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Department of Surgery and Surgical Basic Science [M. J. G-R., S. A., M. F., M. Mi., A. M., K. H., M. Ma., H. F., M. I.] and Division of Gastroenterology and Hepatology, Department of Internal Medicine [Y. K.], Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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76%) in such
tumors. Microangiography revealed massive angiogenesis in control
tumors (mock and vector); however, in MME-transfected primary tumors it
demonstrated a decreased and disrupted vascular network. Western blot
analysis using a specific anti-mouse angiostatin antibody demonstrated
a strong 38-kDa immunoreactive band in MME-transfected tumors and in
the serum of mice bearing those tumor cells. These
results show that placing MME gene directly into B16-BL6 melanoma cells
is an effective approach to suppress primary tumor growth in
vivo because it halts angiogenesis. Our data
provide a feasible and promising strategy for gene therapy of cancer by
targeting tumor vasculature. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Macrophage metalloelastase (MMP-12) 3 was first detected in mouse peritoneal macrophage-conditioned media by Werb and Gordon in 1975 (10) , and, in 1992, Shapiro et al. (11) reported its cDNA sequence. MME has been correlated with the generation of angiostatin in a murine model of Lewis lung cell carcinoma (12) . In our previous report (13) , we demonstrated that macrophage metalloelastase is also produced by human tumor cells. Namely, 62.5% of the HCCs showed high expression of steady-state HME mRNA, which was significantly associated with the presence of elevated levels of a 40–45-kDa protein that reacted with an antibody directed against the kringle 1–3 elastase fragment of human plasminogen, suggesting the generation of angiostatin within these tumors (13) . In addition, we found that those HCCs that showed HME gene expression and angiostatin generation were hypovascular, explaining, in part, the worst clinical outcome in this group of HCCs (14) . Recently, Cornelius et al. (15) proved that among several MMPs, macrophage metalloelastase is the most efficient angiostatin-producing MMP. Moreover, these investigators found that the angiostatin generated from recombinant MME-treated plasminogen is biologically active enough to inhibit human microvascular endothelial cell proliferation and differentiation in vitro (15)
From this point of view, to evaluate the potential role of the MME gene in suppression of primary tumor growth by targeting angiogenesis, we transfected a MME cDNA into a rapidly growing murine melanoma cell line and studied its antitumor and antiangiogenic activities in a syngeneic murine tumor model. Here, we will present the evidence indicating that the overexpression of MME in B16-BL6 melanoma cells effectively suppresses tumor growth in vivo, and it is directly correlated with an inhibition of angiogenesis in the tumors.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Expression Vector Construction and Transfection.
The 888-bp cDNA fragment coding for domains I and II of MME and the Myc
epitope tag was cloned into the XhoI restriction enzyme site
of pCAG-BSD, a novel eukaryotic expression vector, constructed with two
parts of pCAGGS (kindly given by Dr. J. Miyazaki, Department of
Nutrition and Physiological Chemistry, Osaka University Medical School,
Osaka, Japan) and pMAM2-BSD (Kaken, Tokyo, Japan). In the pCAG-BSD-MME
plasmid, the transcription of MME and Myc epitope sequences was
constitutively driven by the CAG enhancer-promoter, and the
drug-resistant selection gene BSD was present. The orientation of the
MME insert was confirmed by restriction mapping, and a sense-type
pCAG-BSD-MME was selected to transfect a murine B16-BL6 melanoma cell
line. Monoclones from wild type B16-BL6 melanoma cells were obtained by
the limiting dilution method and were cultured in six-well plates. DNA
transfections were performed by 4 µl of LipofectAMINE (Life
Technologies, Inc., Gaithersburg, MD) using 1 µg of pCAG-BSD-MME or 1
µg of pCAG-BSD vector alone, as control, per one well of a six-well
plate. The cells were cultured in 1 ml of serum-free OPTI-MEM (Life
Technologies) medium containing DNA-LipofectAMINE complex for 4 h,
and then 1 ml of DMEM containing 20% FBS was added. After 48 h,
the cells in each well were dispersed in trypsin and replated into two
10-cm culture dishes (1:20) in a complete culture medium containing 10
µg/ml BS (Kaken) to select the transfectants. Cell death was observed
after 3–4 days in culture, and discrete colonies were apparent by 7
days after selection. Six individual colonies of each transfectant were
chosen and pooled, and the cells were maintained in a complete culture
medium with 10 µg/ml BS to get stable transfectants.
Collection of Mouse PEMs.
