterization data, kinetic properties and imaging data are presented.

Author Information Corresponding Author *E-mail: [email protected]. Tel: (+81)358413568. Fax: (+81)358413563. Notes: The authors declare no competing financial interest.

Acknowledgments This research was supported in part by AMED-CREST, by JST, PRESTO, by MEXT/JSPS KAKENHI grant numbers JP16H02606 and JP26111012 (to Y.U.), JP15H05951‘Resonance Bio’ (to M.K.), by JSPS Core-to-Core Program, A. Advanced Research Networks, by a grant from Hoansha Foundation (to Y.U.) and by Japan Foundation for Applied Enzymology (to M.K.). The authors 14 ACS Paragon Plus Environment

Page 21 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

thank H. Takahashi for advice on intracellular imaging and statistical analysis, and K. Yamamoto for kind support of ex vivo imaging experiments.

References (1)

Siegel, R. L., Miller, K. D., and Jemal, A. (2015) Cancer statistics, 2015. CA Cancer J Clin. 65, 5-29.

(2)

Heidenreich, A., Aus, G., Bolla, M., Joniau, S., Matveev, V. B., Schmid, H. P., and Zattoni, F. (2008) EAU guidelines on prostate cancer. Eur. Urol. 53, 68-80.

(3)

Wilt, T. J., Brawer, M. K., Jones, K. M., Barry, M. J., Aronson, W. J., Fox, S., Gingrich, J. R., Wei, J. T., Gilhooly, P., Grob, B. M., Nsouli, I., Iyer, P., Cartagena, R., Snider, G., Roehrborn, C., Sharifi, R., Blank, W., Pandya, P., Andriole, G. L., Culkin, D., and Wheeler, T. (2012) Radical prostatectomy versus observation for localized prostate cancer. N. Engl. J. Med. 367, 203-213.

(4)

Bill-Axelson, A., Holmberg, L., Garmo, H., Rider, J. R., Taari, K., Busch, C., Nordling, S., Häggman, M., Andersson, S.-O., Spångberg, A., Andrén, O., Palmgren, J., Steineck, G., Adami, H.-O., and Johansson, J.-E. (2014) Radical prostatectomy or watchful waiting in early prostate cancer. N. Engl. J. Med. 370, 932-942.

(5)

El-gabry, E. A., Halpern, E. J., Strup, S. E., and Gomella, L. G. (2001) Imaging prostate cancer: current and future applications. Oncology 15, 325-342.

(6)

Picchio, M., Mapelli, P., Panebianco, V., Castellucci, P., Incerti, E., Briganti, A., Gandaglia, G., Kirienko, M., Barchetti, F., Nanni, C., Montorsi, F., Gianolli, L., and Fanti, S. (2015) Imaging biomarkers in prostate cancer: role of PET/CT and MRI. Eur. J. Nucl. Med. Mol. Imaging 42, 644-655.

(7)

Nguyen, Q. T., and Tsien, R. Y. (2013) Fluorescence-guided surgery with live molecular navigation — a new cutting edge. Nat. Rev. Cancer 13, 653-662.

(8)

Constantinou, J., and Feneley, M. R. (2005) PSA testing: an evolving relationship with prostate cancer screening. Prostate Cancer and Prostatic Dis. 9, 6-13.

(9)

Esfahani, M., Ataei, N., and Panjehpour, M. (2015) Biomarkers for evaluation of prostate cancer prognosis. Asian Pac. J. Cancer Prev. 16, 2601-2611.

(10)

Chang, S. S. (2004) Overview of Prostate-Specific Membrane Antigen. Rev. Urol. 6, S13-S18.

(11)

Kelly, K. A., Setlur, S. R., Ross, R., Anbazhagan, R., Waterman, P., Rubin, M. A., and Weissleder, R. (2008) Detection of early prostate cancer using a hepsin-targeted imaging agent. Cancer Res. 68, 2286-2291.

(12)

Saleem, M., Adhami, V. M., Zhong, W., Longley, B. J., Lin, C.-Y., Dickson, R. B., Reagan-Shaw, S., Jarrard, D. F., and Mukhtar, H. (2006) A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 24

Yogo et al.

growth factor activator inhibitor-1. Cancer Epidemiol. Biomarkers Prev. 15, 217-227. (13)

Uhland, K. (2006) Matriptase and its putative role in cancer. Cell. Mol. Life Sci. 63, 2968-2978.

(14)

Dhanasekaran, S. M., Barrette, T. R., Ghosh, D., Shah, R., Varambally, S., Kurachi, K., Pienta, K. J., Rubin, M. A., and Chinnaiyan, A. M. (2001) Delineation of prognostic biomarkers in prostate cancer. Nature 412, 822-826.

