Mouse Macrophage Metalloelastase Gene Transfer …...2 h on a six-well dish at a density of 23 105 cells per well. Two hours later, nonadherent cells were removed, and the resultant

Advances in Brief

Mouse Macrophage Metalloelastase Gene Transfer into a MurineMelanoma Suppresses Primary Tumor Growth byHalting Angiogenesis1

Manuel J. Gorrin-Rivas,2 Shigeki Arii,Masaharu Furutani, Masaki Mizumoto,Akira Mori, Koji Hanaki, Masato Maeda,Hiroaki Furuyama, Yoko Kondo, andMasayuki ImamuraDepartment 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 ofGastroenterology and Hepatology, Department of Internal Medicine[Y. K.], Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

AbstractMouse macrophage metalloelastase (MME) has been

associated with the generation of angiostatin, an internalfragment of plasminogen, which inhibits angiogenesis. Toclarify whether tumor cells that consistently generate MMEcan suppress angiogenesis and, therefore, inhibit the growthof primary tumors in vivo, we transfected a cDNA coding forMME into murine B16-BL6 melanoma cells that grow rap-idly and are MME deficient. The generation of active MMEin MME-transfected clones was confirmed by immunopre-cipitation followed by in vitro cleavage of plasminogen. Sub-cutaneous implantation of these stable clones in C57BL/6mice inhibited primary tumor growth by an average of 73%(P 5 0.00002), which directly correlated with a significantreduction of blood vessel formation (;76%) in such tumors.Microangiography revealed massive angiogenesis in controltumors (mock and vector); however, in MME-transfectedprimary tumors it demonstrated a decreased and disruptedvascular network. Western blot analysis using a specificanti-mouse angiostatin antibody demonstrated a strong 38-kDa immunoreactive band in MME-transfected tumors andin the serum of mice bearing those tumor cells.These resultsshow that placing MME gene directly into B16-BL6 mela-noma cells is an effective approach to suppress primarytumor growth in vivo because it halts angiogenesis.Our data

provide a feasible and promising strategy for gene therapyof cancer by targeting tumor vasculature.

IntroductionTumor growth and tumor invasion need angiogenesis,

which is a complex process requiring a fine balance of stimu-lators and inhibitors (1–4). Many investigations in oncologyhave focused on halting angiogenesis by using specific inhibi-tors (5–8). Angiostatin is one of the most potent endogenousantiangiogenic factors, which is derived from plasminogen andoriginally was purified from the serum and urine of mice bear-ing primary Lewis lung carcinoma tumors (9).

Macrophage metalloelastase (MMP-12)3 was first detectedin mouse peritoneal macrophage-conditioned media by Werband Gordon in 1975 (10), and, in 1992, Shapiroet al. (11)reported its cDNA sequence. MME has been correlated with thegeneration of angiostatin in a murine model of Lewis lung cellcarcinoma (12). In our previous report (13), we demonstratedthat macrophage metalloelastase is also produced by humantumor cells. Namely, 62.5% of the HCCs showed high expres-sion of steady-state HME mRNA, which was significantly as-sociated with the presence of elevated levels of a 40–45-kDaprotein that reacted with an antibody directed against the kringle1–3 elastase fragment of human plasminogen, suggesting thegeneration of angiostatin within these tumors (13). In addition,we found that those HCCs that showed HME gene expressionand angiostatin generation were hypovascular, explaining, inpart, the worst clinical outcome in this group of HCCs (14).Recently, Corneliuset al. (15) proved that among severalMMPs, macrophage metalloelastase is the most efficient an-giostatin-producing MMP. Moreover, these investigators foundthat the angiostatin generated from recombinant MME-treatedplasminogen is biologically active enough to inhibit humanmicrovascular endothelial cell proliferation and differentiationin vitro (15)

From this point of view, to evaluate the potential role of theMME gene in suppression of primary tumor growth by targetingangiogenesis, we transfected a MME cDNA into a rapidlygrowing murine melanoma cell line and studied its antitumorand antiangiogenic activities in a syngeneic murine tumormodel. Here, we will present the evidence indicating that theoverexpression of MME in B16-BL6 melanoma cells effectivelysuppresses tumor growthin vivo, and it is directly correlatedwith an inhibition of angiogenesis in the tumors.