PEMs were collected by peritoneal lavage with PBS from mice that had
been given an i.p. injection of 3% thioglycollate medium (1.5 ml) 4
days previously. The PEM were plated in a serum-free medium for 2 h on a six-well dish at a density of 2 x
105 cells per well. Two hours later, nonadherent
cells were removed, and the resultant adherent population was 98%
pure according to immunological, morphological, and phagocytic
criteria.
RNA Isolation and Northern Blot Analysis.
Total RNA from murine B16-BL6 cultured nontransfected parental
cells (mock) and transfected cells (vector and MME), cultured PEMs, and
s.c. tumors growing in vivo were isolated using TRIZOL (Life
Technologies) essentially as described by the manufacturer. Routinely,
20 µg of total RNA were electrophoresed on a 1% agarose-formaldehyde
RNA gel and transferred to a Hybond N+ nylon
membrane filter (Amersham, Buckinghamshire, UK). Hybridization was
performed at 65°C for 3 h in Rapid Hybridization Buffer
(Amersham) with a labeled probe. The complete cDNA sequence (888 bp)
coding for domains I and II of MME obtained by reverse
transcription-PCR, as described earlier, was used as a probe. It was
labeled with [
-32P]dCTP using the Megaprime
random primer DNA-labeling kit (Amersham). After hybridization, the
filters were washed twice with 2x SSC and 0.1% SDS at room
temperature for 10 min, once with 1x SSC and 0.1% SDS at 65°C for
20 min, and once with 0.1x SSC and 0.1% SDS at 65°C for 20 min.
Western Immunoblotting.
To detect the secreted MME protein, stable MME-, vector-, and
mock-transfected cells were cultured in complete media in 15-cm culture
dishes until they reached a semiconfluent condition, then washed twice
with PBS, and recultured in 15 ml of serum-free media for 48 h.
The supernatants were collected and purified by centrifugation
(1,500G, 4°C, 10 min). To concentrate protein, the
purified supernatants were passed through a Millipore filter (Nihon
Millipore Ltd., Yonezawa, Japan). The filter was then washed with 300
µl of PBS to recover the trapped proteins and loaded onto a 12%
SDS-PAGE gel under reducing conditions. The gel was blotted onto a
polyvinyl difluoride membrane (Immobilon; Millipore, Bedford, MA). The
membrane containing the transferred proteins was blocked with
Tris-buffered saline containing 0.5% Tween 20 and 5% dried
milk fat powder. After blocking for 1 h at room temperature, the
membrane was incubated with a solution of mouse
anti-c-myc monoclonal antibody (Biomol Research
Laboratories, Inc., Plymouth Meeting, PA), which was diluted
(1:1,000) with the blocking solution. After a 1-h incubation at room
temperature, the excess antibody was washed from the membrane with
Tris-buffered saline containing 0.5% Tween 20. Incubation for 1 h
at room temperature with a 1:1,000 dilution of horseradish
peroxidase-conjugated goat anti-mouse IgG (Zymed Laboratories Inc., San
Francisco, CA) followed. After washing, the bound antibody complexes
were detected using an ECL reagent (Amersham) as described by the
manufacturer. To detect mouse angiostatin, 20 µg of protein isolated
from both implanted s.c. dorsal tumors and serum of mice bearing s.c.
tumors were separated by 12.5% SDS-PAGE under reducing conditions and
blotted onto an Immobilon-P membrane as described earlier. The membrane
was incubated first with a rabbit anti-mouse angiostatin monoclonal
antibody (product Y-410; Yanaihara Institute, Inc., Fujinomiya, Japan)
diluted at 1:250 for 1 h at room temperature and then with goat
anti-rabbit IgG (heavy and light chain), conjugated with
horseradish peroxidase (Zymed Laboratories), diluted at 1:1000, for 60
min at room temperature. The washing procedure and the detection of
bound antibody complexes were performed as described above.
In Vitro Cell Proliferation.
Cells of each selected clone (105; mock, vector
3, MME 1, and MME 3) were seeded with 10% FBS-DMEM onto six-well
culture plates in duplicate. Cells were dispersed in trypsin,
resuspended in PBS, and counted every 24 h with a hemocytometer.
Immunoprecipitation and in Vitro Cleavage of
Plasminogen.
