Synthesis of 6 '-O-Lissamine-rhodamine B-Glucosamine as a Novel

Bioconjugate Chem. 2005, 16, 843−851

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Synthesis of 6′-O-Lissamine-rhodamine B-Glucosamine as a Novel Probe for Fluorescence Imaging of Lysosomes in Breast Tumors Kristine Glunde,* Catherine A. Foss, Tomoyo Takagi, Flonne Wildes, and Zaver M. Bhujwalla JHU ICMIC Program, The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Received February 19, 2005; Revised Manuscript Received May 17, 2005

Lysosomes contain multiple proteases, which play a crucial role in breast cancer invasion and metastasis. Noninvasive labeling of lysosomes in breast cancer cells and solid breast tumor models is therefore useful to study lysosomal trafficking and its role in invasion. We have synthesized a novel compound, 6′-O-lissamine-rhodamine B-glucosamine, to fluorescently label lysosomes, and evaluated the compound in human breast cancer cells in cell culture or in orthotopic human breast cancer models. We demonstrated that this novel compound biosynthetically labeled lysosomal proteins following addition to cell culture medium or following intravenous injection into mouse models of breast cancer. Fluorescence from 6′-O-lissamine-rhodamine B-glucosamine colocalized with several well-established lysosomal markers, such as lysosome-associated proteins 1 and 2 (LAMP-1 and -2) and CD63. We also demonstrated the feasibility of performing in vivo fluorescence imaging of 6′-O-lissaminerhodamine B-glucosamine to image lysosomes in human breast cancer models.

INTRODUCTION

Lysosomes are membranous organelles of ∼0.5 µm diameter, which contain several types of hydrolytic enzymes and maintain an acidic lumenal pH of ∼5 (1). Lysosomes are the terminal degradative compartment of mammalian cells but are also involved in antigen processing (2), bone remodeling (3), the regulation of growth factors (4), and plasma membrane repair (5). Tumors with high metastatic potential, such as Lewis lung carcinoma (6), melanoma (7), and several breast cancers (8, 9), exhibit increased lysosomal enzyme expression and activity (10). Breast cancer cells can also contain intracellular large acidic vesicles (LAVs,1 g 5 µM in diameter), carrying endocytosed extracellular matrix (ECM) components (11, 12). Breast cancer invasion and metastasis, which is the most life-threatening aspect of malignant breast cancer, requires proteolytic enzymes to degrade ECM proteins (13-15). Cathepsins D, B, and L, which are sequestered in lysosomal vesicles (16), were shown to play a crucial role in cancer invasion and metastasis (17-19). Lysosomal trafficking may be an important parameter affecting lysosomal enzyme release and endocytosis of ECM proteins. Solid tumors contain hypoxic * Correspondence to Kristine Glunde, Ph.D., Dept. of Radiology, Johns Hopkins University School of Medicine, 212 Traylor Bldg., 720 Rutland Ave., Baltimore, MD 21205. Tel: (410)-6142705, Fax: (410)-614-1948, E-mail: [email protected]. 1 Abbreviations: 6-O-dansyl-GlcNH , 6′-O-dansyl-glucosamine; 2 6-O-liss-GlcNH2, 6′-O-lissamine-rhodamine B-glucosamine; Boc, tert-butyloxycarbonyl; BODIPY, 4,4-difluoro-4-bora-3a,4a-diazas-indacene; d, day(s); DIC, differential interference contrast; DMAP, 4-dimethylaminopyridine; ECM, extracellular matrix; ESI, electrospray ionization; Et3N, triethylamine; FAB, fast atom bombardment; FOV, field of view; h, hour(s); HMEC, human mammary epithelial cell; LAMP, lysosome-associated membrane protein; LAV, large acidic vesicle; LSM, laserscanning microscopy; MeOH, methanol; NMR, nuclear magnetic resonance; SCID, severe combined immune deficient; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TLC, thin-layer chromatography.

and acidic regions, which may affect lysosomal trafficking and the release of degradative enzymes by cancer cells (20, 21). Extracellular acidosis was shown to alter lysosomal trafficking in human breast cancer cells (22), which most likely facilitated lysosomal enzyme release. Effective techniques to fluorescently label lysosomes and optically image lysosomal trafficking in solid tumors are a prerequisite to study the role of lysosomes in breast cancer invasion and metastasis in tumor models. In fixed tissues or cell specimens, lysosomes can be visualized by immunofluorescence staining using antibodies against a lysosomal protein of choice. Three wellestablished lysosomal marker proteins are lysosomeassociated membrane proteins 1 and 2 (LAMP-1 and -2) (23, 24) and CD63 (25). LAMPs are highly abundant in lysosomal membranes and have been detected in most human tissues (26). Lysosomes in live cell cultures have been fluorescently labeled with substrates for lysosomal hydrolytic enzymes, such as fluorescent casein, which carry a fluorophore that is quenched before but fluoresces after cleavage (27, 28). Acidotropic fluorescent probes are a second class of lysosomal markers for live cell imaging, which accumulate in acidic organelles because of their matching pKa values, e.g. neutral red (29), acridine orange (30), LysoTracker (31), and LysoSensor (32). Fluorescent probes such as fluorescent dextrans (33), that are endocytosed and accumulate in lysosomes, have been utilized to visualize lysosomes in live cell cultures. These techniques have limited specificity, since they label compartments based on characteristics not unique to lysosomes (34). We recently developed a technique to specifically label lysosomes biosynthetically in live breast cancer cell cultures using a dansylated glucosamine (34). This labeling technique is based on the fact that most lysosomal proteins, and particularly lysosomal membrane proteins, are highly glycosylated and frequently carry several N-linked glycans (35). These glycans consist of mannose, N-acetylglucosamine, fucose, galactose, and sialic acid moieties and are thought to protect lysosomal

