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A novel hexosaminidase-targeting fluorescence probe for visualizing human colorectal cancer Hiroyuki Matsuzaki, Mako Kamiya, Ryu John Iwatate, Daisuke Asanuma, Toshiaki Watanabe, and Yasuteru Urano Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00037 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 25, 2016

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Bioconjugate Chemistry

Bioconjugate Chemistry (Article)

Title: A novel hexosaminidase-targeting fluorescence probe for visualizing human colorectal cancer Authors: Hiroyuki Matsuzaki,a,c Mako Kamiya,a,d Ryu J. Iwatate,a Daisuke Asanuma,a Toshiaki Watanabe,a,c Yasuteru Urano,a,b,e,* a

Graduate School of Medicine, b Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, c Department of Surgical Oncology, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, d PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, e CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.

Author information Corresponding author *E-mail: [email protected]. Tel: (+81)358413601. Fax: (+81)358413563. Notes The authors declare no competing financial interest.

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Abstract Precise tumor diagnosis and evaluation of disease extent are crucial for treatment of solid cancers. In order to complement the limited ability of the unaided human eye to discriminate tumor tissue and normal tissue, we have developed a series of fluorescence probes activatable specifically in cancer tissues. Here, we describe the design, synthesis and application of a new fluorescence probe targeting hexosaminidase (HMRef-βGlcNAc), which is located in lysosomes and is overexpressed in several carcinomas, including colorectal cancer. This probe could sensitively detect intracellular hexosaminidase activity in human colorectal cancer cell lines, and could visualize tiny metastatic nodules (smaller than 1 mm) in a mouse model of disseminated human peritoneal colorectal cancer (HCT116). In human colorectal cancer specimens obtained at surgery, the probe showed high tumor sensitivity/specificity, together with a high tumor-to-normal signal ratio. HMRef-βGlcNAc is a promising candidate for clinical application during surgical or endoscopic procedures to treat colorectal cancer.

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Introduction Colorectal cancer is the third most common cancer in men and the second in women worldwide1. Colonoscopy is the gold standard for detection of colorectal cancer, and it is well known that early detection and endoscopic removal of colorectal polyps reduce mortality from colorectal cancer

2,3

. On the other hand, a systematic review

indicated that about 20 to 30% of colorectal polyps are overlooked during colonoscopy4. In order to improve tumor detection and characterization, various imaging techniques have been developed. For example, dye-spray-based chromoendoscopy (CE) slightly improved the detection rate of adenoma5,6. Narrow-band imaging (NBI) colonoscopy has also been attracting attention, but a recent systematic review and meta-analysis concluded that NBI colonoscopy does not increase the detection rate of adenoma and polyps7,8. Thus, we require a better imaging technique for diagnosis of colorectal neoplasms. Fluorescence imaging is a promising approach for clinical cancer imaging, because of its high sensitivity, high spatial resolution, rapidity and absence of ionizing radiation. Recently, we have developed an activatable fluorescence probe for γ-glutamyl transpeptidase (GGT), gGlu-HMRG, and reported its practical utility in cancer imaging9–12. However, we subsequently found that gGlu-HMRG was insufficiently effective with surgical specimens of human colorectal carcinoma (unpublished data). This prompted us to search for another potential target for fluorescence imaging of colorectal cancer. Hexosaminidase is a dimeric lysosomal enzyme involved in the hydrolysis of terminal N-acetyl-D-hexosamine residues in GM2 ganglioside13–15. There are three isozymes, formed through the combination of subunits α and β: Hex A (αβ heterodimer), Hex B (ββ homodimer) and Hex S (αα homodimer)16. Subunits α and β are encoded by separate genes, HEXA and HEXB, respectively. Mutations in HEXA or HEXB that cause functional impairment are associated with hereditary neurodegenerative diseases (Tay-Sachs disease, Sandhoff disease) due to accumulation of GM2 gangliosides in nerve cells17–19. On the other hand, hexosaminidase is overexpressed in various carcinomas and tumor animal models. Mian et al. reported elevated activities of several glycosidases, including hexosaminidase, in dimethylhydrazine-induced colon tumors in rats20,21. In addition, hexosaminidase activity is increased in human breast and colorectal carcinomas22,23. Thus, we considered hexosaminidase to be a promising target for cancer imaging. Therefore, in this work, we designed and synthesized a novel fluorescence probe targeting hexosaminidase, HMRef-βGlcNAc, and examined its usefulness by means of fluorescence imaging studies with cultured cells, and in a mouse model of peritoneal metastasis, and human colorectal cancer samples.