Conditioned media from mock- and MME 3-transfected clones were
collected by incubating cells at 80% confluence with DMEM without FBS
for 24 h. These conditioned media were then incubated with
rec-Protein G-Sepharose 4B beads (Zymed Laboratories) previously
conjugated with a mouse anti-c-myc monoclonal antibody
(Biomol Research Laboratories) in a rotating wheel for 1 h at
4°C. The beads were collected by centrifugation (2000 x
G, 4°C, 2 min) and washed three times with a buffer
containing 300 mM Tris, 60
mM CaCl2, and 90
mM NaCl (pH 7.5). Human Glu-plasminogen (Enzyme
Research Laboratories, Inc., South Bend, IN) at a final concentration
of 0.8 µM in the same buffer was added to the
collected beads (immunoprecipitates) and incubated for 12 h at
37°C on a rotating wheel. Human Glu-plasminogen alone in the buffer
at the same final concentration was also incubated for 12 h at
37°C on a rotating wheel, as a control. The reaction was stopped by
addition of SDS sample buffer. Samples were subjected to Western
immunoblotting as described earlier. The membrane was probed with a
1:500 dilution of a murine monoclonal antibody against the kringle 1–3
elastase fragment of human plasminogen, subclass IgG1 (product 3642;
American Diagnostica Inc., Greenwich, CT). Then, a 1:1,000 dilution of
horseradish peroxidase-conjugated goat anti-mouse IgG (Zymed
Laboratories) was used as a secondary antibody.
Animal Studies.
Tumor cells from each selected clone (2 x
106) were implanted s.c. into the dorsal area of
6- to 8-week-old male C57BL/6 syngeneic mice purchased from Japan SLC
(Hamamatsu, Japan; n = 10 in each group). Primary
tumors were measured using a caliper on the days indicated, and tumor
volume was calculated using the formula width2 x
length x 0.52 (length > width). At the end of the
measurement period (21 days), the mice were sacrificed with an overdose
of diethyl ether, their tumors were excised, and a portion of each
tumor was fixed in 4% paraformaldehyde-PBS and embedded in paraffin
according to standard histological procedures. The remaining portion
was stored at -70°C until used for Northern blot analysis. All
in vivo experiments were performed according to the
Guidelines for Animal Experiments of Kyoto University.
Immunohistochemical Staining and Microvessel Counting.
Endothelial cells were stained to examine microvessel density.
Sections from specimens fixed in 4% paraformaldehyde-PBS and embedded
in paraffin were processed and immunohistochemically stained with a
rabbit antiserum against vWf (Dako, Carpinteria, CA) using a Vectastain
Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). Briefly, the
sections were incubated overnight at 4°C with rabbit antiserum
against vWf in PBS containing 1% BSA (1:50). After rinsing in PBS
three times for 5 min, the sections were then incubated for 40 min at
room temperature with biotinylated anti-rabbit IgG, followed by six
washes with PBS, and reacted with an avidin-biotin system using 0.03%
3,3'-diaminobenzide tetrahydrochloride, as chromogen, for 5 min.
Sections were counterstained with Mayer’s hematoxylin. Negative
controls were prepared by substituting normal rabbit serum for the
primary antibody.
To evaluate microvessel quantitation, slides were scanned at low-power magnification (x40–x100) to identify the areas with the highest number of vessels. The five areas considered to have the highest densities were selected and counted at x200 power magnification, and mean values ± SEM were recorded. Any brown-staining endothelial cell or cluster of endothelial cells with or without a lumen, clearly separated from adjacent microvessels, tumor cells, and other connective tissue elements, was considered to be individual vessels. All counts were performed by two investigators in a blinded manner.
Microangiography.
To evaluate angiogenesis of tumor allografts, microangiography was
performed. Cells (106/0.2 ml of PBS) of either
stable MME-transfected tumor cells (MME 3) or stable vector- and
mock-transfected tumor cells, as controls, were injected into the s.c.
space of the dorsal area of 4-week-old male C57BL/6 mice. Six days
later, the mice were anesthetized using diethylether, and a 24-gauge
cannula for perfusion was inserted into the exposed thoracic aorta.
Filtered barium sulfate solution (0.25 g/ml) was perfused at a pressure
of 150 mm Hg after flushing the circulatory system with warmed
heparinized saline. The whole body of the mice was fixed with 20%
buffered formalin, and the tumors in the dorsal area were sliced 1 mm
thick by a Microtome through their center with surrounding normal
tissue. The slices from each tumor were subjected to contact
radiograph, and the X-ray film was examined with a microscope.
Corresponding histological sections (4 µm thick) were prepared for
each tumor slice and were stained with H&E (H&E).
Statistics.
Statistical differences regarding in vitro cell growth,
primary s.c. tumor growth, and microvessel density among mock, vector,
and MME groups were analyzed using Student’s unpaired two-tailed
t test. All results are expressed as mean ± SEM.
