Cancer Therapy: Preclinical
A Disaccharide-Based Inhibitor of Glycosylation Attenuates
MetastaticTumor Cell Dissemination
Jillian R. Brown,1 Mark M. Fuster,2 Ruixia Li,1 Nissi Varki,3 Charles A. Glass,1 and Jeffrey D. Esko1
Abstract
Purpose: The binding of hematogenously borne malignant cells that express the carbohydrate
sialyl Lewis X (sLeX) to selectin adhesion receptors on leukocytes, platelets, and endothelial cells
facilitates metastasis.The glycosylation inhibitor, per-O-acetylated GlcNAch1,3Galh-O-naphthalenemethanol (AcGnG-NM), inhibits the biosynthesis of sLeX in tumor cells.To evaluate the efficacy of AcGnG-NM as an antimetastatic agent, we examined its effect on experimental metastasis
and on spontaneous hematogenous dissemination of murine Lewis lung carcinoma and B16BL6
melanoma cells.
Experimental Design: Tumor cells were treated in vitro with AcGnG-NM, and the degree of
selectin ligand inhibition and experimental metastasis was analyzed in wild-type and P-selectindeficient mice. Conditions were developed for systemic administration of AcGnG-NM, and the
presence of tumor cells in the lungs was assessed using bromodeoxyuridine labeling in vivo. The
effect of AcGnG-NM on inflammation was examined using an acute peritonitis model.
Results: In vitro treatment of Lewis lung carcinoma cells with AcGnG-NM reduced expression of
sLeX- and P-selectin-dependent cell adhesion to plates coated with P-selectin. Treatment also
reduced formation of lung foci when cells were injected into syngeneic mice. Systemic administration of the disaccharide significantly inhibited spontaneous dissemination of the cells to the lungs
from a primary s.c. tumor, whereas an acetylated disaccharide not related to sLeX in structure had
no effect. AcGnG-NM did not alter the level of circulating leukocytes or platelets, the expression of
P-selectin ligands on neutrophils, or sLeX-dependent inflammation.
Conclusion: Taken together, these data show that AcGnG-NM provides a targeted glycosidebased therapy for the treatment of hematogenous dissemination of tumor cells.
Metastasis is a multistep cascade involving the migration of
tumor cells from their site of origin, evasion of host defense
systems, subsequent seeding of distant organs, and growth of
secondary tumors. During metastatic dissemination, cells shed
from the primary tumor enter the blood circulation and interact
with platelets and leukocytes forming neoplastic emboli that
can arrest in the microvasculature and adhere to the endotheAuthors’ Affiliations: 1Department of Cellular and Molecular Medicine,
Glycobiology Research and Training Center; 2Department of Medicine, Division of
Pulmonary and Critical Care; and 3 Department of Pathology, University of
California, San Diego, La Jolla, California
Received 12/15/05; revised 2/8/06; accepted 2/27/06.
Grant support: NIH grants CA46462 and CA112278 (J.D. Esko), University
Biotechnology Research and Training Grant (J.R. Brown),Tobacco-Related Disease
Research Program fellowship grant TRDRP #8FT-0118 (M.M. Fuster), and U.S.
Department ofVeterans Affairs Research Career Development Award (M.M. Fuster).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: C.A. Glass is currently at Zacharon Pharmaceuticals, 505 Coast Boulevard
South, La Jolla, CA 92037.
R. Li is currently at the Department of Ophthalmology, University of California, San
Diego, La Jolla, CA 92093-0946.
Requests for reprints: Jeffrey D. Esko, Department of Cellular and Molecular
Medicine, Glycobiology Research and Training Center, University of California, San
Diego, 9500 Gilman Drive, La Jolla, CA 92093-0687. Phone: 858-822-1100; Fax:
585-534-5611; E-mail: jesko@ ucsd.edu.
F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-2745
Clin Cancer Res 2006;12(9) May 1, 2006
lium. Tumor cell adhesion is mediated in part through
selectins, a family of cell surface carbohydrate-binding proteins
(1 – 3). P-selectin on platelets facilitates platelet attachment to
tumor cells forming a protective cloak and preventing tumor
cell lysis by elements of the innate immune system (4 – 6).
L-selectin-mediated adhesion of leukocytes results in formation
of leukocyte-tumor cell emboli as well as local secretion of
cytokines and growth factors thought to aid in secondary tumor
growth (7 – 9). P-selectin and E-selectin on the endothelium
facilitate tumor cells and emboli to anchor in the microvasculature (1, 2, 10, 11). The combined action of the selectins
provides a mechanism that enables tumor cells to survive and
to populate distant organs.
Sialylated, fucosylated tetrasaccharides, such as sLeX (Siaa2,3
Galh1,4(Fuca1,3)GlcNAch-) or sLea (Siaa2,3Galh1,3(Fuca1,4)
GlcNAch-), are two major carbohydrate determinants common
to many selectin ligands (12, 13). A number of clinical studies
show that expression of sLeX and sLea on tumor cell mucins
correlates directly with metastasis, tumor progression, and poor
prognosis (see ref. 3 and references therein). Thus, therapeutic
agents that target these carbohydrate ligands would offer a
promising avenue for intervention (reviewed in refs. 14, 15).