Received 11/15/99; revised 2/10/00; accepted 2/11/00.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisementin accordance with 18 U.S.C. Section 1734 solely toindicate this fact.1 This work was supported in part by Grant 07457271 from the Ministryof Education, Science, Sports and Culture of Japan.2 To whom requests for reprints should be addressed, at Department ofSurgery 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:[email protected].

3 The abbreviations used are: MMP, matrix metalloproteinase; MME,mouse macrophage metalloelastase; HCC, hepatocellular carcinoma;HME, human macrophage metalloelastase; BSD, blasticidin S deami-nase; BS, blasticidin S; FBS, fetal bovine serum; PEM, peritonealexudate macrophage; vWf, von Willebrand factor.

1647Vol. 6, 1647–1654, May 2000 Clinical Cancer Research

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Materials and MethodsConstruction of a cDNA Coding for MMM. A cDNA

coding for domains I and II of MME was prepared as follows.Five micrograms of total RNA from thioglycollate-stimulatedmouse PEMs were reverse transcribed with lower primer 1(59-CAG CTC GAG TCA CAG GTC CTC CTC GGA GATCAG CTT CTG CTCAGT TGA TGG TGG ACT GCT AGGTTT-39), including anXhoI restriction enzyme site (underlined),a stop codon (bold), and a Myc epitope tag sequence (italic), byusing a First Strand cDNA Synthesis Kit (Pharmacia, Piscat-away, NJ). The resulting cDNA was then subjected to 30 cycles(60 s at 94°C, 90 s at 50°C, and 90 s at 72°C) of PCRamplification using a DNA thermal cycler (Astec, Tokyo, Ja-pan),Taq DNA polymerase (Wako, Nippon Gene, Tokyo, Ja-pan), and the following oligonucleotide primers: upper primer(59-TTG CTC GAG TGA CTC AGA CCG TGC ATCATGAAA TTT CTC ATG ATG ATT GTG-39), including anXhoIrestriction enzyme site (underlined) a stop codon (bold), andKozak’s motif (italic; Refs. 16, 17); and lower primer 2 (59-CGA CTC GAG TCA CAG GTC-39). The PCR-amplifiedcDNA fragment (888 bp) was subcloned into pBluescript IISK(2) (Stratagene, La Jolla, CA) and confirmed the sequenceby ALFred DNA sequencer (Pharmacia Biotech, Uppsala,Sweden).

Expression Vector Construction and Transfection.The 888-bp cDNA fragment coding for domains I and II ofMME and the Myc epitope tag was cloned into theXhoI restric-tion enzyme site of pCAG-BSD, a novel eukaryotic expressionvector, constructed with two parts of pCAGGS (kindly given byDr. J. Miyazaki, Department of Nutrition and PhysiologicalChemistry, Osaka University Medical School, Osaka, Japan)and pMAM2-BSD (Kaken, Tokyo, Japan). In the pCAG-BSD-MME plasmid, the transcription of MME and Myc epitopesequences was constitutively driven by the CAG enhancer-promoter, and the drug-resistant selection gene BSD was pres-ent. The orientation of the MME insert was confirmed byrestriction mapping, and a sense-type pCAG-BSD-MME wasselected to transfect a murine B16-BL6 melanoma cell line.Monoclones from wild type B16-BL6 melanoma cells wereobtained by the limiting dilution method and were cultured insix-well plates. DNA transfections were performed by 4ml ofLipofectAMINE (Life Technologies, Inc., Gaithersburg, MD)using 1mg of pCAG-BSD-MME or 1mg of pCAG-BSD vectoralone, as control, per one well of a six-well plate. The cells werecultured in 1 ml of serum-free OPTI-MEM (Life Technologies)medium containing DNA-LipofectAMINE complex for 4 h, andthen 1 ml of DMEM containing 20% FBS was added. After48 h, the cells in each well were dispersed in trypsin andreplated into two 10-cm culture dishes (1:20) in a completeculture medium containing 10mg/ml BS (Kaken) to select thetransfectants. 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 andpooled, and the cells were maintained in a complete culturemedium with 10mg/ml BS to get stable transfectants.

Collection of Mouse PEMs. PEMs were collected byperitoneal lavage with PBS from mice that had been given ani.p. injection of 3% thioglycollate medium (1.5 ml) 4 days

previously. The PEM were plated in a serum-free medium for2 h on a six-well dish at a density of 23 105 cells per well. Twohours later, nonadherent cells were removed, and the resultantadherent population was;98% pure according to immunolog-ical, morphological, and phagocytic criteria.