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Figure 1. Schematic of the three step chemical synthesis of 6′-O-lissamine -rhodamine B-glucosamine (6-O-liss-GlcNH2) (4).

proteins from degradation by lysosomal proteases (36). We previously synthesized and characterized 6-O-dansylGlcNH2 (34), which localized to lysosomes by forming dansylated glycoproteins in live human mammary epithelial cells (HMECs) and live breast cancer cells. We demonstrated that biosynthetic labeling of lysosomes using 6-O-dansyl-GlcNH2 is feasible for imaging lysosomes in live HMECs or breast cancer cells by confocal fluorescence microscopy (34). In the present study, we synthesized and biologically characterized a novel fluorescently labeled glucosamine: 6′-O-lissamine-rhodamine B-glucosamine (6-Oliss-GlcNH2) for in vivo studies in mice. This newly synthesized fluorescent compound is nontoxic and can be administered intravenously in tumor-bearing mice and subsequently localizes to lysosomes in tumors by accumulating in lysosomal proteins. The emission maximum of 6-O-liss-GlcNH2 at 570 nm is suitable for in vivo optical imaging, since it lies within the window of low tissue absorption and autofluorescence. We assessed the uptake, relative distribution, and lysosomal localization of 6-O-liss-GlcNH2 in MCF-7 and MDA-MB-231 breast cancer cells in cultures and in severe combined immune deficient (SCID) mice bearing MCF-7 and MDA-MB-231 tumors in the mammary fat pad. MATERIALS AND METHODS

Cell Culture. Three human mammary epithelial cell (HMEC) lines representing different stages of malignancy were used in this study. MCF-12A, a spontaneously immortalized nonmalignant cell line established from MCF-12M mortal cells (37), was obtained from American Type Culture Collection (ATCC, Rockville, MD) and cultured in DMEM-Ham’s F12 medium (Invitrogen Corporation, Carlsbad, CA) supplemented as described previously (37). MCF-7, an estrogen-positive lowly metastatic mammary epithelial cancer cell line, was obtained from ATCC (Rockville, MD) and cultured in EMEM medium (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum and antibiotics (38). An invasive and metastatic human mammary epithelial cell line, MDA-MB-231 was provided by Dr. R. J. Gillies

(Arizona Health Sciences Center, Tuscon, AZ) and maintained in RPMI-1640 medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin (Invitrogen Corporation, Carlsbad, CA). The human breast cancer cell lines were originally isolated from pleural effusions of patients with breast cancer. All cells were kept at 37 °C in a humidified atmosphere of 5% CO2 in air. Tumor Model and Inoculations. MCF-7 or MDAMB-231 cells were inoculated in the upper left thoracic mammary fat pad of five female severe combined immune deficient (SCID) mice. A quantity of 2 × 106 tumor cells was inoculated in a volume of 0.1 mL of Hanks balanced solution (HBSS, Sigma, St. Louis, MO) at a concentration of 106 cells/0.05 mL. An amount of 0.18 mg of 60-day release 17β-Estradiol Pellets (Innovative Research of America, Sarasota, FL) was subcutaneously implanted near the left shoulder of SCID mice inoculated with MCF-7 cells. Mice weighed between 19- 24 g, and tumor sizes ranged between 75 mm3 and 375 mm3 when experiments were performed. All experimental animal protocols were approved by the Institutional Animal Care and Use Committee. Chemical Synthesis of 6′-O-Lissamine-rhodamine B-Glucosamine. All chemical reactions are shown in Figure 1. All reagents were purchased from Sigma unless otherwise noted and were of p.a. grade. 1-O-Boc, 2-N-Boc-β-D-glucosamine (2). Powdered sodium methoxide (500 mg, 9.98 mmol) and 6-(2-amino2-deoxy-β-D-glucopyranosyl) ether (2.0 g, 9.28 mmol) were added to a baked 25 mL round-bottom flask along with 10 mL of dry methanol. The resulting suspension was stirred for 5 min at room temperature under argon and then filtered over scintered glass. The solid was washed with an additional 10 mL of dry methanol and transferred to a clean 25 mL round-bottom flask. Triethylamine (1.03 g, 10.2 mmol) and di-tert-butyl pyrocarbonate (2.23 g, 10.2 mmol) were added to the flask along with 10 mL of dry methanol, and the thick, white suspension was stirred at room temperature under argon for 10 h. Progress was monitored by silica gel thin-layer chroma-