(15)

Landers, K. A., Burger, M. J., Tebay, M. A., Purdie, D. M., Scells, B., Samaratunga, H., Lavin, M. F., and Gardiner, R. A. (2005) Use of multiple biomarkers for a molecular diagnosis of prostate cancer. Int. J. Cancer 114, 950-956.

(16)

Magee, J. A., Araki, T., Patil, S., Ehrig, T., True, L., Humphrey, P. A., Catalona, W. J., Watson, M. A., and Milbrandt, J. (2001) Expression Profiling Reveals Hepsin overexpression in prostate cancer. Cancer Res. 61, 5692-5696.

(17)

Stephan, C., Yousef, G. M., Scorilas, A., Jung, K., Jung, M., Kristiansen, G., Hauptmann, S., Kishi, T., Nakamura, T., Loening, S. A., and Diamandis, E. P. (2004) Hepsin is highly over expressed in and a new candidate for a prognostic indicator in prostate cancer. J. Urol. 171, 187-191.

(18)

Klezovitch, O., Chevillet, J., Mirosevich, J., Roberts, R. L., Matusik, R. J., and Vasioukhin, V. (2004) Hepsin promotes prostate cancer progression and metastasis. Cancer Cell 6, 185-195.

(19)

Wu, Q., and Parry, G. (2007) Hepsin and prostate cancer. Front. Biosci. 12, 5052-5059.

(20)

Warren, M., Twohig, M., Pier, T., Eickhoff, J., Lin, C.-Y., Jarrard, D., and Huang, W. (2009) Protein expression of matriptase and its cognate inhibitor HAI-1 in human prostate cancer: a tissue microarray and automated quantitative analysis. Appl. Immunohistochem. Mol. Morphol. 17, 23-30.

(21)

Riddick, A. C. P., Shukla, C. J., Pennington, C. J., Bass, R., Nuttall, R. K., Hogan, A., Sethia, K. K., Ellis, V., Collins, A. T., Maitland, N. J., Ball, R. Y., and Edwards, D. R. (2005) Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues. Br. J. Cancer 92, 2171-2180.

(22)

Dorn, J., Beaufort, N., Schmitt, M., Diamandis, E. P., Goettig, P., and Magdolen, V. (2014) Function and clinical relevance of kallikrein-related peptidases and other serine proteases in gynecological cancers. Crit. Rev. Clin. Lab. Sci. 51, 63-84.

(23)

Förbs, D., Thiel, S., Stella, M. C., Stürzebecher, A., Schweinitz, A., Steinmetzer, T., Stürzebecher, J., and Uhland, K. (2005) In vitro inhibition of matriptase prevents invasive growth of cell lines of prostate and colon carcinoma. Int. J. Oncol. 27, 1061-1070.

(24)

Napp, J., Dullin, C., Müller, F., Uhland, K., Petri, J. B., van de Locht, A., Steinmetzer, T., and Alves, F. (2010) Time-domain in vivo near infrared fluorescence imaging for evaluation of matriptase as a potential target for the development of novel, inhibitor-based tumor therapies. Int. J. Cancer 127, 1958-1974.


Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Yogo et al.

(25)

Darragh, M. R., Schneider, E. L., Lou, J., Phojanakong, P. J., Farady, C. J., Marks, J. D., Hann, B. C., and Craik, C. S. (2010) Tumor detection by imaging proteolytic activity. Cancer Res. 70, 1505-1512.

(26)

Nakajima, T., Mitsunaga, M., Bander, N. H., Heston, W. D., Choyke, P. L., and Kobayashi, H. (2011) Targeted, activatable, in vivo fluorescence imaging of prostate-specific membrane antigen (PSMA) positive tumors using the quenched humanized J591 antibody–indocyanine green (ICG) conjugate. Bioconjugate Chem. 22, 1700-1705.

(27)

Sakabe, M., Asanuma, D., Kamiya, M., Iwatate, R. J., Hanaoka, K., Terai, T., Nagano, T., and Urano, Y. (2013) Rational design of highly sensitive fluorescence probes for protease and glycosidase based on precisely controlled spirocyclization. J. Am. Chem. Soc. 135, 409-414.

(28)

Urano, Y., Sakabe, M., Kosaka, N., Ogawa, M., Mitsunaga, M., Asanuma, D., Kamiya, M., Young, M. R., Nagano, T., Choyke, P. L., and Kobayashi, H. (2011) Rapid cancer detection by topically spraying a γ-glutamyltranspeptidase–activated fluorescent probe. Sci. Transl. Med. 3, 110ra119.