RNA Isolation and Northern Blot Analysis. TotalRNA from murine B16-BL6 cultured nontransfected parentalcells (mock) and transfected cells (vector and MME), cul-tured PEMs, and s.c. tumors growingin vivo were isolatedusing TRIZOL (Life Technologies) essentially as describedby the manufacturer. Routinely, 20mg of total RNA wereelectrophoresed on a 1% agarose-formaldehyde RNA gel andtransferred to a Hybond N1 nylon membrane filter (Amer-sham, Buckinghamshire, UK). Hybridization was performedat 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 reversetranscription-PCR, as described earlier, was used as a probe.It was labeled with [a-32P]dCTP using the Megaprime ran-dom primer DNA-labeling kit (Amersham). After hybridiza-tion, the filters were washed twice with 23 SSC and 0.1%SDS at room temperature for 10 min, once with 13 SSC and0.1% SDS at 65°C for 20 min, and once with 0.13 SSC and0.1% SDS at 65°C for 20 min.

Western Immunoblotting. To detect the secreted MMEprotein, stable MME-, vector-, and mock-transfected cells werecultured in complete media in 15-cm culture dishes until theyreached a semiconfluent condition, then washed twice withPBS, and recultured in 15 ml of serum-free media for 48 h. Thesupernatants were collected and purified by centrifugation(1,500g, 4°C, 10 min). To concentrate protein, the purifiedsupernatants were passed through a Millipore filter (NihonMillipore Ltd., Yonezawa, Japan). The filter was then washedwith 300 ml of PBS to recover the trapped proteins and loadedonto a 12% SDS-PAGE gel under reducing conditions. The gelwas blotted onto a polyvinyl difluoride membrane (Immobilon;Millipore, Bedford, MA). The membrane containing the trans-ferred proteins was blocked with Tris-buffered saline containing0.5% Tween 20 and 5% dried milk fat powder. After blockingfor 1 h at room temperature, the membrane was incubated witha solution of mouse anti-c-mycmonoclonal antibody (BiomolResearch Laboratories, Inc., Plymouth Meeting, PA), which wasdiluted (1:1,000) with the blocking solution. After a 1-h incu-bation at room temperature, the excess antibody was washedfrom the membrane with Tris-buffered saline containing 0.5%Tween 20. Incubation for 1 h at room temperature with a1:1,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Zymed Laboratories Inc., San Francisco, CA) fol-lowed. After washing, the bound antibody complexes weredetected using an ECL reagent (Amersham) as described by themanufacturer. To detect mouse angiostatin, 20mg of proteinisolated from both implanted s.c. dorsal tumors and serum ofmice bearing s.c. tumors were separated by 12.5% SDS-PAGEunder reducing conditions and blotted onto an Immobilon-Pmembrane as described earlier. The membrane was incubatedfirst 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 goatanti-rabbit IgG (heavy and light chain), conjugated with horse-

1648Antiangiogenesis and Tumor Suppression Induced by MME



radish peroxidase (Zymed Laboratories), diluted at 1:1000, for60 min at room temperature. The washing procedure and thedetection of bound antibody complexes were performed asdescribed above.

In Vitro Cell Proliferation. Cells of each selected clone(105; mock, vector 3, MME 1, and MME 3) were seeded with10% FBS-DMEM onto six-well culture plates in duplicate. Cellswere dispersed in trypsin, resuspended in PBS, and countedevery 24 h with a hemocytometer.

Immunoprecipitation and in Vitro Cleavage of Plasmin-ogen. Conditioned media from mock- and MME 3-transfectedclones were collected by incubating cells at 80% confluencewith DMEM without FBS for 24 h. These conditioned mediawere then incubated with rec-Protein G-Sepharose 4B beads(Zymed Laboratories) previously conjugated with a mouse anti-c-mycmonoclonal antibody (Biomol Research Laboratories) ina rotating wheel for 1 h at 4°C. The beads were collected by

centrifugation (20003 g, 4°C, 2 min) and washed three timeswith a buffer containing 300 mM Tris, 60 mM CaCl2, and 90 mM