Imaging Lysosomes in Breast Tumors

tography (TLC) using 2-propanol/water/ammonium hydroxide (7:2:1 v/v/v) with ninhydrin (Fisher Scientific, Pittsburgh, PA) detection followed by baking. The starting material 1 displayed an Rf of 0.18. The N-Boc derivative displayed an Rf of 0.79. The reaction mixture was concentrated under reduced pressure and dissolved in 20 mL of dioxane/water (1:1 v/v). Triethylamine (1.03 g, 10.2 mmol), 4-(dimethylamino)pyridine (50 mg, 0.41 mmol), and di-tert-butyl pyrocarbonate (1.1 g, 5.1 mmol) were added, and the clear solution was stirred at room temperature under argon for 24 h. During this time, additional triethylamine (0.51 g, 5.1 mmol) and di-tertbutyl pyrocarbonate (1.1 g, 5.1 mmol) were added to the reaction. Progress was monitored by TLC using chloroform/ethyl acetate/methanol (5:2:1 v/v) with ninhydrin detection followed by baking. The N-Boc derivative displayed an Rf of 0.14, while the Rf of product 2 was 0.37. The reaction was concentrated under reduced pressure and 2 was purified by silica gel chromatography using chloroform/ethyl acetate/methanol (6:3:1 v/v) and resulted in a 48.2% yield. 6-O-Lissamine-rhodamine B-1-O-Boc,2-N-Boc-glucosamine (3). Dry 1-O-Boc,2-N-Boc-β-D-glucosamine (500 mg, 1.37 mmol) and Lissamine Rhodamine B sulfonyl chloride, mixed isomers (9-[4-(chlorosulfonyl)-2sulfophenyl]-3, 6-bis(diethylamino)-xanthylium internal salt) (1 g, 1.78 mmol, Molecular Probes, Eugene, OR), were added to a baked 50 mL round-bottom flask along with dry pyridine (20 mL). The resulting purple suspension was stirred at room temperature under argon for 10 h. The reaction was quenched by addition of a piece of ice followed by removal of the pyridine under reduced pressure. Analysis of this material by TLC using methanol/ methylene chloride (1:9 v/v) revealed the presence of product 3, which exhibits an Rf of 0.6. The product was purified by silica gel chromatography using methanol/ methylene chloride (2:8 v/v) to yield pure 3 at 62% yield. 6′-O-Lissamine-rhodamine B-glucosamine (4). Dry 3 (500 mg, 0.98 mmol) and dry trifluoroacetic acid (TFA) (10 mL) were stirred in a 50 mL round-bottom flask for 4 h at room temperature. The resulting bright red solution was then concentrated under reduced pressure to give a bright red oil. Analysis by TLC using methanol/ methylene chloride (2:8 v/v) revealed the presence of 4, which exhibits an Rf of 0.5-0.62 (mixed isomers). This oil was purified by silica gel chromatography using methanol/methylene chloride (2:8 v/v) and afforded final product 4 in 60% yield. 1H NMR (500 MHz, CD3OD): δ 8.68 (d, J ) 0.91 Hz, 1H), δ 8.08 (dd, J ) 7.94 Hz, 1.53 Hz, 1H), δ 7.38 (d, J ) 7.94 Hz, 1H), δ 7.15 (d, J ) 9.46 Hz, 2H), δ 7.01 (dd, J ) 9.46 Hz, 2.44 Hz, 2H), δ 6.93 (d, J ) 2.44 Hz, 2H), δ 3.67 (ddd, J ) 7.33 Hz, 7.32 Hz, 7.02 Hz, 2H), δ 3.21 (ddd, J ) 7.33 Hz, 7.32 Hz, 7.02 Hz, 5H), δ 1.30 (m, 20H). 13C NMR (100 MHz, CD3OD): δ 163.31 (q, JC-F ) 39.5 Hz), 159.87, 159.14, 157.58, 148.51, 146.48, 134.29, 133.52, 132.12, 129.02, 127.41, 115.91, 115.39, 97.41, 48.32, 47.28, 13.34, 9.71. Mass spectrum (FAB): m/z ) 720 Da. Absorbance max (Beckman DU 7500) ) 574 nm. In Vitro Incubation with 6′-O-Lissamine-rhodamine B-Glucosamine. In cell cultures, different concentrations of 6-O-liss-GlcNH2 (Figure 1, compound 4) were tested in the range of 250 µM to 2 mM. A concentration of 500 µM 6-O-liss-GlcNH2 was found to be sufficient for effective labeling. The time-course of 6-Oliss-GlcNH2 uptake and localization to lysosomes was assessed in MCF-12A, MCF-7, and MDA-MB-231 cells grown on chamber slides. Cells were incubated for time periods of 2-96 h in cell culture medium containing 500

