
[Cancer Research 61, 1022-1028, February 1, 2001]
© 2001 American Association for Cancer Research
Experimental Therapeutics |
Metastatin: A Hyaluronan-binding Complex from Cartilage That Inhibits Tumor Growth1
Ningfei Liu2,
Randall K. Lapcevich2,
Charles B. Underhill,
Zeqiu Han,
Feng Gao,
Glenn Swartz,
Stacy M. Plum,
Lurong Zhang2 and
Shawn J. Green2, 3
Department of Oncology, Georgetown University Medical Center, Washington, D.C. 20007 [N. L., C. B. U., Z. H., F. G., L. Z.], and EntreMed, Inc., Rockville, Maryland 20902 [R. K. L., G. S., S. M. P., S. J. G.]
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ABSTRACT
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In this study, a hyaluronan-binding complex, which we termed Metastatin,
was isolated from bovine cartilage by affinity chromatography and found
to have both antitumorigenic and antiangiogenic properties. Metastatin
was able to block the formation of tumor nodules in the lungs of mice
inoculated with B16BL6 melanoma or Lewis lung carcinoma cells. Single
i.v. administration of Metastatin into chicken embryos inhibited the
growth of both B16BL6 mouse melanoma and TSU human prostate cancer
cells growing on the chorioallantoic membrane. The in
vivo biological effect may be attributed to the antiangiogenic
activity because Metastatin is able to inhibit the migration and
proliferation of cultured endothelial cells as well as vascular
endothelial growth factor-induced angiogenesis on the chorioallantoic
membrane. In each case, the effect could be blocked by either heat
denaturing the Metastatin or premixing it with hyaluronan, suggesting
that its activity critically depends on its ability to bind hyaluronan
on the target cells. Collectively, these results suggest that
Metastatin is an effective antitumor agent that exhibits antiangiogenic
activity.
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INTRODUCTION
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A potential therapeutic target on angiogenic endothelial cells is
hyaluronan, a large negatively charged glycosaminoglycan that plays a
role in the formation of new blood vessels (1)
.
Particularly high concentrations of hyaluronan are associated with
endothelial cells at the growing tips or sprouts of newly forming
capillaries (2
, 3)
. Similarly, when cultured endothelial
cells are stimulated to proliferate by cytokines, their synthesis of
hyaluronan is significantly increased (4)
. Interestingly,
this stimulation is restricted to endothelial cells derived from the
small blood vessels and is not seen in endothelial cells derived from
larger ones (4)
. In the case of mature blood
vessels, hyaluronan is present in perivascular regions and in the
junctions between the endothelial cells (5
, 6)
. Earlier
studies have shown that exogenously applied hyaluronan has different
effects on angiogenesis depending on its size, with macromolecular
hyaluronan inhibiting vascularization in chicken embryos, and
oligosaccharide fragments of hyaluronan stimulating vascularization in
the chorioallantoic membrane (7, 8, 9)
. Thus, hyaluronan
appears to be specifically associated with the endothelial cells of
newly forming blood vessels and can influence their behavior.
In addition to hyaluronan, endothelial cells involved in
neovascularization also express CD44 and other cell surface receptors
for hyaluronan (10, 11, 12)
. In particular, endothelial cells
associated with tumors express large amounts of CD44 (11)
.
In previous studies, we have shown that CD44 allows cells to bind
hyaluronan so that it can be internalized into endosomal compartments,
where the hyaluronan is degraded by the action of acid hydrolases
(13
, 14) . Thus, the expression of CD44 by endothelial
cells allows them to bind and internalize hyaluronan as well as any
associated proteins. The fact that both hyaluronan and CD44 are
up-regulated in endothelial cells involved in neovascularization
suggests that the turnover of hyaluronan by these cells is much greater
than that by cells lining mature blood vessels.
The increased turnover of hyaluronan in tumor-associated endothelial
cells suggested a possible mechanism to specifically target these
cells. Our initial idea was to use a hyaluronan-binding complex
isolated from cartilage to deliver chemotherapeutic agents specifically
to these endothelial cells. Purified by affinity chromatography, this
hyaluronan-binding complex consists of tryptic fragments of the link
protein and aggrecan core protein (5
, 15
, 16)
. We intended
to couple the hyaluronan-binding complex to a chemotherapeutic agent
such as methotrexate and use this derivative to attack endothelial
cells. We hoped that this derivative would bind to the hyaluronan on
the endothelial cells and then be internalized into lysosomes, where
the methotrexate would be released by the action of acid hydrolyses.