NaCl (pH 7.5). Human Glu-plasminogen (Enzyme ResearchLaboratories, Inc., South Bend, IN) at a final concentration of0.8 mM in the same buffer was added to the collected beads(immunoprecipitates) and incubated for 12 h at 37°C on arotating wheel. Human Glu-plasminogen alone in the buffer atthe same final concentration was also incubated for 12 h at 37°Con a rotating wheel, as a control. The reaction was stopped byaddition of SDS sample buffer. Samples were subjected toWestern immunoblotting as described earlier. The membranewas probed with a 1:500 dilution of a murine monoclonalantibody against the kringle 1–3 elastase fragment of humanplasminogen, subclass IgG1 (product 3642; American Diagnos-tica Inc., Greenwich, CT). Then, a 1:1,000 dilution of horserad-ish peroxidase-conjugated goat anti-mouse IgG (Zymed Labo-ratories) was used as a secondary antibody.

Animal Studies. Tumor cells from each selected clone(2 3 106) were implanted s.c. into the dorsal area of 6- to8-week-old male C57BL/6 syngeneic mice purchased from Ja-pan SLC (Hamamatsu, Japan;n 5 10 in each group). Primarytumors were measured using a caliper on the days indicated, andtumor volume was calculated using the formula width2 3

Fig. 1 Construction and expression of a cDNA coding for MME.a,structure of the MME protein consisting of three domains: domain I orthe NH2-terminal signal peptide and proenzyme (8 kDa), domain II orthe catalytic domain (22 kDa), and domain III or the COOH-terminalportion (23 kDa). Activation and processing of MME would result inloss of both domains I and III, reaching a mature, processed, active22-kDa metalloproteinase. A cDNA coding for domains I and II ofMME and for themycepitope tag was amplified by reverse transcrip-tion-PCR. Amycepitope sequence coding for 10 amino acids (EQKLI-SEEDL) derived from the human pp62 c-mycpeptide was fused to the39 end, and a sequence coding for the Kozak motif was fused to the 59end of the resultant cDNA. The MME cDNA fragment was flanked inboth ends byXhoI restriction enzyme sites.b, Northern blot analysis forMME mRNA expression of stable transfectants. The MME transcriptposition is marked by thearrow. PEM, thioglycollate-stimulated mousePEM, which was included as a positive control. The amount of totalRNA loaded in the gel (20mg/lane) was monitored by ethidium bromidestaining (bottom panel).

Fig. 2 Western blot analysis of recombinant MME secretion by stabletransfectants and evaluation of its catalytic activity.a, detection ofrecombinant MME protein by Western blot analysis in serum-free mediaconditioned for 48 h of mock, vector 3, and MME 3 stable clones byusing an anti-c-mycmonoclonal antibody. Thefilled arrow indicates theposition of a pro-MME (domains I and II), and theopen arrowindicatesthe position of a mature and active MME (domain II).b, immunopre-cipitation of recombinant MME followed byin vitro cleavage of humanplasminogen.Lanes 1and 2, immunoprecipitates from vector 3 clonewithout or with plasminogen, respectively (incubation time, 12 h);Lanes 3and4, immunoprecipitates from MME 3 clone without or withplasminogen, respectively (incubation time, 12 h). Plasminogen incu-bated for 12 h in buffer was included as a control (Lane 5). Sampleswere examined by Western blotting with the mAb against the kringle1–3 elastase fragment of human plasminogen, andbracketsindicate the;50-kDa cleavage products arranged as a double band. Molecular massstandards are indicated on theleft. rMME, recombinant MME;h Plg,human plasminogen.

1649Clinical Cancer Research



length3 0.52 (length. width). At the end of the measurementperiod (21 days), the mice were sacrificed with an overdose ofdiethyl ether, their tumors were excised, and a portion of eachtumor was fixed in 4% paraformaldehyde-PBS and embedded inparaffin according to standard histological procedures. The re-maining portion was stored at270°C until used for Northernblot analysis. Allin vivoexperiments were performed accordingto the Guidelines for Animal Experiments of Kyoto University.