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µM 6-O-liss-GlcNH2. Cells were fixed with 3% paraformaldehyde as described previously (22, 34). In Vivo Treatment with 6′-O-Lissamine-rhodamine B-Glucosamine. Female SCID mice bearing MCF-7 or MDA-MB-231 tumors were intravenously injected with 7.8 mg 6-O-liss-GlcNH2 (7.8 mg in 0.1 mL saline, sterile filtered). At 2, 24, 48, and 72 h postinjection, tumors were optically imaged in vivo using a home-built small animal fluorescene imaging system, following which mice were sacrificed and tumor, blood, liver, kidney, and muscle tissue were harvested. Freshly cut 1-mm thick tissue sections of harvested organs and blood were placed on microscope slides (Fisher Scientific, Pittsburgh, PA), covered with CoverWell imaging chambers (Grace BioLabs, Bend, OR) and optically imaged using fluorescence microscopes. The remaining tumors were immediately frozen using Tissue-Tek O.C.T. Compound (Sakura Finetek U.S.A. Inc, Torrance, CA) and Tissue-Tek cryomolds (Miles Inc., Elkhart, IN) and liquid nitrogen. In Vivo Optical Imaging of Breast Tumor Models. For in vivo imaging, the tumor region was shaved in anesthetized mice and the animal was lightly taped to minimize motion. A home-built small animal fluorescene imaging system equipped with a Mini Repro Camera Stand (Industria Fototecnica Firenze, Florence, Italy), a 300 W Gemini 300 high-intensity short arc light source with universal adaptor (Chiu Technical Corporation, Kings Park, NY), an optical fiber (Edmunds, Barrington, NJ), a filter cube containing a 510-560 nm band-pass exitation filter and a 575 nm long pass emission filter (Omega Optical, Inc., Brattleboro, VT), and a Nikon Coolpix 990 or Coolpix 5000 digital camera (Nikon, Tokyo, Japan) was used to obtain optical images of mice with tumor xenografts. Ex Vivo Optical Imaging and Relative Distribution. Fresh tissue sections (1-mm thick) of tumor, liver, kidney, and muscle as well as blood were imaged ex vivo in imaging chambers using bright-field and epi-fluorescence (528-553 nm excitation, 565 nm emission) microscopy using a Nikon eclipse E400 or TS100 attached to a Nikon Coolpix 990 or 5000 digital camera. Exposure times were kept constant for all organ sections and blood. Low power images of whole organ sections were acquired using a 1× lens. High power images were acquired using a 40× or 100× lens. Image analysis was performed using ImageJ (National Institutes of Health, Bethesda, MD). Images were converted to 8-bit gray-scale, the organ section was outlined, fluorescence intensity was measured, and the median fluorescence intensity per pixel was calculated. Immunofluorescence Staining. Tumors frozen in O.C.T. compound (Sakura Finetek U.S.A. Inc.) were sectioned at 15-µm thickness using a microtome cryostat (Microm Int., Walldorf, Germany) and attached to charged microscope slides (Fisher Scientific, Pittsburgh, PA). The 15-µm tumor sections were fixed using 3% paraformaldehyde. Fixed cells in chamber slides and fixed tumor sections were stained using monoclonal antibodies to LAMP-1 (H4A3), LAMP-2 (H4B4), or CD63 (ab1318, Abcam Inc., Cambridge, MA). hLAMP-1 and hLAMP-2 antibodies (26) were kindly provided by Dr. J. Thomas August, Johns Hopkins University, Baltimore, MD. LAMP-1, LAMP-2, and CD63 are three specific marker proteins localized in lysosomal membranes. Tumor sections and cells were incubated with a Cy5-labeled donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), and cell nuclei were counterstained with Hoechst H-33342 (Molecular


Glunde et al.

Figure 2. (a) Human MDA-MB-231 breast cancer cells exposed to 500 µM 6-O-liss-GlcNH2 for 24 h. Fluorescence from (a) 6-Oliss-GlcNH2 is displayed in green, and (b) CD63 immunofluorescence is displayed in red. Nuclei are displayed in blue. (c) The overlay of both images demonstrates the colocalization of 6-O-liss-GlcNH2 and CD63, evident by the yellow color. The field of view of all images is 56.2 µm × 56.2 µm.