P < 0.05 was considered statistically significant.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. The recombinant plasmid,
called pCAG-BSD-MME, and vector plasmid alone (pCAG-BSD) were
transfected into a murine B16-BL6 melanoma cell line. Stable
transfectants were selected by BS and MME mRNA expression was analyzed
by Northern blot. Nontransfected parental cells (mock) and cells
transfected with vector plasmid alone (vectors 3 and 4) were used as
controls, showing no signal for MME. Simultaneously, three different
MME-transfected clones (MME 1, MME 2, and MME 3) were also evaluated by
Northern blot. MME mRNA was detected in MME 1 and MME 3 clones, as well
as in mouse PEMs that were included as a positive control (Fig. 1B)
. Although MME 1 and MME 3 clones expressed MME at high
levels, there was no significant difference between the proliferation
rates of MME clones and control (mock or vector) tumor cells in
vitro.
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. Two bands
(38 and 30 kDa) were detected in the MME 3 clone. The 38-kDa protein
corresponded to the domains I and II of MME, and the 30-kDa protein
corresponded to the active form of MME after cleavage with loss of
domain I. It should be noted that the molecular mass of our recombinant
active MME was higher than that originally reported (18)
,
possibly due to both the presence of a myc tag in our
construct and the result of different glycosylation in the recombinant
protein.
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50-kDa fragments (double-band arrangement) only by
the MME 3 clone (Fig. 2B)
.
Inhibition of Primary Tumor Growth in Vivo.
The stable transfectants (MME 1, MME 3, and vector 3) as well as
nontransfected parental cells were s.c. implanted into the dorsal area
of C57BL/6 syngeneic mice to evaluate antitumor and antiangiogenic
activities of MME. Three independent experiments were performed (first
and second experiments, n = 5 mice per group; third
experiment, n = 10 mice per group). Representative
results are shown in Fig. 3
. Three weeks
after tumor cell implantation, MME-transfected clones dramatically
inhibited the growth of primary tumors by either 76% (MME 3) or 69%
(MME 1) compared with control tumors of mock- and vector 3
plasmid-transfected clones (MME 3 versus controls,
P = 0.000001; MME 1 versus controls,
P = 0.00002). Conversely, primary tumors grew rapidly
to volumes of >8500 mm3 in control animals
implanted with either vector 3 or mock (Fig. 3A)
. In
addition, s.c. tumors derived from mock- and vector 3-transfected
clones enlarged and vascularized, with visible hemorrhagic focus on
their surface (Fig. 3B
, top and middle
panels). In contrast, tumors derived from MME-transfected clones
appeared small and pale, which is a characteristic of tumors with
diminished neovascularization (Fig. 3B
, bottom
panel).
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) compared with control tumor tissues of
mock-transfected (17.28 ± 1.19; Fig. 4, A and D
) and vector 3-transfected (21.3 ± 2.22; Fig. 4, B and D
) clones (MME 3 versus mock,
P < 0.0001; MME 3 versus vector 3,
P < 0.0005). The microvessel density was quantified by
using five mice in each type of tumor group.
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. Data are representative of three
independent experiments. Control tumors derived from mock (Fig. 5B)
and vector 3 (Fig. 5D)
showed strong
neovascularization with a well-formed vascular network. Conversely,
tumors derived from the MME 3-transfected clone showed a hypovascular
pattern with a disrupted vascular network; the vessels shortened in
length and diminished in caliber (Fig. 5F)
. Vigorous
angiogenesis was only seen in the connective tissue around the tumors
and in the underlying muscle but not in the tumors (Fig. 5F)
. These tumors, in addition, showed extensive central
necrosis (Fig. 5E)
.
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The expression of transfected MME mRNA was only detected in
primary s.c. tumors developed from MME clones; in contrast, primary
tumors derived from vector and mock showed no signal for MME. Fig. 6A
shows representative
results of Northern blot analysis. Simultaneously, Western blot
analysis of the same tumor tissues using a specific anti-mouse
angiostatin antibody detected a stronger 38-kDa band in those tumors
derived from the MME 3 clone than in those tumors derived from mock or
vector 3 clones (Fig. 6B)
. According to the molecular
mass, this band represents mouse angiostatin (9)
.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Previous reports have found that metalloelastase is not responsible for
the generation of angiostatin from plasminogen (20, 21, 22)
;
however, the experimental evidence regarding the implication of MME in
angiostatin generation (12
, 15)
and the significant
correlation among HME gene expression, angiostatin production, and
hypovascularity found in some HCCs (13
, 14)
impelled our
group to clarify the potential role of MME in growth suppression of
primary tumors by halting angiogenesis. Our results provide clear
experimental evidence that the overexpression of MME suppresses the
primary tumor growth by down-regulation of tumor angiogenesis. Indeed,
76% inhibition of primary tumor growth by the MME 3 clone was seen 21
days after tumor cell inoculation, which directly correlated with a
reduction of blood vessel formation (76%) and a generation of mouse
angiostatin (38-kDa plasminogen fragment) in the tumors. Moreover,
microangiography studies in mice carrying 6-day s.c. tumors confirmed a
reduction of tumor vessels as well as a disruption of the vascular
network in those tumors derived from MME-transfected clones compared
with control tumors derived from either mock- or vector
plasmid-transfected clones (Fig. 5)
. Thus, the suppression of primary
tumor growth is mainly determined from early stages of tumor
development by both the blockage of neovascularization and the
disruption of already-formed capillary vessels.