Immunohistochemical Staining and MicrovesselCounting. Endothelial cells were stained to examine mi-crovessel density. Sections from specimens fixed in 4%paraformaldehyde-PBS and embedded in paraffin were pro-cessed and immunohistochemically stained with a rabbit an-tiserum against vWf (Dako, Carpinteria, CA) using a Vec-tastain Elite ABC kit (Vector Laboratories, Inc., Burlingame,CA). Briefly, the sections were incubated overnight at 4°Cwith rabbit antiserum against vWf in PBS containing 1%BSA (1:50). After rinsing in PBS three times for 5 min, thesections were then incubated for 40 min at room temperaturewith biotinylated anti-rabbit IgG, followed by six washeswith PBS, and reacted with an avidin-biotin system using0.03% 3,39-diaminobenzide tetrahydrochloride, as chromo-gen, for;5 min. Sections were counterstained with Mayer’shematoxylin. Negative controls were prepared by substitutingnormal rabbit serum for the primary antibody.

To evaluate microvessel quantitation, slides werescanned at low-power magnification (340 –3100) to identifythe areas with the highest number of vessels. The five areasconsidered to have the highest densities were selected andcounted at3200 power magnification, and mean values6SEM were recorded. Any brown-staining endothelial cell orcluster of endothelial cells with or without a lumen, clearlyseparated from adjacent microvessels, tumor cells, and otherconnective tissue elements, was considered to be individualvessels. All counts were performed by two investigators in ablinded manner.

Microangiography. To evaluate angiogenesis of tumorallografts, microangiography was performed. Cells (106/0.2 mlof 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-oldmale C57BL/6 mice. Six days later, the mice were anesthetizedusing diethylether, and a 24-gauge cannula for perfusion wasinserted into the exposed thoracic aorta. Filtered barium sulfatesolution (0.25 g/ml) was perfused at a pressure of 150 mm Hgafter flushing the circulatory system with warmed heparinizedsaline. The whole body of the mice was fixed with 20% bufferedformalin, and the tumors in the dorsal area were sliced 1 mmthick by a Microtome through their center with surroundingnormal tissue. The slices from each tumor were subjected tocontact radiograph, and the X-ray film was examined with amicroscope. Corresponding histological sections (4mm thick)were prepared for each tumor slice and were stained with H&E(H&E).

Statistics. Statistical differences regardingin vitro cellgrowth, primary s.c. tumor growth, and microvessel densityamong mock, vector, and MME groups were analyzed usingStudent’s unpaired two-tailedt test. All results are expressed asmean6 SEM.P , 0.05 was considered statistically significant.

Fig. 3 Suppression of primary tumor growth.a, in vivo growth rate ofs.c. primary tumor derived from MME-transfected clones (MME 1 andMME 3), vector-transfected clone 3, and untransfected B16-BL6 paren-tal cells (Mock). Tumor volumes were determined at several time pointsusing the formula width2 3 length 3 0.52 (length. width), and allvalues are represented as mean6 SEM (n 5 10). At 3 weeks, micecarrying mock- or vector 3-transfected cells formed large primary tu-mors with volumes of.8500 mm3; in contrast, mice implanted withMME-transfected cells significantly formed smaller tumors (22326673 mm3 for MME 3 and 2991.276 1203 mm3 for MME 1). p, P 50.000001;pp, P 5 0.00002, by Student’s unpaired two-tailedt test.b,C57BL/6 syngeneic mice bearing s.c. primary B16-BL6 melanomatumors transfected with mock (top panel), vector (middle panel), orMME (bottom panel). Tumor location is denoted by thearrows. Mac-roscopic characteristics of the tumors are shown 3 weeks after implan-tation.Scale bar, 3 cm.




ResultsGene Construction and Expression. A PCR-amplified

cDNA fragment coding for the signal peptide-NH2-terminalproenzyme domain (domain I) and the catalytic domain (domainII) of MME, as well as for an antigenic epitope tag derived fromthe human pp62 c-myc(amino acids 410–419) peptide, fused tothe 39end, was placed into an expression vector (pCAG-BSD),downstream of the cytomegalovirus enhancer-chickenb-actinpromoter (Fig. 1a). The recombinant plasmid, called pCAG-BSD-MME, and vector plasmid alone (pCAG-BSD) were trans-fected into a murine B16-BL6 melanoma cell line. Stable trans-fectants were selected by BS and MME mRNA expression wasanalyzed 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. Simulta-neously, 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, aswell as in mouse PEMs that were included as a positive control(Fig. 1b). Although MME 1 and MME 3 clones expressed MMEat high levels, there was no significant difference between theproliferation rates of MME clones and control (mock or vector)tumor cellsin vitro.