Results Development of novel fluorescence activatable probe for hexosaminidase We first designed and synthesized a new fluorescence probe for hexosaminidase, HMRef-βGlcNAc, by replacing the β-galactoside moiety of our recently developed probe for β-galactosidase, HMRef-βGal24, with

N-acetyl-D-hexosamine (Figure 1). HMRef-βGlcNAc exhibited almost no fluorescence at pH 7.4 due to a strong preference for the spirocyclized form, as expected, and showed a drastic fluorescence enhancement of up to 1000-fold upon reaction with hexosaminidase (Figure 2, Table 1, Figure S1). Furthermore, we confirmed that HMRef-βGlcNAc works as a fluorescent probe for hexosaminidase even in the presence of mouse serum or BSA (Figure S2, Table S1). HPLC analyses confirmed that HMRef-βGlcNAc is converted to the highly fluorescent 3



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Bioconjugate Chemistry (Article) hydrolysis product HMRef by the enzymatic reaction (Figure S3). We also confirmed that HMRef-βGlcNAc is stable under physiological conditions in the absence of hexosaminidase (Figure 2, Figure S2).

Figure 1. (a) Enzymatic reaction of our newly developed fluorescence probe for hexosaminidase, HMRef-βGlcNAc. (b) Proposed mechanism of HMRef-βGlcNAc activation in lysosomes. HMRef-βGlcNAc reacts with up-regulated hexosaminidase in lysosomes of cancer cells, yielding a highly fluorescent product, HMRef.

Table 1. Photochemical properties of HMRef-βGlcNAc. Absorption maximum (nm) pH 2.0

a

a

pH 7.4

Fluorescence quantum yield

Emission maximum (nm)

b

pH 2.0

a

pH 7.4

c b

pH 2.0

a

pKa

pKcycl

pH 7.4 b

HMRef24

479

498

515

518

0.716

0.777

4.4

10.2

HMRef-βGlcNAc

482

n.d.

519

n.d.

0.664

n.d.

n.d.

4.4

b

c

Measured in 0.2 M sodium phosphate buffer, pH 2.0. Measured in 0.2 M sodium phosphate buffer, pH 7.4. Absolute fluorescence

quantum efficiency. n.d.: not determined.

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Figure 2. (a) Absorption and fluorescence spectra of HMRef-βGlcNAc before and after enzymatic reaction with hexosaminidase. (b) Time course of fluorescence intensity change accompanying enzymatic reaction of HMRef-βGlcNAc with hexosaminidase. Hexosaminidase (2 U) was added at 100 sec to a 1 µM solution of HMRef-βGlcNAc in phosphate-buffered saline, pH 7.4, at room temperature.

Live-cell imaging of hexosaminidase activity To examine whether HMRef-βGlcNAc can detect hexosaminidase activity in living cancer cells, we applied it to four cultured human colorectal cancer cell lines (HCT116, HT29, SW480 and DLD1) and conducted confocal fluorescence imaging. Time-dependent increase of fluorescence was observed in each of the cell lines, although the degree of increase differed among them (Figure 3a,b). No apparent cytotoxicity was observed during incubation with HMRef-βGlcNAc for 30 minutes. We also conducted spectral scanning imaging of each cell line and observed that the fluorescence spectra corresponded with those of cells loaded with HMRef (Figure S4). Thus, we confirmed that the fluorescence signal is caused by the enzyme-catalyzed hydrolysis of HMRef-βGlcNAc, not by a mere pH drop in lysosomes. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that the fluorescence intensities obtained in live-cell imaging of these cell lines were consistent with the relative mRNA levels of HEX (Figure 3c). Next, to confirm substrate selectivity of HMRef-βGlcNAc for hexosaminidase, we silenced the HEXA or HEXB gene with small interfering RNA (siRNA), and confirmed a decrease in fluorescence intensity in each cell line, ranging from 60 to 80% compared with control negative siRNA-transfected cells (Figure S5). These results suggested that the fluorescence signal obtained in live-cell imaging with HMRef-βGlcNAc reflects the level of hexosaminidase activity in colorectal cancer cells.