Towards this goal, we have developed carbohydrate-based
inhibitors of sLeX expression (6, 16 – 18). These agents consist
of a disaccharide conjugated to a hydrophobic moiety, the most
potent being acetylated per-O-acetylated GlcNAch1,3Galh-Onaphthalenemethanol (AcGnG-NM; Fig. 1). Peracetylation
facilitates passive diffusion of the disaccharide across cell
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Inhibition of Metastasis
Fig. 1. The synthetic disaccharide decoyAcGnG-NM.
membranes, allowing entry into the Golgi, where sLeX assembly
takes place. The acetylated disaccharide undergoes deacetylation
by endogenous esterases, which allows it to act as an
intermediate in the biosynthesis of sLeX. Assembly of oligosaccharides on the disaccharide ‘‘decoys’’ the assembly of sLeX from
cellular glycoconjugates; thus, AcGnG-NM acts as an inhibitor of
selectin-ligand formation. We have previously shown that
AcGnG-NM diminishes tumor seeding in an experimental
metastasis model in which human tumor cells pretreated with
disaccharide were injected i.v. into immunocompromised mice
(6). Although these data showed the antimetastatic potential of
AcGnG-NM, the experimental conditions did not mimic the
relevant clinical setting of spontaneous metastasis in which
tumor cells seed and colonize sites distant from the primary
tumor.
As a prelude to clinical trials, we have developed a model for
systemic administration of the acetylated disaccharide in mice
using syngeneic tumor cell lines to study spontaneous
metastasis. Herein, we show that systemic treatment with
AcGnG-NM attenuates spontaneous pulmonary metastatic
seeding. The effect was remarkably selective because the
inhibitor had no effect on leukocyte infiltration in response
to an experimental inflammatory stimulus, which also depends
on selectin-carbohydrate interactions. These findings suggest
that treatment with acetylated disaccharide uniquely targets the
metastatic seeding behavior of tumors cells, setting the stage to
explore the use of disaccharides as adjuvant therapy for
blocking hematogenous tumor metastasis.
Materials and Methods
Disaccharides and cell lines. AcGnG-NM and per-O-acetylated
Galh1,3Galh-O -naphthalenemethanol were prepared as described
(19). Murine Lewis lung carcinoma (LLC) cells were purchased from
the American Type Culture Collection (LLC1, CRL-1642; Manassas,
VA). Murine B16BL6 melanoma cells were a kind gift from J.I. Fidler
(University of Texas, M.D. Anderson Cancer Center). Cells were grown
in DMEM (4.5 g glucose/L) supplemented with sodium bicarbonate
(1.59 g/L for LLC and 3.7 g/L for B16BL6), 10% (v/v) fetal bovine
serum (Hyclone Laboratories, Logan, UT), glutamine (0.3 g/L),
streptomycin sulfate (100 Ag/mL), and penicillin (100 units/mL). Cells
were maintained at 37jC in a humidified incubator containing 5% CO2
and 95% air and passaged every 4 days using ATV solution (Invitrogen,
Carlsbad, CA).
ELISA, flow cytometry, cell adhesion, and enzyme assays. To measure
the presence of selectin ligands, cells were grown to confluence for 4 to
7 days in six-well plates with and without AcGnG-NM. Binding of
monoclonal antibody (mAb) CSLEX-1 was measured by ELISA as
described (18). Sialylated and fucosylated cell surface glycans were
measured by reacting cells with biotinylated Maackia amurensis
hemaglutinin (10 Ag/mL) or biotinylated Aleuria aurantia lectin
(2 Ag/mL; Vector Laboratories, Burlingame, CA), respectively. After
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incubation with streptavidin-R-phycoerythrin (1.85 Ag/mL; Jackson
ImmunoResearch Laboratories, Inc., West Grove, PA), the cells were
analyzed by flow cytometry (FACScan; BD Biosciences, San Jose, CA).
Cell adhesion to immobilized P-selectin was done in 96-well plates
coated overnight at 4jC with recombinant P-selectin (2 Ag/mL; R&D
Systems, Minneapolis, MN) and blocked with 1% bovine serum
albumin in PBS as described (6, 18).
Total fucosyltransferase and sialyltransferase activities were assayed
in cell lysates prepared from LLC cells as described (18).