Surprisingly, however, in the course of these experiments, we found
that the hyaluronan-binding complex by itself (i.e., in the
absence of a chemotherapeutic agent) inhibited angiogenic activity.
Functionally, we termed the hyaluronan-binding complex, which inhibits
tumor growth, Metastatin.
In the present study, we demonstrate that Metastatin has a number of
intriguing biological activities, including inhibition of endothelial
cell proliferation and migration, inhibition of angiogenesis, and
suppression of tumor cell growth in chicken embryos and pulmonary
metastasis in mice. These effects are blocked by preincubating
Metastatin with hyaluronan, suggesting that the activity of Metastatin
depends on its ability to bind hyaluronan on the target cells.
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MATERIALS AND METHODS
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Preparation of Metastatin.
The hyaluronan-binding complex was prepared by a modified version of
the method originally described by Tengblad (15
, 16)
. Briefly, bovine nasal cartilage (Pel-Freez, Rogers,
AR) was shredded with a Sure-Form blade (Stanley), extracted overnight
with 4 M guanidine-HCl and 0.5 M sodium acetate
(pH 5.8), and dialyzed against distilled water to which 10x PBS
was added to a final concentration of 1x PBS (pH 7.4). The protein
concentration was measured, and for each 375 mg of protein, 1 mg of
trypsin (type III; Sigma, St. Louis, MO) was added. After digestion for
2 h at 37°C, the reaction was terminated by the addition of 2 mg
of soybean trypsin inhibitor (Sigma) for each milligram of trypsin. The
digest was dialyzed against 4 M guanidine-HCl and 0.5
M acetate (pH 5.8), mixed with hyaluronan coupled to
Sepharose, and then dialyzed against a 10-fold volume of distilled
water. The hyaluronan-Sepharose beads were placed into a chromatography
column and washed with 1.0 M NaCl, followed by a gradient
of 1.03.0 M NaCl. Metastatin was eluted from the
hyaluronan affinity column with 4 M guanidine-HCl and 0.5
M sodium acetate (pH 5.8), dialyzed against saline, and
sterilized by passage through a 0.2-µm-pore filter. For SDS-PAGE
analysis, the purified preparation was loaded onto a 10% BisTris
nonreducing gel (Novex, Inc.) and subsequently stained with Coomassie
Blue. To identify the link protein by Western blotting, the proteins on
the gel were transferred to a sheet of nitrocellulose and immunostained
with the 9/30/8-A-4 monoclonal antibody. (The monoclonal antibody,
developed by Dr. B. Caterson, was obtained from the Developmental
Studies Hybridoma Bank under the auspices of the NICHD and maintained
by the University of Iowa, Department of Biological Sciences, Iowa
City, IA 52242.) This identity was further confirmed by
NH2-terminal sequencing (Fig. 1)
. In tests of the biological activity of Metastatin, controls consisted
of Metastatin mixed with an excess mass of hyaluronan (Lifecore,
Chaska, MN) or a heat-inactivated preparation made by placing
it in a boiling water bath for 30 min.

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Fig. 1. SDS-PAGE and NH2-terminal analysis of
Metastatin. Lane 1, molecular mass markers;
Lane 2, Metastatin stained with Coomassie blue;
Lane 3, Western blot of Metastatin immunostained with an
antibody against the link protein. The fragment of aggrecan migrated as
a diffuse band at 85 kDa, whereas the truncated link protein was at
38 kDa. NH2-terminal sequence analysis of the 38-kDa band
indicated that the first 24 amino acids of the link protein have been
cleaved and is indicated on the schematic diagram.
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Endothelial and Tumor Cell Lines.
HUVECs4
were obtained from the Tumor Bank of the Lombardi Cancer Center
(Georgetown University, Washington, DC). ABAEs were kindly provided by
Dr. Luyuan Li (Lombardi Cancer Center), and BRECs were provided by Dr.