Secretion of Active Metalloelastase by Stable MME-transfected Clones of B16-BL6 Melanoma Cells and Plas-minogen Cleavage. MME secretion into the serum-free con-ditioned medium was confirmed by Western blot analysis usingthe anti-c-mycmonoclonal antibody (Fig. 2a). Two bands (38and 30 kDa) were detected in the MME 3 clone. The 38-kDaprotein corresponded to the domains I and II of MME, and the30-kDa protein corresponded to the active form of MME aftercleavage with loss of domain I. It should be noted that themolecular mass of our recombinant active MME was higherthan that originally reported (18), possibly due to both thepresence of amyctag in our construct and the result of differentglycosylation in the recombinant protein.

In vitro cleavage of plasminogen performed after immuno-precipitation of proteins containing amyctag from conditionedmedia of stable MME 3 and mock-transfected clones revealedthe generation of;50-kDa fragments (double-band arrange-ment) only by the MME 3 clone (Fig. 2b).

Inhibition of Primary Tumor Growth in Vivo. Thestable transfectants (MME 1, MME 3, and vector 3) as well asnontransfected parental cells were s.c. implanted into the dorsalarea of C57BL/6 syngeneic mice to evaluate antitumor andantiangiogenic activities of MME. Three independent experi-ments were performed (first and second experiments,n 5 5mice per group; third experiment,n 5 10 mice per group).Representative results are shown in Fig. 3. Three weeks aftertumor cell implantation, MME-transfected clones dramaticallyinhibited the growth of primary tumors by either 76% (MME 3)or 69% (MME 1) compared with control tumors of mock- andvector 3 plasmid-transfected clones (MME 3versuscontrols,P 5 0.000001; MME 1versuscontrols,P 5 0.00002). Con-versely, 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 vector3-transfected clones enlarged and vascularized, with visible

Fig. 4 Immunohistochemical staining for endothelial cells in s.c. pri-mary tumors using a polyclonal antibody against vWf.a–c, represent-ative sections showing neovascularization (arrowheads) in 21-day tu-mor tissues of mock-transfected (a), vector 3-transfected (b), and MME3-transfected (c) clones. Original magnification,3200.d, microvesselsstained by the anti-vWf antibody were randomly counted from fivedifferent high-power fields (magnification,3200), and the values arerepresented as mean6 SEM of microvessel density per high-powerfield (n 5 5). p, MME 3 versusmock, P , 0.0001; MME 3versusvector 3,P , 0.0005, by Student’s unpaired two-tailedt test.




hemorrhagic focus on their surface (Fig. 3b, top and middlepanels). In contrast, tumors derived from MME-transfectedclones appeared small and pale, which is a characteristic oftumors with diminished neovascularization (Fig. 3b, bottompanel).

Microvessel Density of Primary s.c. Tumors. Immuno-histochemical analysis of vascularization of s.c. tumors per-formed by staining with polyclonal antibody against endothelialcell-specific von Willebrand factor demonstrated a decreasedmicrovessel density, counted from five different high-powerfields (3200), in 21-day tumor tissues obtained from the MME3-transfected clone (4.586 0.79; Fig. 4,c andd) compared withcontrol tumor tissues of mock-transfected (17.286 1.19; Fig. 4,a andd) and vector 3-transfected (21.36 2.22; Fig. 4,b andd)clones (MME 3versusmock,P , 0.0001; MME 3versusvector3, P , 0.0005). The microvessel density was quantified byusing five mice in each type of tumor group.

Spatiotemporal Evaluation of Neovascularization inTumor Isografts and Histological Findings. Microangiogra-phy was performed to evaluate angiogenesis in syngeneic tumorgrafts of mice bearing 6-day s.c. tumors (Fig. 5). Data arerepresentative of three independent experiments. Control tumorsderived from mock (Fig. 5b) and vector 3 (Fig. 5d) showedstrong neovascularization with a well-formed vascular network.Conversely, tumors derived from the MME 3-transfected cloneshowed a hypovascular pattern with a disrupted vascular net-work; the vessels shortened in length and diminished in caliber

(Fig. 5f). Vigorous angiogenesis was only seen in the connectivetissue around the tumors and in the underlying muscle but not inthe tumors (Fig. 5f). These tumors, in addition, showed exten-sive central necrosis (Fig. 5e).

MME mRNA Expression and Mouse Angiostatin Gen-eration in s.c. Primary Tumors. We examined whether pri-mary s.c. tumors can constitutively produce mRNA levels ofMME. All primary tumors were resected 21 days after implan-tation and subjected to both Northern and Western blot analyses.