In some experiments, cells or whole blood were incubated with mAbs
to the following antigens: CA19-9 (anti-sLea, 120 Ag/mL; Chemicon,
Temecula, CA), Gr-1 (myeloid differentiation antigen, 12 Ag/mL), CD45
(leukocyte common antigen, 26 Ag/mL), CD41 (integrin aIIb chain
expressed on platelets, 10 Ag/mL), CD62P (P-selectin, 10 Ag/mL; BD
Biosciences). Murine selectin/IgM chimeras were used to probe the
tumor cells for selectin ligands (Ps-IgM, Ls-IgM, and Es-IgM, a kind gift
from J.B. Lowe, University of Michigan). The cells were washed
and incubated with goat antibodies against mouse or human IgM or
IgG (30 Ag/mL, Sigma, St. Louis, MO) conjugated to FITC and analyzed
by flow cytometry.
Experimental metastasis using AcGnG-NM-pretreated cells. LLC cells
were grown for 4 to 5 days in the presence or absence of 50 Amol/L
AcGnG-NM. The cells were then harvested with EDTA, resuspended in
sterile 0.9% saline, and injected (2 105 per 150 AL) into the lateral
tail vein of anesthetized (inhaled isoflurane, Janssen Pharmaceuticals,
Titusville, NJ) 8- to 12-week-old Es1(e) mice (bred in a C57Bl/6
background, a kind gift from P. Potter, University of North Carolina
via Charles River Laboratories; ref. 20). These mice carry a mutant
allele of the esterase locus Es-1 designated Es1(e) (21) that results in
reduced plasma esterase activity (22). After 10 days, lung metastases
were detected by visible nodules on the surface of the lungs and
quantitated under a dissecting microscope. Representative pictures
were taken.
Spontaneous metastatic seeding of LLC cells after systemic AcGnG-NM
treatment. To measure the effect of systemic administration of AcGnGNM on metastasis, Alzet osmotic minipumps (Durect Corp., Cupertino,
CA) containing vehicle (DMSO/propylene glycol, 1:1, v/v) or AcGnGNM (150 mg/mL) were surgically implanted in a dorsal skin fold of
Es1(e) mice (23). The dose rate of compound was f0.9 mg/d/mouse
(0.25 AL/h). LLC cells (5 105) were injected s.c. in the hindquarter a
day after implantation of the pump. After 4 weeks, each animal was
injected i.p. with 1 mg of bromodeoxyuridine (BrdUrd) to measure
DNA synthesis (24). Two hours later, the animals were sacrificed and
perfused with PBS, and the lungs were removed. To detect BrdUrdlabeled cells, sections were overlaid with anti-BrdUrd mAb (Becton
Dickinson, San Jose, CA) and horseradish peroxidase – labeled goat
anti mouse IgG. Specific binding was detected with the 3-amino-9ethylcarbazole (Vector Labs, Burlingame, CA). Nuclei were counterstained using Mayer’s hematoxylin. Standard histologic sections were
also prepared and stained with H&E.
To measure the extent of BrdUrd labeling, lungs were incubated with
collagenase (10 mg/mL, 1 hour, 37jC), dispersed by repeated passage
through an 18-gauge needle, and filtered through a 40-Am pore nylon
filter. The cells were fixed (70% ethanol, 1 106 cells/mL), and the
relative number of BrdUrd-labeled cells was determined by reacting the
cells with mouse FITC-labeled anti-BrdUrd antibody followed by flow
cytometry. Each lung dispersion was incubated with FITC-conjugated
isotype control antibody to set the lower threshold for BrdUrd-positive
cells, and we gated on labeled cells that were positive for propidium
iodide staining. In some experiments, the animals received a control
disaccharide, per-O-acetylated Galh1,3Galh-O-naphthalenemethanol.
To determine how metastatic seeding depended on P-selectin, LLC cells
(6 105) were implanted s.c. in the hindquarter of P-selectin-deficient
mice (P / ) bred on a C57Bl/6 background (The Jackson Laboratory, Bar
Harbor, ME).
The effect of systemic administration of AcGnG-NM on experimental
metastasis was measured in animals 2 weeks after implantation of
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pumps. LLC cells (2 105 cells per 150 AL) were injected into the
lateral tail vein, and 2 weeks later, each animal was injected i.p. with
BrdUrd and processed as described above.
Thioglycollate inflammation model. Acute peritoneal inflammation
(peritonitis) was induced by injection of 3% thioglycollate broth
(2 mL), with sterile pyrogen-free saline as control (25). Groups of five
animals per experimental condition were injected and sacrificed after 3
hours. The peritoneal cavities were lavaged with ice-cold saline
containing 3 mmol/L EDTA (8 mL) to prevent aggregation, and cells
were counted in the lavage fluid using a particle counter. The cells were
also stained with FITC-conjugated rat anti-mouse Gr-1 mAb and
counted by flow cytometry.
Statistics. All statistical analyses were done using Prism software.
Statistics were calculated by either one-way ANOVA test comparing three
groups of four to seven animals or by Student’s t test comparing two
groups of four to seven animals as indicated in the individual
experiments.