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Figure 3. (a) Fluorescence confocal imaging of human colorectal cancer cell lines (HCT116, HT29, SW480, and DLD1) incubated with HMRef-βGlcNAc for 1, 10, 20 and 30 minutes. Differential interference contrast (DIC) image were captured. Scale bars represent 100 µm. (b) Quantified fluorescence intensities of human colon cancer cell lines loaded with HMRef-βGlcNAc at each time point. Data are shown as mean ± SD (n=10). (c) Relative HEXA and HEXB expression levels measured by qRT-PCR in human colorectal cancer cell lines. Data are shown as mean + SD (n=3).

Lysosomal hexosaminidase activities in cancer cells Next, we performed immunofluorescence staining of these cell lines to confirm the subcellular localization of hexosaminidase protein. We observed bright small vesicles in the cytoplasm, confirming that hexosaminidase protein is well localized in lysosomes (Figure 4a). Comparison of the enzymatic activities in cell lysates at different pH values (pH 5.0 and 7.4) showed that hexosaminidase activity was higher at acidic pH (Figure 4b; note that the pH was adjusted to 7.4 before measuring fluorescence intensities, even after incubation at pH 5.0). Since lysosome is an acidic compartment, it is reasonable that hexosaminidase exhibits higher activity at pH 5.0 than pH 7.4. These results confirmed that HMRef-βGlcNAc reacts with hexosaminidase in lysosomes of colorectal cancer cells, and that its fluorescence intensity reflects the expression level of hexosaminidase.

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Figure 4. (a) Immunofluorescence staining of human colorectal cancer cell lines by anti-HEXA and anti-HEXB antigens with negative control. Scale bars represent 25 µm. (b) Fluorescence intensities of human colorectal cancer cell lysates prepared 30 minutes after application of 100 µM HMRef-βGlcNAc. The background intensities of the control cell-lysate-free wells were subtracted. Data are shown as mean + SD (n=3).

Ex vivo fluorescence imaging of mouse model In order to examine the suitability of HMRef-βGlcNAc for cancer imaging, we prepared a mouse model of peritoneal metastasis of colon cancer. Although we tried to establish mouse models of all four cell lines, peritoneal nodules developed well only in mice injected with HCT116 cells, and not in mice injected with HT29, SW480 or DLD1 cells. Therefore, we used the HCT116 mouse model for further imaging experiments. At 30 minutes after intraperitoneal administration of HMRef-βGlcNAc to a mouse model of peritoneal metastasis of HCT116 cells, peritoneal metastases inside the peritoneal cavity were visualized clearly and specifically, including tiny nodules of less than 1 mm in diameter on the mesentery, which cannot be detected with the naked eye (Figure 5a,b). Further, we confirmed that the fluorescence-positive peritoneal nodules were histopathologically cancerous (Figure 5c). Thus, HMRef-βGlcNAc has clear potential for fluorescence-guided tumor diagnosis.

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Figure 5. Fluorescence imaging of HCT116 xenograft model in nude mouse: (a) gross appearance of internal surface of peritoneal wall (b) resected intestine and mesentery. A: white light, B: fluorescence image, C: pseudo real color image. Green and red circle indicate ROIs used for spectral unmixing D: unmixed image. E: spectra of ROIs for unmixing. (c) Hematoxylin-eosin staining of a resected fluorescence-positive peritoneal nodule of mouse. Scale bars indicate 0.25 mm. A: Low-power field. B: Magnified image of the red square in A.