Rosemary Higgins (Pediatrics, Georgetown University). These endothelial
cells were cultured in 90% DMEM, 10% fetal bovine serum, and 10 ng/ml
bFGF. The B16BL6 melanoma tumor line was obtained from the National
Cancer Institute Central Repository (Frederick, MD), TSU cells were
obtained from the American Type Culture Collection (Rockville, MD), and
Lewis lung carcinoma cells were kindly supplied by Dr. Michael
OReilly (Childrens Hospital, Boston, MA). The tumor cells were
grown in 90% DMEM, 10% fetal bovine serum, and 2 mM
L-glutamine. For the mouse metastasis assays, cells were
generally used between passages 6 and 18.
Mice.
Specific pathogen-free, male, 68-week-old C57Bl/6 mice were obtained
from The Jackson Laboratory (Bar Harbor, ME). Animals were cared for
and treated in accordance with the procedures outlined in the Guide for
the Care and Use of Laboratory Animals (NIH Publication No. 86-23).
Animals were housed in a pathogen-free environment and provided with
sterilized animal chow (Harlan Sprague Dawley, Indianapolis, IN) and
water ad libitum.
Mouse Metastasis Model System.
For the experimental melanoma model, mice were inoculated i.v. in the
lateral tail vein with B16Bl6 cells (5 x 104
cells/animal) on day 0. Treatment was
initiated on day 3 with 5 (0.2 mg/kg), 15 (0.6 mg/kg), and 49 µg (2
mg/kg) of Metastatin and continued daily until animals were sacrificed
on day 14. After euthanasia, the lungs were removed, and surface
metastatic lesions were enumerated under a dissecting microscope.
Mice were also inoculated with Lewis lung carcinoma cells, which
aggressively form pulmonary metastases. Mice were injected i.v. in the
lateral tail vein with 2.5 x 105
cells/animal (day 0), and beginning on day 3, the Metastatin was
administered by daily i.p. injections of 15 (0.6 mg/kg) and 49 µg (2
mg/kg) or by three i.v. injections of 100 µg (4 mg/kg) on days 1, 3,
and 5. Animals were euthanized, and their lungs were removed and
weighed. To obtain the lung weight gain, the average lung weight of
nontreated mice (0.2 g) was subtracted from that of the treated
animals.
The number of pulmonary metastases and lung weight gains were reported
as mean ± SD, and the differences were compared using
Students t test. The groups were considered to be
different when the probability (P) value was <0.05.
Chicken Chorioallantoic Membrane Assays.
To measure angiogenesis, a chick chorioallantoic membrane assay was
performed using a modification of the methods of Brooks et
al. (17)
. For this, holes were drilled in the tops of
10-day-old chicken eggs to expose the chorioallantoic membranes, and
filter discs (0.5 cm in diameter) containing 20 ng of human recombinant
VEGF [20 µl (1 µg/ml); Pepro, Rocky Hill, NJ] were placed on the
surface of each chorioallantoic membrane (day 0). The holes were
covered with parafilm, and the eggs were incubated at 37°C in a
humidified atmosphere. One day later, the eggs were given injections
(via a blood vessel in the chorioallantoic membrane using a 30-gauge
needle) of the various substances [Metastatin (80 µg/egg) or
controls consisting of PBS or heat-inactivated Metastatin]. Three days
later (day 4), the chorioallantoic membranes and associated discs were
cut out and immediately immersed in 3.7% formaldehyde. For
computer-assisted image analysis, the discs were divided into quarters
with fine wires, and the blood vessels in each quarter were digitally
photographed and analyzed by an Optimas 5 program to calculate the
vessel area and length normalized to the total area measured. The means
and the SEs were calculated from all quadrants within each group, and
the statistical significance was determined by Students t
test. Twelve or more eggs were used for each sample point.
For the growth of xenografts on the chorioallantoic membrane, holes
were cut into the sides of 10-day-old eggs exposing the membrane (day
0), and then 1 x 106 B16BL6 or
TSU cells were applied to the membranes. Two days later, the eggs were
given i.v. injections of the various substances. On day 7, the tumor
masses were fixed in formalin, dissected free from the normal membrane
tissue, and weighed.
Cell Growth Assays.
To determine the effects of Metastatin on cell growth, the cell lines
were subcultured into 24-well dishes at a density of approximately
5 x 105 cells/well for the
endothelial cell lines (HUVEC, ABAE, and BREC) and 5 x 104
cells/well for tumor cell lines
(B16BL6, TSU, and Lewis lung carcinoma). For the dose-response
experiments, the medium was changed every other day, and at the end of
6 days, the cells were released with 0.5 mM EDTA in PBS,
and the cell number was determined with a Coulter counter (Hialeah,
FL).