Results
Sensitivity of LLC to AcGnG-NM. LLC was derived from a
C57BL/6 mouse and spontaneously metastasizes from primary
s.c. tumors to the lung, mimicking the aggressiveness of clinical
pulmonary metastatic tumors (26 – 28). LLC cells also reacted
strongly in vitro with a chimeric selectin composed of the
carbohydrate recognition domain of murine P-selectin and the
Fc region of human IgM (ref. 29; Fig. 2A). In contrast, the cells
only weakly reacted with an L-selectin-IgM chimera and not at
all with an E-selectin-IgM chimera. The cells also bound mAbs
that recognize sLeX (CSLEX-1) and sLea (CA19-9; data not
shown).
Treatment of LLC cells with AcGnG-NM decreased binding of
CSLEX-1 in a dose-dependent manner, with partial inhibition
occurring as low as f10 Amol/L (Fig. 2B). In contrast, the
compound had no effect on sLea expression, which only differs
from sLe X in the linkage arrangement of the sugars
[Siaa2,3Galh1,3(Fuca1,4)GlcNAc- versus Siaa2,3Galh1,
4(Fuca1,3)GlcNAc-]. Treatment of cells with low concentrations of AcGnG-NM also significantly reduced cell adhesion to
plates coated with recombinant P-selectin (Fig. 2B). Control
experiments using sialidase, which removes the crucial sialic
acid determinant for selectin binding, decreased adhesion to
the same extent as AcGnG-NM. Addition of a P-selectin
antibody and EDTA blocked adhesion confirming the specificity of the interaction. These agents decreased adhesion to a
greater extent than AcGnG-NM, suggesting that a second ligand
insensitive to AcGnG-NM might be present, such as sulfatide
(3-sulfoGalh1,4Glch-O-ceramide; ref. 30). Disaccharide inhibition of adhesion also depended on the structure of the
Fig. 2. Selectin ligands on LLC cells. A, LLC cells were incubated for
with mouse P-selectin, L-selectin, and E-selectin IgM chimeras and
analyzed by flow cytometry (solid lines ; Materials and Methods).
The shaded peaks represent samples incubated without chimeras.
Incubation with 5 mmol/L EDTA prevented binding of P-selectin
chimera to the cells (dashed line). B, treatment of LLC cells with
AcGnG-NM showed a concentration-dependent decrease in binding
of mAb CSLEX-1as measured by ELISA (Materials and Methods).
Adhesion to immobilized P-selectin was also altered. The extent of
inhibition was comparable with that achieved by sialidase treatment.
Adhesion was blocked by anti-P-selectin mAb (20 Ag/mL) or by the
inclusion of 5 mmol/L EDTA in the incubation. Columns, average of
triplicate measurements, which varied by <10%. C, treatment of LLC
cells with 50 Amol/L AcGnG-NM reduced binding of A. aurantia
lectin (AAL), which reacts with a1,3/6-linked fucose, but had no
effect on binding of M. amurensis hemaglutinin (MAH), which reacts
with a2,3-linked sialic acid. Cells were analyzed by flow cytometry,
and the average fluorescence value was normalized to that obtained
for a sample of cells that had not been treated with AcGnG-NM. D,
total sialyltransferase (SiaT) and fucosyltransferase (FucT) activity
was measured in LLC cells by assaying the transfer of radiolabeled
sialic acid to Galh1,4GlcNAch-O-NM (SiaT) and radiolabeled fucose
to Galh1,4GlcNAch-O-NM (FucTa) or NeuAca2,3Galh1,4GlcNAc
(FucTb). Columns, average of duplicate measurements, which varied
by <10%.
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Inhibition of Metastasis
Fig. 3. AcGnG-NM inhibits experimental metastasis. A, LLC cells were treated in
culture with vehicle (DMSO) or AcGnG-NM. A single-cell suspension (2 105)
was injected in the tail-vein of mice. After 10 days, the mice were sacrificed, and their
lungs were analyzed for surface tumor foci. B, counting the number of tumors
showed a strong inhibition of tumor formation by the disaccharide (n = 5-7).
C, P-selectin deficiency reduced experimental lung metastasis. LLC cells were
injected in the tail-vein of wild-type or P-selectin-deficient mice (P-sel / ; n = 8-9),
and the number of lung tumors was counted. Treatment of cells with AcGnG-NM
and P-selectin deficiency did not have an additive effect, suggesting that the
disaccharide worked through a P-selectin-dependent pathway.
compound because per-O-acetylated Galh1,3Galh-O-naphthalenemethanol, which is not an intermediate in sLeX biosynthesis, did not affect binding to CSLEX-1 or adhesion to P-selectincoated plates (data not shown).
To determine how AcGnG-NM inhibited P-selectin ligand
formation in LLC cells, cells were reacted with FITC-conjugated
M. amurensis hemaglutinin, which recognizes a2,3-linked sialic
acid and 3-O-sulfated galactose containing glycans (31) and A.
aurantia lectin, which recognizes a1,3/6-linked fucose (32).