Ex vivo fluorescence imaging of human colorectal cancer specimen Next, we applied HMRef-βGlcNAc to surgical specimens from colorectal cancer patients in order to further examine its potential for clinical usage. In the example shown in Figure 6a, the tumor lesion was clearly visualized; fluorescence intensity started increasing within 3 minutes after the probe application, and kept increasing during the observation (Figure 6a,C). The tumor lesion was clearly imaged with a commercial digital camera under blue-light excitation (Figure 6a,D). Immunohistochemistry showed positive staining at the tumor site (Figure 6b). The ROC curve of fluorescence intensity increments demonstrated 80% sensitivity and 90% specificity (n=10) (Figure S6). Though some autofluorescence was seen in normal mucosa, the fluorescence increase in tumor lesions during 30 minutes was about 2-6 times higher than that in normal mucosa (Table S2), suggesting that this probe has potential 8


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Bioconjugate Chemistry (Article) for clinical use.

Figure 6. (a) Application of HMRef-βGlcNAc to surgical specimen of colorectal cancer. A: Preoperative endoscopic finding. A 15-mm tumor was seen at the patient’s lower rectum. B: Gross appearance of the specimen under white light. Tumor location is marked with India ink. Scale bar indicates 1 cm. C: Fluorescence images 3, 10 and 20 minutes after the probe application. D: Gross appearance of the specimen 20 minutes after HMRef-βGlcNAc application, pictured with a commercial digital camera fitted with a 515 nm long-pass filter under blue light (445-490 nm). The red line indicates the cut line in the preparation of the pathological specimen shown in (b). (b) Immunohistochemical staining of the surgical specimen. Scale bars indicate 1 mm. A: Hematoxylin-eosin staining. B: Staining with anti-HEXA antibody. C: Magnified image of the blue square in B.

Discussion In this study, we focused on a lysosomal enzyme, hexosaminidase, which is overexpressed in several cancer tissues including human colorectal cancer20–23, and developed a new fluorescence probe, HMRef-βGlcNAc. This probe could detect different intracellular hexosaminidase activities in human colorectal cancer cell lines, and we succeeded in applying it for fluorescence imaging of cancer cells not only in an animal model, but also in human colorectal cancer specimens. There are few reports on enzyme-targeting fluorescence imaging of tumors in actual clinical specimens11,12, and as far as we know, this is the first report of fluorescence imaging of human spontaneous colorectal cancer. Although the number of colorectal cancer specimens was limited (10 cases), our new probe showed relatively high sensitivity (80%) and specificity (90%); it appears to be a promising candidate for clinical use. 9



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Bioconjugate Chemistry (Article) In the treatment of colorectal cancer, early detection and resection are very important for prevention of cancer death. The prognosis of early colorectal cancer is good, with the 5-year survival rate exceeding 90%; in particular, intramucosal carcinoma (Stage 0) can be radically treated by endoscopic resection, which is far less invasive than surgical operation. Therefore, practical diagnostic techniques are urgently needed for early detection of colorectal cancer. Although various endoscopic imaging techniques have been reported, including CE, NBI, endomicroscopy, etc.25, they each have significant limitations. On the other hand, our new fluorescence probe should easily reach the pan-colonic mucosa if topically administered with a bowel-cleansing agent before colonoscopy. This method of administration would ensure sufficient time for the probe to react with the target enzyme so that cancerous regions can be fluorescently visualized during operation. Thus, our probe is expected to offer clear advantages over existing endoscopic imaging methods as a simple, noninvasive tool for early detection of colorectal cancer and as an aid to accurate resection. On the other hand, advanced cancer also presents diagnostic problems. Lymph node and peritoneal metastases are clinically diagnosed by means of preoperative imaging examinations such as CT, MRI and PET-CT. However, it is not necessarily easy to translate such preoperative diagnostic information to the actual operative situation, using only the surgeon’s unaided eye and tactile sensation. In particular, advanced low rectal cancer is associated with lateral pelvic lymph node (LPLN) metastasis in approximately 15% of cases26–29, and radical surgery with LPLN dissection has a rather high urogenital complication rate, because of damage to the hypogastric nerves or pelvic nerve plexuses during surgery30. Peritoneal metastasis exists in approximately 7% of colorectal cancer patients at primary surgery, and is found in approximately 4% to 19% of patients during follow-up after curative surgery31. The prognosis of peritoneal metastasis has been improving thanks to modern systemic chemotherapy, including molecular target drugs, but long-term survival is still limited in patients with diffuse dissemination. This is partly because tiny metastatic nodules are hard to detect with existing diagnostic imaging methods. A few institutions have reported cytoreductive surgery combined with total peritonectomy and perioperative intraperitoneal chemotherapy; however, the operative procedure is highly invasive32,33. If our fluorescence probe could distinguish cancerous lesions from surrounding normal tissue intraoperatively, it would enable more accurate and less invasive selective resection of metastatic lesions with improved curability, in line with our present observations of peritoneal metastasis. Another diagnostic problem arises in colitis-associated cancer. Ulcerative colitis (UC) patients have an increased risk of colorectal cancer, because of chronic persistent colitis. A recent meta-analysis found that the overall risk of colorectal cancer in 181,923 UC patients was 1.69/1,000/year34, and colonoscopic surveillance is recommended for high-risk UC patients with a large extent and long duration of active colitis35–39. The problem here is that colitis-associated cancer or dysplasia sometimes takes the form of non-polypoid, non-adenoma-like lesions, which are hard to detect by conventional white-light endoscopy. At present, random biopsies of pan-colonic mucosa combined with targeted biopsy using dye-based chromoendoscopy are usually conducted40, but a definitive diagnostic method has yet to be developed. Once colitis-associated cancer or high-grade dysplasia is diagnosed, total proctocolectomy, which is highly invasive and requires changes in diet and bowel habits, is indicated. Our fluorescence probe might bring additional information to existing diagnostic modalities and hence, be of benefit to UC patients. 10