Probes, Eugene, OR) and mounted using Faramount aqueous mounting medium (DakoCytomation, Carpinteria, CA). Confocal Laser-Scanning Fluorescence Microscopy. Fluorescence microscopy of fixed specimen was performed on a Zeiss LSM 510 META confocal laserscanning microscope (Carl Zeiss, Inc., Thornwood, NY) using a Plan-Apochromat 63x/1.4 oil immersion lens (Zeiss) (The Johns Hopkins University School of Medicine Microscope Facility, Director Dr. Douglas B. Murphy). 6-O-liss-GlcNH2 was excited with a 543 nm laser, and fluorescence emission was detected by a photomultiplier tube using a 565-595 nm band-pass filter. Simultaneously, H-33342 was excited with a 405 nm laser and the fluorescence emission was detected with a second photomultiplier by applying a 490 nm dichroic beam splitter and a 420-480 nm band-pass filter. Cy5 was excited simultaneously with a 633 nm laser and the fluorescence emission was detected with a third photomultiplier by using a 635 nm dichroic beam splitter and a 650 nm long pass filter, to achieve simultaneous detection of all three fluorophores. Confocal Z-sections of 1 µm thickness were imaged. Colocalization coefficients were measured using the LSM Software Release 3.2 (Zeiss). Western-Blot Analysis. Cells were cultured in 100mm tissue culture dishes until they reached 60-70% confluence. Following treatment with 500 µM 6-O-lissGlcNH2 for 2 or 4 days, cells were scraped and homogenized with lysis buffer containing protease inhibitors as previously described (34). A 50 µg amount of total protein was loaded in each lane of a 9% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and proteins were resolved by electrophoresis (34). Approximate protein molecular weights were determined by comparison with a molecular weight marker (BenchMark, Life Technologies, Rockville, MD). Fluorescence and bright-field images were acquired for the resulting polyacrylamide gels using our homebuilt small animal fluorescence imaging system. RESULTS

Chemistry. The novel compound 6′-O-lissaminerhodamine B-glucosamine (6-O-liss-GlcNH2, Figure 1, compound 4) was synthesized in a three-step procedure analogous to the previously published synthesis of 6-Odansyl-GlcNH2 (34). As summarized in Figure 1, a Lissamine Rhodamine B group (39) was regiospecifically introduced to the 6′-hydroxyl group of 6-(2-amino-2deoxy-β-D-glucopyranosyl)ether hydrochloride while the 2′-amino and 1′-hydroxyl groups were protected with tertbutyloxycarbonyl (Boc) groups (34, 40). The structure of

Figure 3. Time course of the colocalization coefficient. The colocalization coefficient (y-axis) of 6-O-liss-GlcNH2 fluorescence with CD63 immunofluorescence increased gradually with increasing incubation time (x-axis) in MCF-12A HMECs, MCF-7, and MDA-MB-231 breast cancer cells. Colocalization was most rapid in MDA-MB-231 cells, followed by MCF-7 and MCF-12A cells. Values are mean ( standard error, n ) 15, for each time point (five fields of view for three independent experiments each).

the final product 6-O-liss-GlcNH2 was verified by 1H and NMR spectroscopy and FAB-mass spectrometry (ppm- and m/z-values are given in Materials and Methods). Uptake and Localization of 6-O-Liss-GlcNH2 in Breast Epithelial and Cancer Cells. 6-O-liss-GlcNH2 was taken up by MCF-12A HMECs and human MCF-7 and MDA-MB-231 breast cancer cells and significantly localized to lysosomes starting from ∼8 h of incubation in MDA-MB-231 and MCF-7 breast cancer cells, and from ∼24 h of incubation in MCF-12A HMECs. To assess the degree of lysosomal labeling from 6-O-dansyl-GlcNH2, cells were fixed following 6-O-liss-GlcNH2 incubation and costained with lysosomal markers CD63, LAMP-1, or LAMP-2. Representative fluorescence images of MDAMB-231 breast cancer cells incubated with 6-O-lissGlcNH2 for 24 h in Figure 2a-c show that labeling from 6-O-liss-GlcNH2 colocalized with the lysosomal marker CD63, as evident from the yellow color in the overlay image in Figure 2c. In Figure 3, the colocalization coefficient from 6-O-liss-GlcNH2 and CD63 fluorescence was quantified from images derived from three independent experiments with each cell line, demonstrating the gradual increase in lysosomal 6-O-liss-GlcNH2 staining. The fastest incorporation of fluorescent 6-O-liss-GlcNH2 label into lysosomes occurred in MDA-MB-231 breast cancer cells, followed by MCF-7 cells and MCF-12A HMECs (Figure 3). Following 2-4 h of incubation, 6-Oliss-GlcNH2 fluorescence was distributed over the cytoplasm of cells and concentrated in some bright nonlysosomal areas close to the nucleus (data not shown). Following 8 h of incubation in MDA-MB-231 cells, or 2448 h in MCF-7 and MCF-12A cells, lysosomes were labeled up to a colocalization coefficient of 0.8-0.95 with 13C



Figure 4. Fluorescence images of SDS-PAGE gels of protein lysates obtained from (a) MCF-12A, (b) MCF-7, and (c) MDA-MB-231 cells incubated with 6-O-liss-GlcNH2 for 2 or 4 days. Bright-field images were acquired from the same field of view, and a molecular weight marker lane from bright-field images (lane 1) was copied into the fluorescence image to assign the molecular mass. Lane 1 contains molecular weight marker, lane 2 contains control cell lysate, lane 3 contains cell lysate following 2 days of incubation of cells with 6-O-liss-GlcNH2, and lane 4 contains cell lysate following 4 days of incubation of cells with 6-O-liss-GlcNH2. 6-O-lissGlcNH2 fluorescence is enriched in protein(s) of an apparent mass of 26-30 kDa following 2-4 days 6-O-liss-GlcNH2 incubation.