Binding of M. amurensis hemaglutinin did not change after
treatment with AcGnG-NM, whereas binding of A. aurantia
lectin decreased by f40% (Fig. 2C), suggesting that fucosylation was selectively altered. In previous studies of human LS180
colon carcinoma cells, we showed that the disaccharide can also
affect sialylation. The different modes of inhibition of sLeX
formation in different cell types (i.e., fucosylation versus
sialylation) seems to correlate inversely with the relative
activities of fucosyltransferases and sialyltransferases involved
in sLeX formation (18). In LLC cells, fucosyltransferase activity
using Galh1,4GlcNAch-O-NM (FucTa; 33.4 pmol/mg/h) or
NeuAca2,3Galh1,4GlcNAc (FucTb; 13.2 pmol/mg/h) as the
acceptor was lower than the sialyltransferase activity (65.6
pmol/mg/h) measured with Galh1,4GlcNAch-O-NM as the
acceptor (Fig. 2D), consistent with idea that the compound
inhibits P-selectin ligand formation by blocking fucosylation.
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AcGnG-NM inhibits experimental pulmonary metastasis of LLC
cells. To test whether inhibition of sLeX by AcGnG-NM
diminished experimental pulmonary metastasis, mice were
injected i.v. with AcGnG-NM-treated or vehicle-treated LLC
cells. After 10 days, the animals were sacrificed, and the lungs
were analyzed for surface tumor foci. Disaccharide treatment
dramatically reduced the incidence of macroscopic tumor foci
[16 F 6 (n = 7) versus 77 F 12 (n = 5); P = 0.003; Fig. 3]. Some
animals injected with disaccharide-treated cells had only one to
two nodules, showing the strong antimetastatic effect of the
compound in this model.
Prior studies have shown that genetic deletion of P-selectin
can reduce experimental metastasis of tumor cells by blocking
platelet-tumor cell aggregation and tumor cell-endothelial cell
interactions (33, 34). To determine if LLC cell metastasis also
depended on P-selectin, cells were i.v. injected in wild-type and
P-selectin-deficient mice (Fig. 3C). The number of tumor foci
was dramatically reduced in P-selectin deficient mice (12 F 6
versus 73 F 27, P < 0.001). When disaccharide-treated cells
were injected into P-selectin-deficient mice, no further reduction in lung metastases was observed. The lack of additive or
synergistic effects of P-selectin deficiency and AcGnG-NM
suggested that the disaccharide inhibited experimental pulmonary metastasis through a pathway involving P-selectin.
AcGnG-NM inhibits spontaneous metastatic seeding of the
lung. The blockade of experimental metastasis by AcGnG-NM
encouraged us to examine the effect of the disaccharide on
spontaneous pulmonary metastasis. Because these studies
would require systemic administration of the compound to
mice, we first examined the stability of AcGnG-NM. Incubation
of radiolabeled disaccharide with mouse serum for 3 hours
resulted in extensive removal of the acetyl groups due to serum
esterases but no hydrolysis of the glycosidic linkages. Because
uptake of the compound depends on the presence of the acetyl
groups to facilitate passive diffusion (16), we examined the
stability of the compound in serum obtained from an esterasereduced Es1(e) mouse, which has a hypomorphic allele of
serum Es-1 (21). Acetylated disaccharide in serum from Es1(e)
mice was stable for several hours, which approximated
conditions in human serum (22). Thus, all subsequent studies
were done in Es1(e) mice.
We first attempted to administer the compound by tail vein
injection. However, this method of delivery led to the collapse
of the vein and necrosis of the tail after repeated injection of the
vehicle. To circumvent this problem, the compound was
dissolved in DMSO/propylene glycol (1:1. v/v) and placed in
small Alzet osmotic pumps, which were surgically implanted
under a dorsal skin fold. Based on the theoretical delivery rate
of 0.25 AL/h, the animals received an effective dose of f0.9
mg/d/mouse, which translates to a maximum dose of f45 mg/
kg/d. Detailed pharmacokinetic and pharmacodynamic studies
to determine the actual steady-state concentration achieved by
continuous infusion have not been done. Thus, the calculated
concentration should be considered the maximum that could
be achieved under these conditions.
To measure spontaneous pulmonary metastasis, animals
were injected s.c. with LLC cells in the hindquarter. After
4 weeks, large tumors arose at the s.c. site, but secondary
metastatic nodules were rare in the lungs and other tissues. In
histologic sections stained with H&E, we found numerous cells
with characteristics of malignant tumor cells embedded in the
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lung parenchyma (Fig. 4A; ref. 28). These cells exhibited
nuclear pleomorphism, increased nuclear/cytoplasmic ratios,
irregular nuclear membranes, and in some cases prominent
nucleoli. Many of the metastatic cells were within capillaries,
and some had already entered the alveolar spaces. Because
humane treatment of the animals required euthanasia when
the primary tumors reached about 20% of the animal mass,
we could not extend the duration of the experiments to
determine if micrometastatic foci formed in the lungs.