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Bioconjugate Chemistry (Article) One of the possible limitations in clinical application of our new probe is that the fluorescence signal might be subject to interference from autofluorescence of background tissue in the VIS range. However, the two signals can be distinguished by spectral unmixing, since the fluorescence spectrum of the probe is different from that of autofluorescence, as shown in Figure 5. In addition, the probe targets enzymatic activity, and therefore a time-dependent fluorescence increase is observed at lesions, and the fluorescence intensity becomes much greater than that of autofluorescence. Indeed, we have previously reported successful fluorescence imaging in vivo by using probes operating in the VIS range and targeted to other enzymes

9,10,41

. In future clinical application during

colonoscopy, options for topical delivery of the probe might include oral co-administration with the bowel-cleansing agent or spraying onto the internal surface of the intestinal tract from the forceps hole of an endoscope. In conclusion, we have developed a novel activatable probe, HMRef-βGlcNAc, and confirmed its usefulness in cell experiments and in an animal model. Its potential usefulness for clinical cancer imaging was also supported by the results of studies on human colorectal cancer specimens, though the number of cases was limited. We are acquiring a larger number of clinical specimens, including endoscopic submucosal dissection (ESD) samples, for further evaluation of the usefulness of this probe for early detection of carcinoma and adenoma.

Experimental Procedures Optical properties and in vitro enzyme reaction UV-visible spectra were obtained on a Shimadzu UV-2450 or a Shimadzu UV-1800. Fluorescence spectroscopic studies were performed on a Hitachi F-7000. Absolute fluorescence quantum efficiency was determined with an absolute PL quantum yield spectrometer, Quantaurus-QY (Hamamatsu Photonics). The probe was dissolved in dimethyl sulfoxide (DMSO, fluorometric grade, Dojindo) to obtain stock solutions. Optical properties of probes were examined in 0.2 M sodium phosphate buffer containing 0.1% (v/v) DMSO as a cosolvent. These buffers were prepared by mixing 0.2 M solutions of disodium hydrogenphosphate (Na2HPO4), sodium dihydrogenphosphate (NaH2PO4), and phosphoric acid. For in vitro enzyme reaction, DMSO stock solution was diluted with phosphate-buffered saline (PBS), pH 7.4, to a final concentration of 1 µM. Fluorescence of the solution before/after addition of hexosaminidase (2 U) was measured every 1 second (excitation wavelength 498 nm, emission wavelength 518 nm). Hexosaminidase from Canavalia ensiformis (Jack bean) was used for this assay.