Figure 5. In vivo (a) bright-field and (b) fluorescence images of a representative MCF-7 tumor 2 h following injection of 7.8 mg of 6-O-liss-GlcNH2 into the tail vein. Efficient delivery of 6-O-liss-GlcNH2 to the tumor (tumor marked by arrow) is evident in these images.

respect to CD63 immunofluorescence (Figure 3), which corresponds to almost complete lysosomal staining. Comparable results were obtained with LAMP-1 and -2 immunofluorescence staining. Protein Analysis. SDS-PAGE electrophoresis of lysates from 6-O-liss-GlcNH2-treated cells was performed, followed by fluorescence imaging of these gels to determine the molecular weight of the protein(s) containing covalently bound 6-O-liss-GlcNH2. As displayed in Figure 4, fluorescence imaging of these gels revealed that proteins with an apparent molecular weight of ∼26-30 kDa contained 6-O-liss-GlcNH2-fluorescence. This fluorescent protein band was detected in MCF-12A HMECs as well as MCF-7 and MDA-MB-231 breast cancer cells (Figure 4). Uptake and Localization of 6-O-Liss-GlcNH2 in Tumor-Bearing SCID Mice. MCF-7 or MDA-MB-231 tumors in the mammary fat pad of female SCID mice were optically imaged in vivo at 2, 24, and 48 h following intravenous administration of 7.8 mg of 6-O-liss-GlcNH2 via the tail vein. Figure 5 demonstrates that fluorescence from 6-O-liss-GlcNH2 was detected in MCF-7 tumors at 2 h postinjection but not at 24 or 48 h postinjection. Similar results were obtained with MDA-MB-231 tumors. To assess the relative distribution of 6-O-liss-GlcNH2 in selected tissues, bright-field and fluorescence microscopy were performed on freshly cut 1-mm sections from MCF-7 tumors, MDA-MB-231 tumors, liver, kidney, and muscle tissue as well as on blood. Although there was no detectable fluorescence at 24 and 48 h postinjection of 6-O-liss-GlcNH2 in vivo, high-resolution fluorescence images of fresh tissue sections revealed that MCF-7 and MDA-MB-231 tumors contained 6-O-liss-GlcNH2-fluores-

cence at 2, 24, and 48 h postinjection, as shown in Figure 6. Fluorescence intensity declined at later time points postinjection (Figures 6, 7). Tumoral 6-O-liss-GlcNH2 fluorescence intensity was most pronounced in necrotic tumor areas, but also present in all other tumor regions, as evident from comparison of fluorescence with brightfield images (Figure 6a,b,f,g). High-power fluorescence images revealed that the staining pattern in viable tumor areas changed over time. At 2 h postinjection, 6-O-lissGlcNH2 was detected in the cytoplasm of tumor cells in viable tumor regions (Figure 6c,h). At 24 and 48 h postinjection, a punctate staining pattern from 6-O-lissGlcNH2 fluorescence was observed in viable tumor cells (Figure 6d,i). The liver contained detectable fluorescence intensity from 6-O-liss-GlcNH2 only at 2 h postinjection but not at later time points (Figure 6j,k). A small amount of fluorescence intensity was detected in the kidneys at 2 h postinjection. Muscle and blood did not contain any detectable fluorescence from 6-O-liss-GlcNH2 at the time points tested (data not shown). Figure 7 displays the time course of 6-O-liss-GlcNH2 fluorescence intensity in all tested organs. MCF-7 and MDA-MB-231 tumors retained higher levels of 6-O-liss-GlcNH2 fluorescence for a longer time than blood, liver, kidney, and muscle. Lysosomal Localization of 6-O-Liss-GlcNH2 in Tumor-Bearing SCID Mice. The subcellular localization of the tumoral 6-O-liss-GlcNH2 staining pattern was assessed by immunofluorescence microscopy of MCF-7 and MDA-MB-231 tumor sections obtained following 6-Oliss-GlcNH2 injection. Lysosomal staining with 6-O-lissGlcNH2 was evaluated by colocalization of 6-O-lissGlcNH2 with well-established lysososmal markers, such as CD63, LAMP-1, and LAMP-2. Figure 8 demonstrates


Glunde et al.

Figure 6. Ex vivo images of freshly cut 1-mm tissue sections (1× lens). Bright-field images of a representative (a) MCF-7 tumor section, (f) MDA-MB-231 tumor section, and (j) liver section and the corresponding 6-O-liss-GlcNH2 fluorescence images of the (b) MCF-7 tumor section, (g) MDA-MB-231 tumor section, and (k) liver section are displayed. High-power (100× lens) fluorescence images of a representative (c) MCF-7 tumor section 2 h postinjection and (d) MCF-7 tumor section 24 h postinjection, as well as a representative (h) MDA-MB-231 tumor section 2 h postinjection and (i) MDA-MB-231 tumor section 24 h postinjection are displayed. DISCUSSION

Figure 7. The time course of the mean 6-O-liss-GlcNH2 fluorescence intensity per pixel is shown for MCF-7 tumors, MDA-MB-231 tumors, liver, kidney, muscle, and blood. Values are mean ( standard error, n ) 2-4 for each time point.