Attempts to surgically resect the primary tumor to extend the
experiments were unsuccessful because the primary tumors
were invasive and grew back rapidly before nodule formation
in other organs.
To determine the effect of AcGnG-NM on tumor cell seeding
of the lungs, we injected BrdUrd and stained tissue sections
with anti-BrdUrd mAb. Numerous brown-red – stained cells
were present in samples obtained from animals with a tumor
(Fig. 4A, arrows). The brown-red stain left by the substrate
highlights the sharp but irregularly shaped nuclear membranes
of the malignant cells. Examination of lung sections from
BrdUrd-labeled mice without a tumor did not show any
malignant cell infiltrates (Fig. 4B). Sections were prepared from
the primary tumor as well (Fig. 4C). H&E staining of the tumor
mass showed malignant cells with pleomorphic nuclei, altered
nuclear/cytoplasmic ratios, and many mitotic figures. Immunohistochemical staining for incorporated BrdUrd showed
mitotically active nuclei scattered within the tumor but did
not label all of the tumor cells presumably because they were at
different stages of the cell cycle.
To determine whether BrdUrd labeling was suitable for quantitative measurement of tumor cells, the lungs were removed
and treated with collagenase. The resulting cell suspension was
then analyzed by flow cytometry after permeabilizing the cells
and staining them with anti-BrdUrd mAb. The percentage of
BrdUrd-positive cells in the lungs from animals bearing tumors
was 3.1 F 0.8%, whereas animals that had no tumor contained
0.7 F 0.2% BrdUrd-positive cells (n = 4, P = 0.0018). Because
not all tumor cells took up BrdUrd, this measurement actually
underestimates the tumor cell burden in the lungs (Fig. 4C).
Nevertheless, the labeling method provides a way to estimate
metastatic seeding of the lungs and the effect of potentially
antimetastatic agents (28, 35).
To determine the antimetastatic effect of AcGnG-NM, pumps
containing vehicle or disaccharide were surgically implanted,
and 1 day later, LLC cells were injected s.c. in the hindquarter.
After 4 weeks, the animals were labeled with BrdUrd, and the
lungs were processed for flow cytometry. As shown in Fig. 5A,
continuous systemic administration of disaccharide resulted
in a decreased proportion of BrdUrd-labeled cells compared
with animals receiving only vehicle. The experiment was
repeated with three sets of mice on different days, with consistently the same result (average values: 1.4 F 0.2% BrdUrdlabeled cells in disaccharide-treated animals versus 2.9 F 0.3%
in animals treated with vehicle; P < 0.001). Subtracting the
background of BrdUrd-labeled cells in naive animals from the
data (0.7 F 0.1%) showed that the disaccharide decreased
seeding by f3-fold. Averaging the data in this way underestimates the efficacy of AcGnG-NM because several of the
Fig. 4. BrdUrd labeling of tumor cells.
A, lung sections from an animal bearing a
s.c. tumor were immunostained for BrdUrd
(Materials and Methods). Arrows indicate
tumor cells with pleomorphic nuclei,
increased nuclear/cytoplasmic ratio, sharp
irregular nuclear membranes, and frequent
prominent nucleoli. Photomicrographs
were taken at 400 and 1,000 (oil
immersion) as indicated. B, BrdUrd-positive
tumor cells were not detected in lung
sections from mice that did not bear a tumor.
C, for comparison, a section of a primary
s.c. tumor was analyzed, showing cells
that took up BrdUrd in the 2-hour labeling
period.
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Fig. 5. AcGnG-NM treatment of mice inhibits spontaneous tumor metastasis. A, osmotic pumps containing vehicle or AcGnG-NM were surgically implanted in a dorsal
skin fold of Es1(e) mice. LLC cells (5 105) were implanted s.c. in the hindquarter. After 4 weeks, each animal was injected i.p. with BrdUrd, and the relative number of
BrdUrd-labeled cells in the lungs was determined by flow cytometry (Materials and Methods). Control animals (no tumor) did not receive tumor cells. The experiment was
repeated with three sets of mice independently (different shaded data points). Statistics were calculated by a one-way ANOVA test comparing three groups of four to seven
animals. B, one set of animals were dosed with a control disaccharide, per-O-acetylated Galh1,3Galh-O-naphthalenemethanol (AcGG-NM), and compared with a set
treated with vehicle (n = 5). C, LLC cells (6 105) were implanted s.c. in the hindquarters of wild-type and P-selectin-deficient (P-sel / ) mice (n = 5) to test for the
P-selectin-dependent seeding of the lungs with tumor cells.
animals had values below the naive control and did not have
metastatic tumor cells by histologic analysis.
Several control experiments were conducted to determine the
specificity and mechanism of inhibition of tumor cell seeding
of the lungs by AcGnG-NM. First, continuous systemic
administration of the control disaccharide, per-O-acetylated
Galh1,3Galh-O-naphthalenemethanol, using the same delivery
method had no effect on metastasis (Fig. 5B). Second,
spontaneous pulmonary metastasis, like experimental metastasis, depended on P-selectin expression, as shown by reduction
of BrdUrd-labeled cells in P-selectin-deficient mice (Fig. 5C).