ELISA Assay for Hyaluronan.
Cells were grown to confluence in 24-well dishes, and the conditioned
medium was collected, incubated with a biotinylated version of the
Metastatin (16)
, and then transferred to plates precoated
with hyaluronan (umbilical cord; Sigma). The hyaluronan present in the
conditioned medium interacts with the biotinylated Metastatin so that
less of it will be left to bind to hyaluronan attached to the plate. At
the end of the incubation, the plates were washed, and the amount of
biotinylated Metastatin remaining attached was determined by incubating
the plates with streptavidin coupled to peroxidase (Kirkegard &
Perry, Gaithersburg, MD) followed by a soluble substrate for
peroxidase. The amount of hyaluronan in the conditioned medium was
calculated by comparison with a standard curve with known amounts of
hyaluronan (16)
.
Wound Migration Assay.
A suspension of HUVECs (5 x 105
cells in 5 ml of 98% M199 and 2% fetal bovine serum) was added to
60-mm tissue culture plates that had been precoated with gelatin (2 ml
of 1.5% gelatin in PBS, 37°C, overnight) and allowed to grow for 3
days to confluence. An artificial "L"-shaped wound was generated in
the confluent monolayer with a sterile razor blade by moving the blade
down and across the plate. Plates were then washed with PBS, and
2 ml of PBS were added to each plate along with 2 ml of sample in M199
and 2% fetal bovine serum in the presence and absence of 5 ng/ml bFGF.
After an overnight incubation, the plates were treated with Diff-Quik
for 2 min to fix and stain the cells. The number of cells that migrated
were counted under x200 magnification using a 10-mm micrometer over a
1 cm distance along the wound edge. Ten fields for each plate were
counted, and an average for the duplicate was calculated.
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RESULTS
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Characterization of Metastatin.
Metastatin was isolated from bovine nasal cartilage by affinity
chromatography on hyaluronan-Sepharose. As shown in Fig. 1
, Metastatin
consisted of two molecular factions as determined by SDS-PAGE, a sharp
band at 38 kDa that corresponds to the link protein, and a diffuse band
at approximately 85 kDa that represents a tryptic fragment of the
aggrecan core protein (5
, 15
, 16)
. The diffuse
nature of this latter fraction is probably due to variations in the
degree of glycosylation and glycosaminoglycan content. The identity of
the link protein was verified by immunoblotting with a specific
monoclonal antibody against this protein (Fig. 1)
. In addition,
NH2-terminal sequence analysis of the 38-kDa band
revealed that the purified protein was missing the first 24 amino
acids. Previous studies have shown that this complex binds to
hyaluronan with high affinity and specificity (5
, 16)
.
Indeed, a biotinylated version of the preparation has been widely used
as a histochemical stain to localize hyaluronan in tissue sections
(5
, 16)
.
Because cartilage is known to contain various protease inhibitors,
which may contribute to its antitumor properties (18)
, we
wanted to determine whether Metastatin possessed such attributes. For
this reason, we used a chromogenic assay (Diapharma Group, Inc., West
Chester, OH) to assess the effect of Metastatin on the following
enzymes: (a) trypsin; (b) chymotrypsin;
(c) plasmin; and (d) elastase. At concentrations
as high as 100 µg/ml, Metastatin did not inhibit the activity of any
of the enzymes tested (data not shown).
Effect of Metastatin on Metastatic Tumors.
In initial experiments, we found that Metastatin was effective at
inhibiting pulmonary metastases of B16BL6 cells. When mice were given
daily i.p. injections of Metastatin 3 days after tumor inoculation,
lung metastases were strikingly reduced (Fig. 2
A). Fig. 2
B shows that the number of surface lung
metastases (>0.5 mm) in the mice treated with 15 and 49 µg
Metastatin/day were reduced by more than 80%. The doseresponse
curve shown in Fig. 2C
was constructed from two independent
experiments and shows that Metastatin decreased the number of
metastatic colonies in a dose-dependent manner with an ED50
of approximately 10 µg (0.4 mg/kg). Significantly, when Metastatin
preparations were premixed with macromolecular hyaluronan, the
antimetastatic activity was blocked, and the mean number of surface
pulmonary metastases was comparable to that seen in control mice
(Fig. 2B)
. This suggests that the ability of Metastatin to
bind hyaluronan is required for its anti-tumor activity.