The expression of transfected MME mRNA was onlydetected in primary s.c. tumors developed from MME clones;in contrast, primary tumors derived from vector and mockshowed no signal for MME. Fig. 6ashows representativeresults of Northern blot analysis. Simultaneously, Westernblot analysis of the same tumor tissues using a specificanti-mouse angiostatin antibody detected a stronger 38-kDaband in those tumors derived from the MME 3 clone than inthose tumors derived from mock or vector 3 clones (Fig. 6b).According to the molecular mass, this band represents mouseangiostatin (9).

Detection of Mouse Angiostatin in Serum of Mice Bear-ing s.c. Tumors. Western blot analysis was used to examineangiostatin levels in serum of mice. The angiostatin band wasdetected in the serum of those mice bearing MME-transfecteds.c. tumors. In contrast, the control group (vector 3 and mock)showed no band or a very faint band for angiostatin (Fig. 7).

Fig. 5 Tumor angiogenesis 6days after s.c. inoculation ofB16-BL6 stable clones inC57BL/6 syngeneic mice. Cells(106) of untransfected parentalB16-BL6 clone (mock;a andb)and vector 3 (cand d) andMME 3 (e and f) stable cloneswere implanted s.c. On day 6after tumor cell inoculation, mi-croangiography was performedas described in “Materials andMethods” (b, d, and f). Parallelsections were also stained withH&E (a, c, ande). Tumor loca-tion is denoted by thearrows.n,necrotic area. Original magnifi-cation,340.




DiscussionThe delivery of nutrients and oxygen through capillaries is

necessary for survival, growth, and invasion of tumors. There-fore, angiogenesis appears an attractive target in the fightagainst cancer. Several reports have focused on antiangiogenicagents. More recently, angiostatin and endostatin (19), proteo-lytic cleavage fragments of larger nonangiogenic precursorsshowing a well-known antiangiogenic activity, have gainedmajor attention among oncologists. However, there are twopotential limitations to clinical trials using these proteins: first,the requirement for long-term systemic drug delivery; and sec-ond, the relatively high dosages required to control the tumorgrowth. These would increase the treatment costs in patientssuffering from cancer. Thus, one attractive approach, to over-come these limitations, is deliveryin vivo of antiangiogenicgenes, which could further improve the treatment outcome inaggressive tumors.

Previous reports have found that metalloelastase is notresponsible for the generation of angiostatin from plasminogen(20–22); however, the experimental evidence regarding the

implication of MME in angiostatin generation (12, 15) and thesignificant correlation among HME gene expression, angiostatinproduction, and hypovascularity found in some HCCs (13, 14)impelled our group to clarify the potential role of MME ingrowth suppression of primary tumors by halting angiogenesis.Our results provide clear experimental evidence that the over-expression of MME suppresses the primary tumor growth bydown-regulation of tumor angiogenesis. Indeed, 76% inhibitionof primary tumor growth by the MME 3 clone was seen 21 daysafter tumor cell inoculation, which directly correlated with areduction of blood vessel formation (;76%) and a generation ofmouse angiostatin (38-kDa plasminogen fragment) in the tu-mors. Moreover, microangiography studies in mice carrying6-day s.c. tumors confirmed a reduction of tumor vessels as wellas a disruption of the vascular network in those tumors derivedfrom MME-transfected clones compared with control tumorsderived from either mock- or vector plasmid-transfected clones(Fig. 5). Thus, the suppression of primary tumor growth ismainly determined from early stages of tumor development byboth the blockage of neovascularization and the disruption ofalready-formed capillary vessels.

Immunoprecipitation of recombinant MME protein con-taining amyc tag by using rec-Protein G-Sepharose 4B beads,previously conjugated with a mouse anti-c-myc monoclonalantibody, and then followed byin vitro cleavage of humanplasminogen, confirmed the generation of active MME by ourMME-transfected clones. Both thein vitro capability of MME tocleave human plasminogen into;50-kDa fragments, whichwould correspond to human angiostatin according to previousreports (Refs. 9, 12, 20; Fig. 2b), and thein vivo localization ofhigh levels of angiostatin in s.c. tumors derived from MME-transfected clones (Fig. 6b) strengthen, without any doubt, theimportant role of MME in antiangiogenesis mediated by an-giostatin. These data agree with a previous report that demon-strated the augmentation of angiostatin production by up-regu-lating expression of MME in tumor-infiltrating macrophagesthrough granulocyte-macrophage colony-stimulating factor se-creted by tumor cells, which were engineered to produce it (23).Now we demonstrate that up-regulation of MME gene expres-sion directly in tumor cells is also a feasible alternative approachto 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 tumorcells most likely cleaves the circulating plasminogen seques-tered into the tumor stroma into active angiostatin. Therefore,