Measurement of the primary tumor mass in these experiments
did not show any significant differences (3,500 F 1,500 mm3
in animals receiving vehicle versus 3,700 F 1,800 mm3 in
animals treated with AcGnG-NM), suggesting a selective effect
of the compound on metastatic seeding as opposed to tumor
growth per se. As an additional control, animals were
implanted for 2 weeks with pumps containing disaccharide or
vehicle and then injected i.v. with untreated LLC cells. No
significant reduction in the number of metastatic tumor foci
was noted in AcGnG-NM-treated mice (P = 0.22), showing that
systemic administration of AcGnG-NM specifically affected
seeding of the lungs by tumor cells.
We also examined the metastatic properties of murine
B16BL6 melanoma cells. Only two of five animals receiving
vehicle survived 25 days after injection of the tumor. Histologic
examination of the lungs showed numerous malignant cells
within capillaries. In contrast, four of five animals treated with
AcGnG-NM survived, and no malignant cells were present (data
not shown). Although the low number of animals did not
allow us to draw statistically firm conclusions from the data,
the trend of reduced metastatic seeding of the lungs matched
that seen with LLC cells.
AcGnG-NM has no effect on acute inflammation. Because
leukocytes express selectin ligands, it was possible that
AcGnG-NM treatment could inhibit leukocyte trafficking and
cause leukocytosis, a phenomena that occurs in selectindeficient mice (36). To determine if AcGnG-NM also affected
leukocytes, we analyzed blood samples from animals sys-
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temically treated with AcGnG-NM for 4 weeks. No change
occurred in total leukocyte or platelet counts compared with
vehicle-treated animals (Table 1). Systemic treatment with
AcGnG-NM did not affect expression of P-selectin ligands on
neutrophils (Fig. 6A) and lymphocytes (Fig. 6B), or Pselectin expression on platelets (Fig. 6C). No thrombotic
events occurred or excessive bleeding was noted. Additionally, treatment of animals with AcGnG-NM had no effect on
acute peritoneal inflammation (peritonitis) induced with thioglycollate (Fig. 6D).
Discussion
The majority of therapeutic strategies for treating cancer focus
on cytotoxic agents designed to alter growth and/or differentiation of the primary tumor (37). These agents have helped
reduce the death rate from many types of cancer, but the overall
cancer mortality rate has not changed significantly since the
1950s due to changes in cancer demographics (38). An open
area for therapeutic development includes agents that target
aspects of tumor progression required for metastasis, such as
invasion, angiogenesis, and survival of metastatic cells in the
microvasculature. These steps constitute the major cause of
morbidity and mortality in patients with epithelial tumors,
suggesting that antimetastatic agents in conjunction with
cytotoxic agents could greatly improve survival. One recently
successful approach involves inhibition of tumor angiogenesis.
Avastin (bevacizumab) targets endothelial growth factors and
their receptors and has proven effective in combination with
cytostatic agents for increasing progression-free survival and
decreasing overall mortality (39). However, at present, the
ability to intervene in other steps of metastasis is extremely
limited.
A potential approach to block hematogenous metastasis
involves targeting selectins and their ligands, which facilitate
the aggregation of tumor cells with platelets and leukocytes and
tumor cell adhesion to the endothelium. These cell-cell interactions enhance tumor cell resistance to cytolytic leukocytes
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Cancer Therapy: Preclinical
Table 1. Blood cell counts
Cell type
Vehicle
Neutrophils (103/AL)
Lymphocytes (103/AL)
Platelets (103/AL)
Red blood cells (106/AL)
Untreated (n = 375)
Vehicle treated (n = 15)
AcGnG-NM treated (n = 13)
6.5 F 2.8
1.1 F 0.7
5.0 F 2.5
961 F267
8.64 F 1
7.9 F 1.6
1.9 F 1.0
5.6 F 0.7
1146 F 251
10.8 F 1.8
7.8 F 2.6
1.5 F 0.5
6.1 F2.1
1031 F174
10.5 F 0.9
present in the circulation (4 – 6) and provide growth factors
that enhance tumor growth (7 – 9), suggesting that inhibitors
of selectins would decrease tumor cell survival and metastasis.
In this report, we showed that systemic administration of an
acetylated synthetic disaccharide will inhibit spontaneous
metastatic seeding of the lungs from cells shed from a primary
s.c. tumor. To our knowledge, this is the first report showing
that pharmacologic inhibition of tumor glycosylation in this
way can reduce spontaneous metastatic seeding of the lungs.