Cell lines and culture conditions Four human colorectal cancer adenocarcinoma-derived cell lines (HCT116, HT29, SW480, and DLD1) were purchased from American Type Culture Collection (VA, USA). HCT116 and HT29 were cultured in McCoy’s 5A (Gibco, Life Technologies, NY, USA) and maintained at 37 °C in a humidified incubator with 5% CO2 in air. DLD-1 was cultured in RPMI-1640 (11875093 Gibco BRL, Life Technologies, NY, USA). SW480 was cultured in Leibovitz’s L-15 (11415064 Gibco BRL, Life Technologies, NY, USA) without CO2. All media were supplemented with 10% fetal bovine serum (Bioscience Pty., Australia) and 1% penicillin/streptomycin (Gibco, Life Technologies, NY, USA).

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Bioconjugate Chemistry (Article) Live cell fluorescence microscopy For fluorescence microscopy, about 10,000 cells in 100 µL of medium were seeded in each well of an 8-well chamber (µ-Slide 8 well; ibidi) and cultured overnight. The medium was replaced with phenol red- and serum-free RPMI 1640 containing 10 µM HMRef-βGlcNAc. Cells were incubated at 37 °C, and fluorescence images were obtained at 1, 10, 20 and 30 minutes after probe application. Differential interference contrast (DIC) images and fluorescence images were captured with a confocal fluorescence microscope SP5 equipped with a white-light laser and an objective lens (HCX PL APO CS 63x/1.40 Oil, Leica). Excitation and emission wavelengths were 488 nm/500-600 nm.

Immunofluorescence staining of colorectal cancer cell lines Cells of the four cancer lines were seeded on 8-well µ-Slides (Ibidi) and cultured overnight. The medium was removed and cells were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Cells were rinsed with PBS to remove paraformaldehyde, incubated with 0.1% Triton X-100 for 10 minutes, and washed with PBS again to remove the detergent. For blocking, permeabilized cells were incubated in PBS with 1% BSA for an hour at room temperature, and then washed 3 times (5 minutes each) with PBS. Cells were incubated with primary antibody [HEXA antibody (H-40): sc-134577, or HEXB antibody (H-45): sc-134579; Santa Cruz Biotechnology, Inc., USA] (dilution rate: 1:50) for 60 minutes at room temperature, and washed again 3 times (5 minutes each) with PBS. Cells were incubated with secondary antibody [Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)] (dilution rate: 1:500) for 15 minutes at room temperature, and washed with PBS 3 times (5 minutes each). HMRef-βGlcNAc was added to each well (final concentration: 10 µM), and fluorescence microscopy was performed as above.

Lysate preparation Cultured colorectal cancer cells were washed twice with 2 ml of DPBS (Life Technologies), and 1 ml of CelLyticM (Sigma) was added. Incubation was continued for 15 minutes at room temperature on a shaker. The lysed cells were collected and centrifuged for 15 minutes at 12000 × g to pellet cellular debris. The supernatant was aliquoted into chilled test tubes and stored at -80 °C. Protein concentration in cell lysates was measured by Bradford protein assay and adjusted to 1 mg/ ml.

Measurement of hexosaminidase activity in lysate Experiments were performed on 96-well plates (BD Biosciences). To each well were added 10 µl of cell lysate and 90 µl of 0.2 M sodium phosphate buffer (pH 5.0 or pH 7.4) containing 111 µM HMRef-βGlcNAc (final concentration 100 µM). The plate was incubated at 37 °C for 30 minutes. To each well of the pH 5.0 groups, 20 µl of 1.0 M aqueous NaOH was added to quench the reaction (final pH 7.4). To the pH 7.4 groups, 20 µl of 0.2 M sodium phosphate buffer (pH 7.4) was added. Fluorescence intensity (Ex/Em = 498 nm/518 nm) was measured with a microplate reader (SH-8000; Corona) and the background intensity of the control cell-lysate-free wells was subtracted.