Immunoprecipitation of recombinant MME protein containing a
myc tag by using rec-Protein G-Sepharose 4B beads,
previously conjugated with a mouse anti-c-myc monoclonal
antibody, and then followed by in vitro cleavage of human
plasminogen, confirmed the generation of active MME by our
MME-transfected clones. Both the in vitro capability of MME
to cleave human plasminogen into 50-kDa fragments, which would
correspond to human angiostatin according to previous reports (Refs.
9
, 12
, 20
; Fig. 2B
), and the in vivo
localization of high levels of angiostatin in s.c. tumors derived from
MME-transfected clones (Fig. 6B)
strengthen, without any
doubt, the important role of MME in antiangiogenesis mediated by
angiostatin. These data agree with a previous report that demonstrated
the augmentation of angiostatin production by up-regulating expression
of MME in tumor-infiltrating macrophages through granulocyte-macrophage
colony-stimulating factor secreted by tumor cells, which were
engineered to produce it (23)
. Now we demonstrate that
up-regulation of MME gene expression directly in tumor cells is also a
feasible alternative approach to enhance the production of angiostatin
within the tumors.
In our experimental model using B16-BL6 melanoma cells, the MME released directly from the MME-transfected tumor cells most likely cleaves the circulating plasminogen sequestered into the tumor stroma into active angiostatin. Therefore, the switch of tumor vascularity toward a hypovascular phenotype in those tumors derived from MME-transfected clones results from an excess of antiangiogenesis inhibitors (i.e., angiostatin) over stimulators in the tumor microenvironment. In addition to the antiangiogenic mechanism involving angiostatin, MME might also degrade the perivascular matrix of new capillary vessels into the tumors, leading to endothelial cell detachment and disruption of the vascular network, thereby blocking tumor angiogenesis. It has been demonstrated already, for example, that in liver preservation injury, metalloproteases secreted into the preservation media lead to endothelial cell detachment by digesting the perisinusoidal matrix (24) . This would explain the disrupted vascular network observed in the microangiographies of tumors derived from MME-transfected clones.
It should be emphasized that we have also observed a significant
suppression of lung metastases in our MME-transfected B16 melanoma
cells, 21 days after i.v. inoculation through the tail vein, compared
with control tumor
cells.4
This
observation, together with the high levels of angiostatin also detected
in the serum of mice bearing s.c. tumors derived from MME clones (Fig. 7)
, opens a door in the treatment of distant metastasis, which is the
ultimate goal of cancer therapy.
Finally, this study clearly demonstrates that transduction of the MME gene into murine melanoma cells effectively suppresses primary tumor growth by halting angiogenesis. This suggests a novel strategy for cancer gene therapy by targeting tumor vasculature.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by Grant
07457271 from the Ministry of Education, Science, Sports and Culture of
Japan. 
2 To whom requests for reprints should be
addressed, at Department of Surgery and Surgical Basic Science,
Graduate School of Medicine, Kyoto University, 54 Shogoin, Kawara-cho,
Sakyo-ku, Kyoto 606-8507, Japan. Phone: (81) 75-751-3445; Fax:
(81) 75-751-3219; E-mail: mjgorrin{at}kuhp.kyoto-u.ac.jp
3 The abbreviations used are: MMP, matrix
metalloproteinase; MME, mouse macrophage metalloelastase; HCC,
hepatocellular carcinoma; HME, human macrophage metalloelastase; BSD,
blasticidin S deaminase; BS, blasticidin S; FBS, fetal bovine serum;
PEM, peritoneal exudate macrophage; vWf, von Willebrand factor.
4 M. J. Gorrin-Rivas, S. Arii, M. Furutani, M.
Mizumoto, A. Mori, K. Hanaki, M. Maeda, H. Furuyama, Y. Kondo, and M.
Imamura, unpublished data.
Received 11/15/99; revised 2/10/00; accepted 2/11/00.
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