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Fig. 2. Effect of Metastatin on B16BL6 melanoma metastasis. B16BL6
melanoma cells were injected into the tail veins of C57Bl/6 mice, and 3
days later, the mice were injected i.p. with increasing doses of
Metastatin. After 14 days, the lungs were removed, and surface
pulmonary metastases were counted. A, the lungs from
control animals had a greater number of metastases than those from the
Metastatin (49 µg)-treated animals. B, the number of
pulmonary metastases larger than 0.5 mm is plotted against the
concentration of Metastatin injected. Metastatin inhibits the number of
metastases, and the addition of hyaluronan to Metastatin blocked its
inhibitory activity. The values shown are the mean of at least five
mice/group; bars, SD. C, this
dose-response curve was derived from two independent experiments
(n = 5 for each point) and shows the
ratio of pulmonary metastasis in the test and control animals
(T/C) as a function of the Metastatin dose.
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Similar results were obtained with the Lewis Lung carcinoma cell line,
which is a more aggressive mouse tumor model. As shown in Fig. 3
, A and B, Metastatin inhibited pulmonary
metastasis of Lewis lung carcinoma cells in a dose-related fashion, as
reflected in the weight gain of the lungs. Furthermore, Metastatin was
effective when given by two different routes, i.p. and i.v. (Fig. 3
,
B and C).

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Fig. 3. Inhibition of pulmonary metastasis of Lewis lung carcinoma
cells. Lewis lung carcinoma cells were injected into the tail veins of
C57Bl/6 mice, and 3 days later, mice were treated with PBS or
Metastatin. Once the treatment was stopped, animals were euthanized,
and lungs were removed and weighed. A, shows
representative lungs from control and treated animals and illustrates
that Metastatin lowers the tumor burden. B, Metastatin
injected i.p. daily beginning on day 4 decreased the relative lung
weights in a dose-dependent fashion. C, the weight of
the lungs in animals was also decreased when Metastatin was
administered by three separate i.v. injections (100 µg/injection on
days 1, 3, and 5). The values shown are the mean of at least five
mice/group; bars, SD.
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Effect of Metastatin on in Vitro Cell Proliferation
and Migration.
In the next series of experiments, we wanted to determine whether
Metastatin has any effect on the growth of either endothelial or tumor
cells in tissue culture. For these experiments, the cells were grown in
the presence of varying concentrations of Metastatin for 6 days, and
then the final cell numbers were determined. Metastatin inhibited the
proliferation of the endothelial cell lines HUVEC, ABAE, and BREC (Fig. 4
A) and two of the tumor cell lines (B16BL6 and Lewis lung
carcinoma cells) but had no effect on the TSU cells (Fig. 4
B). Similar results were obtained when proliferation was
monitored by incorporation of bromodeoxyuridine (data not shown). It is
important to note that the growth inhibition of B16BL6 cells was
partially blocked when the preparation of Metastatin was premixed with
an excess of hyaluronan (Fig. 4
B).

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Fig. 4. The effects of varying concentrations of Metastatin on the
growth of cultured cells. The cell lines were cultured in 24-well
dishes in complete medium containing the indicated amounts of
Metastatin, with medium changes ever other day. After 6 days, the cells
were harvested with a solution of EDTA in PBS, and the cell numbers
were determined with a Coulter counter. A, a
dose-response curve is shown for the effects of Metastatin on the
growth of the endothelial cell lines HUVEC, ABAE, and BREC. In each
case, the growth of the cells was inhibited by Metastatin.
B, a dose-response plot is shown for the tumor cell
lines B16BL6 and TSU. Whereas Metastatin inhibited the growth of the
B16BL6 cells, it had little or no effect on the TSU cells. The addition
of an equal mass of hyaluronan to the Metastatin significantly reduced
its effect on the proliferation of B16BL6 cells.