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Bioconjugate Chemistry (Article) Quantitative RT-PCR Cells of the four cancer lines were cultured on 10 cm culture dishes. The growth medium was removed and 8 ml TRIzol RNA Isolation Reagent (Gibco BRL, Life Technologies, NY, USA) was added. Cells were retrieved into centrifuge tubes. Chloroform (200 µl per milliliter of sample in TRIzol) was added, and each tube was shaken vigorously by hand for 15 seconds, incubated at room temperature for 3 minutes, and centrifuged at 12000 × g for 15 minutes at 4 °C. The supernatant aqueous phase was transferred to a new tube. Then, 500 µl of 100% isopropanol per 1 mL of TRIzol Reagent was added to the aqueous phase. The mixture was incubated at room temperature for 10 minutes, and then centrifuged at 12000 × g for 10 minutes at 4 °C. The supernatant was removed, and 1 mL of 75% ethanol per 1 mL of TRIzol reagent was added to the residue. The mixture was centrifuged at 7500 × g for 5 minutes at 4 °C. The supernatant was removed and the sample was air-dried at room temperature for 10 minutes. RNAase-free water (10 µl) was added to the RNA pellet, which was then incubated in a heating block set at 60 °C for 10 minutes. The RNA concentration of each sample was measured using NanoDrop 2000 (Thermo Scientific) and adjusted to 0.1 µg/µL. cDNA was synthesized using PrimeScript II 1st standard Synthesis Kit (C6210A; Takara Bio Inc., Japan) according to the manufacturer’s protocol, and reverse transcription reaction was performed as follows: annealing at 30 °C for 10 minutes, extension at 42 °C for 60 minutes, then deactivation at 95 °C for 5 minutes on a PCR thermocycler (Applied Biosystems Veriti® 96 well Thermal cycler, Life Technologies, USA). qRT-PCR assay was performed in triplicate in a 96-well plate with LightCycler 480 SYBR Green I Master (04707516001 Roche, Switzerland) with primers of HEXA, HEXB and GAPDH. The results were expressed as relative mRNA expression level, with GAPDH as the reference gene.

Tumor model of peritoneal implants All experimental protocols were in accordance with the policies of the Animal Ethics Committee of the University of Tokyo. HCT116 was implanted by intraperitoneal injection of 1 × 106 cells suspended in 300 µl of PBS into female nude mice (BALB/cA Jcl-nu/nu, Clea Japan Inc.). Experiments with tumor-bearing mice were performed after 2 weeks, when disseminated peritoneal implants had reached 0.5-3 mm in size.

Ex vivo fluorescence imaging of the animal model A solution of HMRef-βGlcNAc (100 µM) in 300 µL PBS was injected intraperitoneally into the abdominal cavity of HCT116 model mice. After 30 minutes, the mice were sacrificed and the abdominal cavity was exposed. Fluorescence images were obtained with a Maestro In-Vivo imaging system (CRi Inc.). A 300 W xenon lamp was used as the light source (fluence rate: 4-20 mW/cm2). For fluorescence imaging using HMRef-βGlcNAc, the blue-filter setting (excitation filter: 445 to 490 nm band-pass, emission filter: 515 nm long-pass) was used. The tunable filter was automatically stepped in 10 nm increments, from 500 to 800 nm, while the camera sequentially captured images at each wavelength interval (exposure time: 50 ms at each wavelength). For obtaining spectrally unmixed images, fluorescence spectra of the probe and of autofluorescence were assigned and displayed as different colors after unmixing (in Figure 5, the probe and autofluorescence are displayed in green and in red, respectively).

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Bioconjugate Chemistry (Article) Colorectal cancer samples Human colorectal cancer specimens were obtained from the Department of Surgical Oncology, The University of Tokyo Hospital. Before the study, all patients provided written informed consent for joining the ex vivo colorectal cancer fluorescence imaging study and having their medical charts reviewed. The Research Review Board of The University of Tokyo examined and approved our research protocol, which was in accordance with the Declaration of Helsinki.

Ex vivo fluorescence imaging of human colorectal cancer specimens Human colorectal cancer specimens covered by saline gauze were transferred from the operating room to the laboratory within 1-2 hours after resection. Each specimen was washed with lukewarm water to remove blood, feces and mucus from the mucosal surface. The tumor site and adjacent areas of normal mucosal surface were covered with non-woven gauze impregnated with a 50 µM solution of HMRef-βGlcNAc in PBS. The gauze was removed after 2-3 minutes. Fluorescence images were obtained with the Maestro In-Vivo imaging system as described above (in the animal model), before and 1, 3, 5, 10, 20 and 30 minutes after the probe application. Regions of interest (ROIs) in tumor and normal regions were drawn with the Maestro software: the brightest area in the tumor lesion was enclosed as the tumor ROI, and normal mucosa of average brightness as the normal ROI on the fluorescence image at 30 minutes. Change in fluorescence intensity was obtained by subtracting the value before the probe application from that at 30 minutes. Using these numerical values of fluorescence intensity, a receiver operating characteristics (ROC) curve was plotted and a cutoff value was derived from the ROC curve.