that viable tumor regions from MCF-7 and MDA-MB231 tumors contained lysosomal 6-O-liss-GlcNH2-fluorescence at 24 h postinjection. This was evident from colocalization of 6-O-liss-GlcNH2 displayed in green (Figure 8a,d) with CD63-staining displayed in red (Figure 8b,e), resulting in the yellow color from the overlay of both images (Figure 8c,f). Nuclei were counterstained and are displayed in blue. The colocalization coefficient for 6-O-liss-GlcNH2 with CD63 fluroescence was 0.92 ( 0.09 (n ) 15) in viable areas of MCF-7 tumors, as quantified using five sections from three tumors each. In MDA-MB231 tumors, a colocalization coefficient of 0.96 ( 0.08 (n ) 15) was observed in viable regions. Comparable results were obtained with the lysosomal markers LAMP-1 and LAMP-2 (data not shown). Necrotic tumor regions displayed abundant 6-O-liss-GlcNH2 staining (Figure 8g) and, as anticipated, very little nuclear staining, since these tumor regions were not viable (Figure 8g-i). In addition, few yellow areas of colocalization were apparent in these necrotic areas (Figure 8i), indicating lack of lysosomal markers in these regions of dying cells (Figure 8g-i).

Here we have developed a novel compound, 6′-Olissamine-rhodamine B-glucosamine, to perform noninvasive labeling of lysosomes in vivo and in vitro and to follow lysosomal trafficking in live breast cells and solid breast tumor models. This method will be useful to understand the relationship between lysosomal trafficking and the tumor microenvironment and its role in breast cancer invasion and metastasis. The synthesis of 6-O-liss-GlcNH2 is a relatively straightforward three-step procedure analogous to the synthesis of 6-O-dansyl-glucosamine (34). A Lissamine Rhodamine B group was regiospecifically introduced to the 6′hydroxyl group of 6-(2-amino-2-deoxy-β-D-glucopyranosyl)ether hydrochloride while the 2′-amino and 1′hydroxyl groups were protected with Boc (tert-butyloxycarbonyl) groups (34, 40). Cell culture and animal studies are feasible using 6-Oliss-GlcNH2, since it dissolves well in aqueous solutions. In cell culture studies, a concentration of 500 µM of 6-Oliss-GlcNH2 in the cell culture medium resulted in clearly defined lysosomal fluorescence labeling in HMECs and breast cancer cells. In SCID mice bearing orthotopic breast tumor xenografts, administration of 7.8 mg of 6-Oliss-GlcNH2 into the tail vein resulted in robust lysosomal fluorescence labeling of tumors. Lysosomes in HMECs, breast cancer cell lines, and tumors were specifically labeled by 6-O-liss-GlcNH2 as verified by colocalization of 6-O-liss-GlcNH2 fluorescence with CD63, LAMP-1, and LAMP-2, the latter three of which are well-established marker proteins for lysosomes (23-26). Lysosomal labeling of breast cancer cells in culture or solid tumors required relatively long incubation and postinjection uptake times of approximately 24 h. In in vitro cell culture assays, lysosomes in MDA-MB-231 breast cancer



Figure 8. Confocal laser-scanning fluorescence images of (a-c) MCF-7 tumor sections and (d-f) MDA-MB-231 tumor sections. 6-O-liss-GlcNH2 fluorescence images are displayed in green in a representative (a) MCF-7 tumor section and (d) MDA-MB-231 tumor section. The corresponding CD63 immunofluorescence images are displayed in red in the (b) MCF-7 tumor section and (e) MDAMB-231 tumor section. 6-O-liss-GlcNH2 and CD63 fluorescence images were overlaid for (c) MCF-7 and (f) MDA-MB-231 tumor sections, demonstrating strong colocalization of 6-O-liss-GlcNH2 and CD63 as evident from the yellow color. (g-i) Representative confocal fluorescence images of necrotic regions from a MDA-MB-231 tumor, containing (g) strong 6-O-liss-GlcNH2 fluorescence, and very little fluorescence from (h) lysosomes or (i) nuclei.

cells were labeled faster than lysosomes in MCF-7 and MCF-12A cells. At early incubation or postinjection uptake times, breast cancer cells exhibited a nonvesicular staining pattern. This observation is consistent with our earlier studies with the 6-O-dansyl derivative of glucosamine (34), where dansyl fluorescence was distributed over the cytoplasm of the cell and concentrated in some bright areas close to the nucleus following 5 h of exposure, and vesicular lysosomal staining appeared following 22 h or longer time periods of exposure (34). Similar to the analogous compound 6-O-dansyl-GlcNH2, 6-O-liss-GlcNH2 was most likely biosynthetically incorporated into highly glycosylated lysosomal proteins by passing through cellular sites of protein glycosylation. The lysosomal membrane contains several N-linked glycan-carrying proteins (35). N-Linked glycans are added cotranslationally to newly synthesized polypeptides in the endoplasmic reticulum (ER) and are extensively modified, as these glycoproteins mature and move through the ER and Golgi complex to their final destination in the cell (41, 42). Protein lysates of cells exposed to 6-O-liss-GlcNH2 exhibited one major band at 26-30

