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Experimental Therapeutics |
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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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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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Mice.
Specific pathogen-free, male, 6–8-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 Student’s 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 Student’s 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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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 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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, 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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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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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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, 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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. 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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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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