Fig. 6 MME gene expression and mouse angiostatin generation in s.c.tumor tissues.a, expression of MME mRNA in s.c. tumor tissues. Thetotal RNA isolated from s.c. tumors was subjected to Northern blotanalysis. The name of each transfectant from which the tumors origi-nated is indicated above thelane. Crude B16-BL6 melanoma cellsengineered to express MME (clone 3) were used as a positive control.The MME transcript position is marked by thearrow. The amount oftotal RNA loaded in the gel (20mg/lane) was monitored by ethidiumbromide staining (bottom panel).b, Western blot analysis of angiostatingeneration in s.c. tumor tissues. Twenty micrograms of protein per lanefrom s.c. tumor tissues were separated by 12.5% SDS-PAGE, blotted,and probed using a specific anti-mouse angiostatin monoclonal anti-body. A 38-kDa band corresponding to mouse angiostatin was identi-fied. The name of each transfectant from which the tumors were orig-inated is indicated above thelane. Molecular mass standards areindicated on theleft. m Agst, mouse angiostatin.

Fig. 7 Angiostatin detection in serum of mice bearing s.c. tumors byWestern blot. Twenty micrograms of protein per lane isolated fromserum were separated by 12.5% SDS-PAGE, blotted, and probed usinga specific anti-mouse angiostatin monoclonal antibody. A 38-kDa bandcorresponding to mouse angiostatin was identified. Molecular massstandards are indicated on theleft. m Agst, mouse angiostatin.




the switch of tumor vascularity toward a hypovascular pheno-type in those tumors derived from MME-transfected clonesresults from an excess of antiangiogenesis inhibitors (i.e., an-giostatin) over stimulators in the tumor microenvironment. Inaddition to the antiangiogenic mechanism involving angiostatin,MME might also degrade the perivascular matrix of new cap-illary vessels into the tumors, leading to endothelial cell detach-ment and disruption of the vascular network, thereby blockingtumor angiogenesis. It has been demonstrated already, for ex-ample, that in liver preservation injury, metalloproteases se-creted into the preservation media lead to endothelial cell de-tachment by digesting the perisinusoidal matrix (24). Thiswould explain the disrupted vascular network observed in themicroangiographies of tumors derived from MME-transfectedclones.

It should be emphasized that we have also observed asignificant suppression of lung metastases in our MME-trans-fected B16 melanoma cells, 21 days after i.v. inoculationthrough the tail vein, compared with control tumor cells.4 Thisobservation, together with the high levels of angiostatin alsodetected in the serum of mice bearing s.c. tumors derived fromMME clones (Fig. 7), opens a door in the treatment of distantmetastasis, which is the ultimate goal of cancer therapy.

Finally, this study clearly demonstrates that transduction ofthe MME gene into murine melanoma cells effectively sup-presses primary tumor growth by halting angiogenesis. Thissuggests a novel strategy for cancer gene therapy by targetingtumor vasculature.

AcknowledgmentsWe thank Dr. J. Miyazaki for kindly providing the expression

vector pCAGGS, Dr. S. Ito, Y. Yukawa, and S. Kawase (Department ofRadiology, Faculty of Medicine, Kyoto University) for advice andtechnical assistance in microangiography, and Lisa Mahoney Beltranand Beth Chamberlin for editorial assistance in preparation of themanuscript.

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2000;6:1647-1654. Clin Cancer Res Manuel J. Gorrin-Rivas, Shigeki Arii, Masaharu Furutani, et al. Halting AngiogenesisMurine Melanoma Suppresses Primary Tumor Growth by Mouse Macrophage Metalloelastase Gene Transfer into a

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Mouse Macrophage Metalloelastase Gene Transfer …...2 h on a six-well dish at a density of 23 105 cells per well. Two hours later, nonadherent cells were removed, and the resultant