kDa in optically imaged protein SDS-PAGE gels. Apparently, 6-O-liss-GlcNH2 was utilized by breast cells in the biosynthesis of a glycosylated protein highly represented in lysosomes. However, 6-O-dansyl-GlcNH2 was predominantly found in 70 kDa protein(s) (34). Although 6-O-liss-GlcNH2 and 6-O-dansyl-GlcNH2 both localized to lysosomes through biosynthetic incorporation into highly glycosylated lysosomal proteins, the difference in apparent molecular weight suggests that two distinct lysosomal proteins were labeled by these compounds. In contrast to commonly used less specific lysosomal probes utilized for live cell imaging, 6-O-liss-GlcNH2 and 6-Odansyl-GlcNH2 (34) confer the advantage of being specifically targeted to lysosomes. Systemic administration of 6-O-liss-GlcNH2 in SCID mice revealed that this compound is nontoxic in SCID mice. 6-O-liss-GlcNH2 is rapidly cleared from the blood stream within 2 h of intravenous administration, as demonstrated by a lack of 6-O-liss-GlcNH2 fluorescence in the blood at 2 h postinjection. MCF-7 and MDA-MB231 tumors were intensely fluorescent up to 24-48 h, while the liver and kidney contained moderate 6-O-liss-


GlcNH2 fluorescence intensity at similar time points. Thus, 6-O-liss-GlcNH2 was efficiently delivered to MCF-7 and MDA-MB-231 breast tumor xenografts in SCID mice. Although 6-O-liss-GlcNH2 was detectable in these breast tumor models for more than 3 days, its fluorescence intensity declined over time, indicating elimination of 6-O-liss-GlcNH2 from breast tumors. The fluorescence intensity from 6-O-liss-GlcNH2 in these tumors was sufficient for tumor in vivo optical imaging at 2 h postinjection. Subcellular localization of 6-O-liss-GlcNH2 to lysosomes in tumors required longer time periods. The highest colocalization coefficients of 6-O-liss-GlcNH2 fluorescence with lysosomal markers CD63, LAMP-1, and LAMP-2 was detected at 24 h postinjection in viable tumor regions, suggesting that breast cancer cells in these viable tumor regions utilized 6-O-liss-GlcNH2 in the biosynthesis of a glycosylated lysosomal protein. The intratumoral distribution of 6-O-liss-GlcNH2 appeared to be inhomogeneous, exhibiting higher fluorescence intensity in necrotic areas. Necrotic areas did not contain viable cells, as evident from missing nuclear counterstaining in these areas. Thus, 6-O-liss-GlcNH2 accumulation in necrotic tumor regions was nonspecific. While lysosomal localization of fluorescence from 6-O-lissGlcNH2 was most pronounced at 24 h postinjection, in vivo imaging of this fluorescence was not feasible with our system, because the fluorescence intensity was below the detection limit of this system. Images obtained by fluorescence microscopy of fresh tumor slices strongly suggest that intravital fluorescence microscopy or endoscopy of 6-O-liss-GlcNH2-labeled lysosomes in solid breast tumor models will have sufficient resolution and sensitivity to detect lysosomal labeling in vivo. The novel compound 6-O-liss-GlcNH2 described here will be valuable not only to understand the role of lysosomes and lysosomal trafficking in breast cancer invasion and metastasis, but also to study cell and animal models of lysosomal diseases. 6-O-liss-GlcNH2 itself, or analogous compounds, may, in the future, be utilized for detecting aberrant lysosomal trafficking in clinical settings. 6-O-liss-GlcNH2 will prove useful for studying lysosomal storage diseases (43) such as Gauchers disease (44), as well as neurodegenerative diseases such as Alzheimer’s disease (45, 46), which exhibit robust activation of the neuronal lysosomal system, namely, endocytosis and autophagy. Although 6-O-liss-GlcNH2 may not be able to cross the intact blood-brain barrier (BBB), pathogenesis of increased BBB permeability has been demonstrated in Alzheimer’s disease (47). 6-O-lissGlcNH2 may also be helpful for assessing host-parasite relationships such as that of Trypanosoma cruzi trypomastigotes (48), which involve participation of the host’s lysosomes, and for following therapeutic targeting of lysosomes. ACKNOWLEDGMENT

This work was supported by a Career Development Award to Dr. Kristine Glunde through P50 CA103175 (JHU ICMIC Program) and R24 CA92871. We thank Dr. Dmitri Artemov for building the small animal fluorescence imaging system and for technical support with this home-built system. We thank Dr. J. Thomas August for providing us with monoclonal antibodies against hLAMP-1 (H4A3) and hLAMP-2 (H4B4). We thank Dr. Douglas B. Murphy (Director, The Johns Hopkins University School of Medicine Microscope Facility) for technical support. We thank Mr. Gary Cromwell for maintaining the cell lines.

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Synthesis of 6 '-O-Lissamine-rhodamine B-Glucosamine as a Novel

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