In addition to AcGnG-NM, other metabolic inhibitors of the
carbohydrate ligands for selectins have been described. For
example, 4-fluoro-N-acetylglucosamine and N-acetylgalactosaminides inhibit sLeX synthesis (40 – 42). Treating tumor cells
with N-acetylgalactosaminides also prevents tumor cell adhesion (43), but to our knowledge, this compound has not been
tested in models of spontaneous metastasis. Very high
concentrations (usually 1-10 mmol/L) of both 4-fluoro-Nacetylglucosamine and N-acetylgalactosaminides are needed to
alter selectin ligand expression. In contrast, AcGnG-NM acts
in the 10 to 50 Amol/L range in vitro, making it less likely to
have toxic side effects in vivo. Indeed, systemic administration
of the compound for 28 days at f45 mg/kg/d did not cause
acute toxicity, weight loss, obvious changes in behavior, or
alterations of standard blood chemistry and inflammatory
responses.
Other strategies for blocking selectin-carbohydrate interactions may prove useful for treating tumor cell dissemination
and metastasis as well, including (a) competition by soluble
recombinant forms of selectins or their glycopeptide and
glycolipid ligands; (b) competition by peptides derived from
the primary sequence of the carbohydrate binding site in
selectins; (c) anti-selectin antibodies, (d) oligosaccharides
related to sLea and sLeX; (e) inositol polyanions and sulfated
sugars, (f) molecular mimics of sLeX, including oligonucleotides; and (g) unfractionated and low molecular weight
heparins (for a review, see refs. 44 – 47). Heparin also blocks
tumor metastasis probably through multiple mechanisms (e.g.,
blockade of P- and L-selectin and inhibition of chemokine/
cytokine interactions with cell surface proteoglycans expressed
on tumor cells or the vasculature (reviewed in refs. 47, 48).
Unlike heparin, AcGnG-NM has a specific mechanism of
action, blocking selectin ligand formation without causing
any change in hemostasis.
Because AcGnG-NM inhibits the formation of selectin
ligands on a number of tumor cell lines in vitro (6, 16 – 18),
it may also be applicable to blocking metastatic seeding of
organs from a variety of carcinomas in vivo. Several human
tumor lines (e.g., LS180 colon carcinoma, PC-3 prostatic
carcinoma, and A549 and A427 lung adenocarcinomas) possess
selectin ligands sensitive to AcGnG-NM (6, 18). However, mea-
Clin Cancer Res 2006;12(9) May 1, 2006
suring spontaneous metastatic tumor formation using these
models has been difficult due to the low efficiency of tumor
growth from spontaneously shed cells. To circumvent this problem, indirect methods have been used to measure disseminated
tumor cells in tissues [e.g., lacZ expression (28), green fluorescent protein fluorescence of tagged cells (49), and PCR to detect
species specific sequence tags (50)]. The advantages of BrdUrd
labeling include its low cost, the absence of any prior manipulation of the tumor cell or immune response (e.g., by transfection of green fluorescent protein or lacZ; ref. 51), and its
effectiveness for measuring mitotic cells independent of the type
of tumor. Its primary disadvantage is that it underestimates
the extent of seeding because only mitotic cells take up the
nucleoside.
Fig. 6. AcGnG-NM has no effect on leukocytes. Blood samples were collected
from Es1(e) mice 4 weeks after surgical implantation of osmotic pumps containing
vehicle or AcGnG-NM. Gr-1-positive neutrophils (A) and CD45-positive
lymphocytes (B) were stained for P-selectin ligands using P-selectin IgM
chimera. C, CD41-positive platelets were stained for P-selectin using CD62P mAb.
D, acute peritoneal inflammation (peritonitis) was induced by injection of 3%
thioglycollate (ref. 25; Materials and Methods). Cells in the peritoneal lavage fluid
were stained with FITC-conjugated rat anti-mouse Gr-1mAb and counted by
flow cytometry. Statistics were calculated by a Student’s t test comparing two
groups (n = 4-5 animals).
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Research.
Inhibition of Metastasis
The data presented here provide crucial preclinical animal
data needed to begin phased trials in cancer patients. Because
AcGnG-NM is ‘‘first in class,’’ further studies are needed to
determine the optimal formulation, bioavailability, and maximum tolerated dose. Modification of the disaccharide by deoxygenation, fluorination, or by addition of reactive groups
may enhance its pharmacologic properties. Because the formation of selectin ligands depends on a complex biosynthetic
pathway involving several enzymes and oligosaccharide intermediates, other acetylated disaccharide intermediates could
provide additional candidates for drug development (19).
AcGnG-NM may be just the first of a new class of disaccharidebased agents that function as specific inhibitors of cancer
metastasis.
Acknowledgments
We thank David Ditto of the University of California, San Diego, Hematology
Core for blood counts and Drs. Ajit Varki and Jennifer Stevenson for many helpful
discussions.
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A Disaccharide-Based Inhibitor of Glycosylation Attenuates
Metastatic Tumor Cell Dissemination
Jillian R. Brown, Mark M. Fuster, Ruixia Li, et al.
Clin Cancer Res 2006;12:2894-2901.
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