Immunohistochemistry of human colorectal cancer specimen The paraffin-embedded slide was soaked in a xylene bath and subjected to 6 exchanges of xylene (3 minutes each). The slide was rehydrated with 2 exchanges of 95% ethanol and 4 exchanges of 100% ethanol, for 2 minutes each. The slide was soaked in methanol containing 0.3% hydrogen peroxide and incubated for 25 minutes at room temperature, and then washed in pooled running water for 5 minutes, and in PBS 3 times for 5 minutes each. For antigen retrieval, the slide was placed in citric acid buffer, autoclaved at 120 °C for 5 minutes, and cooled slowly at room temperature for 10 minutes. The slide was washed in PBS 3 times and incubated in PBS with 5% BSA for 30 minutes at room temperature in a humidified chamber. Excess fluid was shaken off with a brisk motion. The primary antibody [HEXA antibody (H-40): sc-134577] was diluted 1:200 with PBS. Primary antibody solution (200 µl) was applied to each slide, and the slide was incubated at 4 °C overnight. The next day, the slide was washed in PBS 3 times. Secondary antibody reaction was performed using the EnVision kit (Dako ChemMate) according to the manufacturer’s protocol. The slide was washed in PBS 3 times, counterstained in a bath of Mayer's hematoxylin for 30 seconds, and washed in a pool of warm water. The slide was dehydrated through 6 exchanges of ethanol (95% twice, 100% 4 times) for 2 minutes each. The slide was washed in 6 changes of xylene and coverslipped using mounting solution.

Statistical analysis Statistical analysis was carried out using JMP software (SAS Institute Inc., Cary, NC). For the comparison of 14


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Bioconjugate Chemistry (Article) fluorescence intensities in siRNA knockdown experiments, the Mann-Whitney U test was used. For the ex vivo evaluation of human colorectal cancer specimens, a receiver operating characteristics (ROC) curve was plotted by using numerical values of fluorescence change and a cutoff value was derived from the ROC curve.

Associated content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental procedures. Figure S1. Fluorescence spectra of HMRef-βGlcNAc at various pH values in 0.2 M sodium phosphate buffer. Figure S2. Time course of fluorescence intensity change accompanying enzymatic reaction of HMRef-βGlcNAc with hexosaminidase in the presence of mouse serum. Figure S3. HPLC chromatogram of HMRef-βGlcNAc after reaction with hexosaminidase. Figure S4. Fluorescence spectra of live-cell imaging with HMRef-βGlcNAc or HMRef. Figure S5. Fluorescence imaging of human colon cancer cell lines transfected with HEXA- or HEXB-targeting siRNA or control siRNA. Figure S6. Receiver operating characteristics curve of HMRef-βGlcNAc in human colorectal cancer specimens. Table S1. Photochemical properties of HMRef-βGlcNAc in the presence of 4% (w/v) bovine serum albumin. Table S2. Increase in fluorescence intensity after 30 minutes at normal tissue and tumor lesion sites in surgically obtained human colorectal cancer specimens.

Acknowledgements This research was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research (KAKENHI), grant 26111012 to Y.U., grants 25870180 and 15H05951 to M.K.), by the Basic Research Program from the Japan Science and Technology Agency (to Y.U.), by JSPS Core-to-Core Program, A. Advanced Research Networks (to Y.U.), by The Daiichi-Sankyo Foundation of Life Science (grant to Y.U.), by Japan foundation for applied enzymology (to M.K.), and by stipends from JSPS and the Graduate Program for Leaders in Life Innovation (GPLLI) to R.J.I..

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Novel Hexosaminidase-Targeting Fluorescence ... - ACS Publications

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