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One possible explanation for the lack of TSU cell sensitivity to
Metastatin could be the amount of hyaluronan that they secrete because
it has an inhibitory effect. To test this possibility, conditioned
media from confluent cultures of the different cell lines were
collected and analyzed for hyaluronan by a modified ELISA. TSU cells
were found to secrete significantly larger amounts of hyaluronan into
the medium than the other cell lines (7 µg/ml versus <0.5
µg/ml, respectively). Indeed, this level of hyaluronan would be
sufficient to block the effects of added Metastatin.
We also examined the effects of Metastatin on the migration of
endothelial cells, another important factor in the process of
angiogenesis (19)
. In this assay, we examined the effect
of Metastatin on the migration of HUVECs using the wound migration
assay. Fig. 5
shows that at a concentration of 10 µg/ml, Metastatin inhibited the
migration of HUVECs by 50% as compared with controls treated with bFGF
alone. Again, similar results were obtained when migration was
monitored using Nucleopore filters (data not shown).

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Fig. 5. Dose-dependent inhibitory effect of Metastatin on the
migration of endothelial cells. HUVECs were grown to confluence on
gelatin-coated culture plates, wounded with a sterile razor blade, and
induced to migrate with bFGF in the presence of varying amounts of
Metastatin. The number of cells that migrated were enumerated using a
micrometer and microscope at x200 magnification.
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Effect of Metastatin on VEGF-induced Angiogenesis.
The fact that Metastatin could inhibit both the growth and migration of
endothelial cells in vitro suggested that it might also be
able to block angiogenesis in vivo. To test this
possibility, we examined the effect of Metastatin on angiogenesis
induced in the chick chorioallantoic membrane. In this assay, filter
papers containing recombinant human VEGF were placed on the
chorioallantoic membrane of 10-day-old eggs, which were then given a
single i.v. injection of the Metastatin or control preparations. Three
days later, the extent of vascularization in the region of the filters
was determined by computer-assisted image analysis. As shown in Fig. 6
, treatment with Metastatin reduced both the length and area of vessels
as compared with the control group, suggesting that Metastatin does
indeed have the ability to block VEGF-induced angiogenesis.

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Fig. 6. Effect of Metastatin on VEGF-induced angiogenesis. The top
of air sacs of 10-day-old chicken eggs were opened, exposing the
chorioallantoic membranes, and filter discs containing 20 ng of VEGF
were applied. The treated group was then injected i.v. with Metastatin
(80 µg/egg), and the control group did not receive injections. The
chorioallantoic membranes and associated discs were cut out on day 3
and analyzed by computer-assisted image analysis as described in
"Materials and Methods". Metastatin had a significant inhibitory
effect on both the (A) vessel length index and
(B) vessel area index.
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Inhibition of Tumor Growth on the Chorioallantoic Membrane.
To further explore the antitumor activity of Metastatin, we examined
its effect on the growth of B16BL6 and TSU cells on the chicken
chorioallantoic membrane. Tumor cells were applied to the
chorioallantoic membranes of 10-day-old chicken embryos and allowed to
grow for 1 week. Pilot experiments revealed that after inoculation with
106 cells, the take rate was almost 100% and
resulted in xenografts with weights from 50150 mg in 7 days. However,
when the inoculated eggs were given a single i.v. injection of the
Metastatin, the growth of the B16BL6 and TSU xenografts was greatly
inhibited (Fig. 7)
. Again, this inhibitory effect was abolished if the preparation of
Metastatin was heat inactivated or preincubated with its ligand,
hyaluronan (Fig. 7
B). It is important to note that
Metastatin did not appear to adversely affect the development of the
chicken embryos.

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Fig. 7. Effect of Metastatin on the growth of tumor cells on the
chicken chorioallantoic membrane. The top of the air sacs of 10-day-old
chicken eggs were opened, exposing the chorioallantoic membrane, and
pellets containing B16BL6 melanoma or TSU prostate tumor cells were
placed on the membrane. On day 3, the embryos were given a single i.v.
injection of PBS or Metastatin (80 µg). The tumors and associated
chorioallantoic membranes were removed on day 6 and weighed.
A and C, examples of the B16BL6 and TSU
xenografts from eggs treated with PBS, Metastatin, or heat-denatured
Metastatin. B and D, the weights of
B16BL6 and TSU xenografts from eggs treated with Metastatin and control
preparations are shown.