(29)

Ueo, H., Shinden, Y., Tobo, T., Gamachi, A., Udo, M., Komatsu, H., Nambara, S., Saito, T., Ueda, M., Hirata, H., Sakimura, S., Takano, Y., Uchi, R., Kurashige, J., Akiyoshi, S., Iguchi, T., Eguchi, H., Sugimachi, K., Kubota, Y., Kai, Y., Shibuta, K., Kijima, Y., Yoshinaka, H., Natsugoe, S., Mori, M., Maehara, Y., Sakabe, M., Kamiya, M., Kakareka, J. W., Pohida, T. J., Choyke, P. L., Kobayashi, H., Ueo, H., Urano, Y., and Mimori, K. (2015) Rapid intraoperative visualization of breast lesions with γ-glutamyl hydroxymethyl rhodamine green. Sci. Rep. 5, 12080.

(30)

Hino, H., Kamiya, M., Kitano, K., Mizuno, K., Tanaka, S., Nishiyama, N., Kataoka, K., Urano, Y.,

and

Nakajima,

J.

(2016)

Rapid

cancer

fluorescence

imaging

using

a

γ-glutamyltranspeptidase-specific probe for primary lung cancer. Transl. Oncol. 9, 203-210. (31)

Barré, O., Dufour, A., Eckhard, U., Kappelhoff, R., Béliveau, F., Leduc, R., and Overall, C. M. (2014) Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs) using PICS with proteome-derived peptide libraries. PLoS ONE 9, e105984.

(32)

Webber, M. M., Bello, D., and Quader, S. (1997) Immortalized and tumorigenic adult human prostatic epithelial cell lines: Characteristics and applications part 2. Tumorigenic cell lines. Prostate 30, 58-64.

(33)

Enyedy, I. J., Lee, S.-L., Kuo, A. H., Dickson, R. B., Lin, C.-Y., and Wang, S. (2001) Structure-based approach for the discovery of bis-benzamidines as novel inhibitors of matriptase. J. Med. Chem. 44, 1349-1355.

(34)

Gallagher, F. A., Kettunen, M. I., Day, S. E., Hu, D. -E., Ardenkjær-Larsen, J. H., Zandt, R., Jensen, P. R., Karlsson, M., Golman, K., Lerche, M. H. et al. (2008) Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate. Nature 453, 940-943.

(35)

Petrakis, D., Pentheroudakis, G., Kamina, S., Pappa, L., Papadiotis, E., Malamou-Mitsi, V., and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 24

Yogo et al.

Pavlidis, N. (2015) An unusual presentation of a patient with advanced prostate cancer, massive ascites and peritoneal metastasis: Case report and literature review. J. Adv. Res. 6, 517-521. (36)

Healy, J. C. (2001) Detection of peritoneal metastases. Cancer Imaging 1, 4-12.

(37)

Kyriazi, S., Kaye, S. B., deSouza, N. M. (2010) Imaging ovarian cancer and peritoneal metastases - current and emerging techniques. Nat. Rev. Clin. Oncol. 7, 381-393.

(38)

Sanda, M. G., Dunn, R. L., Michalski, J., Sandler, H. M., Northouse, L., Hembroff, L., Lin, X., Greenfield, T. K., Litwin, M. S., Saigal, C. S., Mahadevan, A., Klein, E., Kibel, A., Pisters, L. L., Kuban, D., Kaplan, I., Wood, D., Ciezki, J., Shah, N., and Wei, J. T. (2008) Quality of life and satisfaction with outcome among prostate-cancer survivors. N. Engl. J. Med. 358, 1250-1261.

(39)

Vaidya, A., Hawke, C., Tiguert, R., Civantos, F., and Soloway, M. (2001) Intraoperative T staging in radical retropubic prostatectomy: is it reliable? Urology 57, 949-954.

(40)

Ficarra, V., Novara, G., Rosen, R. C., Artibani, W., Carroll, P. R., Costello, A., Menon, M., Montorsi, F., Patel, V. R., Stolzenburg, J.-U., Van der Poel, H., Wilson, T. G., Zattoni, F., and Mottrie, A. (2012) Systematic review and meta-analysis of studies reporting urinary continence recovery after robot-assisted radical prostatectomy. Eur. Urol. 62, 405-417.

(41)

Kojima, Y., Takahashi, N., Haga, N., Nomiya, M., Yanagida, T., Ishibashi, K., Aikawa, K., and Lee, D. I. (2013) Urinary incontinence after robot-assisted radical prostatectomy: Pathophysiology and intraoperative techniques to improve surgical outcome. Int. J. Urol. 20, 1052-1063.


Development of an Activatable Fluorescent Probe ... - ACS Publications

Recommend Documents