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DISCUSSION
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In this study we report that Metastatin, a cartilage-derived
hyaluronan-binding complex consisting of proteolytic fragments of
bovine link protein and aggrecan, is able to block the growth and
metastasis of tumor cells under the following conditions:
(a) a single i.v. injection of Metastatin into the
chorioallantoic membrane of chicken embryos inhibited the growth of
B16BL6 mouse melanoma cells and TSU human prostate cancer cells;
(b) multiple i.p. injections of Metastatin prevented the
experimental metastasis of B16BL6 and Lewis lung carcinoma cells to the
lungs of mice; and (c) three i.v. injections of Metastatin
were sufficient to inhibit the formation of Lewis lung carcinoma
metastasis. In each case, Metastatin did not have an obvious
detrimental effect on the host and was neutralized by complexing with
soluble hyaluronan.
Metastatin is a member of a family of hyaluronan-binding proteins that
also includes CD44, tumor necrosis factor-stimulated gene 6 (TSG-6),
versican, neurocan, and brevican (20)
. Interestingly,
Metastatin is similar to other factors that influence angiogenesis in
that it is a fragment of a larger complex. For example, Angiostatin is
a fragment of plasminogen, Endostatin represents a fragment of collagen
XVIII, and serpin consists of a fragment of antithrombin
(21, 22, 23, 24)
. It is possible that the production of the
peptide fragments is part of a feedback loop important in the
down-regulation of angiogenesis.
In addition to Metastatin, a number of other antiangiogenic factors
have been isolated from cartilage. Indeed, cartilage has been
extensively studied as a source of molecules that could account for its
avascular nature. Langer et al. (25)
first
reported a bovine cartilage fraction isolated by guanidine extraction
and purified by trypsin affinity chromatography that inhibited
tumor-induced vascular proliferation. In addition, Moses et
al. (26)
have recently isolated Troponin I from veal
scapulae, which was shown to have antitumor and antiangiogenic
properties. Lee and Langer (27)
have described a
guanidine-extracted factor from shark cartilage that inhibited
angiogenesis and suppressed tumor vascularization. Similarly, Moses
et al. (18)
isolated a factor from cultures of
scapular chondrocytes that inhibited angiogenesis in the chicken
chorioallantoic membrane and appeared to be a protease inhibitor.
However, it is likely that our preparation of Metastatin acts through a
distinct mechanism because it has no detectable antiprotease activity
and is inhibited by the addition of hyaluronan. It is tempting to
speculate that Metastatin may contribute to the avascular nature of
cartilage. Along these lines, we have previously found that
hypertrophic chondrocytes produce large amounts of free hyaluronan,
which may neutralize the effects of Metastatin in this region and
thereby allow blood vessels to invade (28)
.
The results of this study suggest that Metastatin has antiangiogenic
properties as demonstrated by its ability to block VEGF-induced
formation of blood vessels in the chicken chorioallantoic membrane. The
antiangiogenic effect of Metastatin was also consistent with our
finding that it blocked both the proliferation and migration of
cultured endothelial cells. Whereas Metastatin can directly attach
tumor cells, we believe that most of its antitumor activity is due to
its inhibition of angiogenesis because after its injection, the first
cells that it would encounter are the endothelial cells, which would be
exposed to the highest concentration. In addition, this antiangiogenic
mechanism is suggested by the fact that Metastatin blocked the growth
of TSU cells in vivo (i.e., on the chicken
chorioallantoic membrane) but had little or no effect on their
proliferation in vitro. In this particular case, it seems
likely that Metastatin was acting indirectly on the TSU tumor cells by
blocking angiogenesis.
In other cases, the antitumor activity of Metastatin may be due to the
combined action of direct killing of the tumor cells and the inhibition
of angiogenesis. Indeed, Metastatin does appear to partially inhibit
the growth of B16BL6 in tissue culture, and it could presumably have a
similar effect in vivo. Because many blood vessels that are
associated with tumors are leaky (29)
, Metastatin may be
able to escape the circulation to interact directly with the tumor
cells and block their proliferation. Along these lines, a recent study
by Maniotis et al. (30)
has indicated that some
tumors have the ability to form vasculature independent of endothelial
cells. The tumor cells themselves appear to take on the characteristics
of endothelial cells and are responsible for the formation of blood
vessels. It is possible that such dual-acting tumor cells could also
respond to Metastatin.
The biological effects of Metastatin appear to be closely linked to its
ability to bind hyaluronan. If the preparation of Metastatin was
premixed with hyaluronan, then this reversed its inhibitory effects on
tumor growth in vivo and in vitro and its effects
on the growth and migration of cultured endothelial cells. This
indirectly suggests that Metastatin is binding to hyaluronan associated
with the target cell. In the case of endothelial cells, particularly
high levels of hyaluronan are localized to the tips of newly forming
capillaries in the chicken chorioallantoic membrane and rabbit cornea
(2
, 3) . A variety of other cell types show a similar
relationship between proliferation and the production of hyaluronan
(31, 32, 33, 34)
. Whereas the hyaluronan present on proliferating
tumor and endothelial cells could interact directly with Metastatin in
the blood, the hyaluronan in other locations would not be exposed to
high concentrations of the complex. Most normal cells would be
protected by the fact that high concentrations of hyaluronan are
present in connective tissues such as the dermis, lamina propria,
and capsules (5
, 35
, 36)
, which would help to
neutralize the Metastatin that diffused into these regions. It is
important to note that under normal physiological conditions,
hyaluronan in the blood is maintained at low levels by the liver and
lymphatic system (37
, 38)
. Thus, the circulating
Metastatin should retain its hyaluronan binding activity.
Cell surface hyaluronan may serve as a target for other inhibitors of
angiogenesis and tumor growth. For example, Endostatin, a
20-kDa
fragment of the COOH-terminal of collagen XVIII that inhibits
angiogenesis (21
, 22)
, may also be able to bind
hyaluronan, as suggested by the presence of specific structural motifs
(39)
. Secondly, a soluble, recombinant version of
immunoglobulin fused with CD44 that binds to hyaluronan can inhibit the
growth of human lymphoma cells that express CD44 in nude mice
(40
, 41)
. TSG-6, which is secreted by a variety of cells
after stimulation with inflammatory cytokines, is able to both bind
hyaluronan and block tumor cell growth (42)
. In each of
these cases, these factors may be interacting with hyaluronan on the
surfaces of target cells to exert their effects on angiogenesis and
tumor growth.
In preliminary studies, we have found that Metastatin induces apoptosis
in the target cells. However, at present, the mechanism by which
Metastatin is able to do this is unclear. One possibility is that after
Metastatin has bound to hyaluronan on the cell surface, it is taken up
by the cells into lysosomes, where it is broken down into smaller
fragments that enter the cytoplasm and induce apoptosis, perhaps by
interacting with the mitochondrial membrane. Alternatively, Metastatin
could be interacting directly with the plasma membrane of the target
cells, causing damage that in turn induces the apoptotic cascade.
Clearly, future experiments will be directed toward elucidating the
mechanism by which Metastatin induces apoptosis in the target cells.
In conclusion, we have found that Metastatin is able to block tumor
growth in two model systems, and this effect depends on its ability to
bind hyaluronan. Metastatin appears to target both tumor cells and
endothelial cells that are involved in neovascularization. We postulate
that during angiogenesis, the endothelial cells up-regulate their
synthesis of hyaluronan, which then serves as a target for the injected
Metastatin. Thus, Metastatin may represent a new type of antitumor
agent, which targets cell surface hyaluronan.
 |
ACKNOWLEDGMENTS
|
|---|
We are grateful to Dr. Theresa LaVallee and Wendy Hembrough for
their assistance with the migration assay.
 |
FOOTNOTES
|
|---|
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.
1 Supported in part by the United States Army
Medical Research and Materiel Command under DAMD1717-94-J-4284,
DAMD17-98-1-8099, and DAMD17-99-1-9031. Additional support was obtained
from the Susan G. Komen Foundation and NIH Grant R29CA71545. 
2 These authors contributed equally to this
work. 
3 To whom requests for reprints should be
addressed, at EntreMed, Inc., Medical Center Drive, Suite 200,
Rockville, MD 20902. Phone: (301) 738-2494; Fax (301) 217-9594;
E-mail shawng@entremed.com. 
4 The abbreviations used are: HUVEC, human
umbilical vein endothelial cell; ABAE, adult bovine aorta endothelial
cell; BREC, bovine retinal endothelial cell; VEGF, vascular endothelial
growth factor; bFGF, basic fibroblast growth factor. 
Received 7/12/00.
Accepted 11/28/00.
 |
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