A Potential Marker Protease of Invasiveness, Seprase, Is Localized on Invadopodia of Human Malignant Melanoma Cells1

[CANCER RESEARCH 54, 5702-5710, November 1, 19941 A Potential Marker Protease of Invasiveness, Seprase, Is Localized on Invadopodia of Human Malignant Melanoma Cells1

Wayne L. Monsky, Chen-Yong Lin, Atsuko Aoyama,2 Thomas Kelly,3 Steven K. Akiyama, Susette C. Mueller, and Wen-Tien Chen4

Department of Cell Biology and The Lombardi Cancer Research Center, Georgetown University School of Medicine, Washington, DC 21XXJ'71W. L M., C-i'. L, A. A., T. K., S. C. M., W-T. C.], and Laboratory ofDevelopmental Biology, National Institute ofDental Research, NIH, Bethesda, Maryland 20892 [S. K. A.]

ABSTRACT Gelatinase A can bind to the plasma membrane and become active at the invasive front (24). The association of integral membrane pro Seprase, a large, gelatin-degrading membrane-protease complex, is teases with ECM degradation has been reviewed (25), and a number expressed at the invasive front of malignant melanoma cells on invadopo dia, and its surface expression contributes to the invasive phenotype. An of plasma membrane-associated proteases have also been described in vitro assay was used to determine the matrix-degrading activity of four on neoplastic cells (19, 26â€”28). It remains unclear whether these malignant human melanoma cell lines. The lines differ in matrix-degrad plasma membrane proteases, as well as secreted proteases other than ing activity with LOX > RPM17951> A375 > SKMEL28. The seprase and Gelatinase A and plasminogen activators, are localized and activated Gelatinase A activities of these cell lines were also investigated. Seprase at the leading edge of the invading cell. and active gelatinase A are found in cell membranes of LOX and Previously, we defined a specialized membrane region, invadopo RPM17951 cells but not those of SKMEL2S cells. Experiments using dia, at the invasive front, the interface between the cell and the ECM anti-seprase monoclonal antibodies in conjunction with a cell fraction (29â€”31).Proteolytic degradation of various matrix components in ation technique indicate that seprase consists of Mr 97@000 polypeptides cluding fibronectin, laminin, type I collagen, and type IV collagen and is enriched on the ventral membrane of LOX in contact with planar occurs specifically at these sites in a time-dependent manner as the extracellular matrix substratum. Confocal microscopy further substanti cell extends invadopodia into the underlying substratum, suggesting ates our biochemical fmdings that seprase, as well as Gelatinase A, is localized on invadopodia membranes with a 6-fold increase ofseprase and local concentration of a variety of ECM-degrading proteases (32). The 4-fold increase of Gelatinase A intensity over the level expressed on dorsal secreted protease Gelatinase A was found to bind to and localize at membranes. In addition, LOX cells expressing higher levels of seprase at invadopodia of transformed chicken cells (24). Also, these invadopo the cell surface, as selected by fluorescence-activated cell sorting, are dia contain high levels of tyrosine-phosphorylated proteins (31), significantly more degradative than LOX cells with lower seprase expres p6Ã˜@@C(33)13@integrin (30), and microfilament components (34). sion. Taken together, our data show a concordance between seprase and Previously, we reported that a large membrane protease complex, Gelatinase A expression on the cell surface at invadopodia and the matrix termed seprase (surface expressed protease), may be a marker mol degrading activity of human malignant melanoma cells. Seprase and ecule for melanoma cell invasiveness (28). From correlating the major secreted proteases may act in concert to degrade components of the morphologically defmed matrix-degrading activity of tumor cells with extracellular matrix during invasion. the presence of a Mr 170,000 gelatinase by gelatin zymography, we have identified a malignant human melanoma cell line, LOX, that INTRODUCTION invades cross-linked gelatin films in vitro and contains seprase. How Neoplastic cells must attach to adhesion proteins of the ECM5, ever,theproteasewasnotfoundin32othertumorcelllinesthatdid proteolytically degrade the ECM, and migrate to distant sites in order not show extracellular gelatin degradation. The proteolytic activity to invade surrounding tissues and ultimately metastasize (1â€”4).Thus profile suggests that it is a neutral protease, but it is not yet classified far, ECM degradation and tumor invasion have been correlated with (28). Active seprase is associated with melanoma cell membranes, can an increased production of several classes of secreted enzymes, in be extracted from the membranes by detergent, and is a hydrophobic cluding MMPs (5, 6), serine proteases such as plasminogen activators protein. A similar gelatinase was also found in membranes recovered (7, 8), lysosomal cysteine proteases including cathepsin B (9, 10) and from culture media conditioned with LOX. These observations led us cathepsin L (1 1), as well as lysosomal aspartic proteases such as to isolate and characterize seprase from shed membrane vesicles of cathepsin D (12, 13). Many of these proteases can associate with LOX cells.6 The melanoma-seprase consists of novel M1 97,000 receptorson the plasma membrane(14). The plasma membraneas polypeptides, and an inhibitory mAb, V4, blocks activity as well as sociation of the urokinase plasminogen activator is well described the degradation of fibronectin substrata by LOX cells. However, it is (15â€”18).In addition, a growing body of literature indicates that the not yet clear what role seprase plays in the invasive process or how MMPs associate with the plasma membrane of invasive cells (19â€”23). this protease might be directed to the leading edge of the invading cell. In the present study, we examined matrix-degrading activity and Received 5/4/94; accepted 8/26/94. seprase expression in four malignant human melanoma cell lines, The costs of publication of this article were defrayed in part by the payment of page LOX, RPMI7951, A375, and SKMEL28. Invading tumor cells were charges. This article must therefore be hereby marked advertisement in accordance with defined as those that both express invadopodia and degrade ECM 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by USPHS Grant RO1CA39077 (to W-T. C.) and in part by components at invadopodial sites. We then investigated the presence the Lombardi Cancer Research Center Cytochemistry and Morphology Core and Flow of seprase activity in the cell membrane of melanoma cells using a Cytometry Core Facilities, USPHS Grant 1P30-CA-51008. rapid procedure to extract membrane-associated proteins with Triton 2 Present address: Bureau of Internation Cooperation, International Medical Center of Japan, 21-I, Toyama i-chome, Shinjuku-ku, Tokyo 162, Japan. X-1i4, followed by concentration of seprase from these extracts with 3 Present address: Department of Pathology, University of Arkansas for Medical a single step-organomercurial affinity chromatography based on the Sciences, Little Rock, AR 72205. 4 To whom requests for reprints should be addressed, at Department of Cell Biology, initial isolation procedure (28). Cell lines having higher levels of Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, DC invadopodial activity also have more seprase than other cells. More 20007. 5 The abbreviations used are: ECM, extracellular matrix; MMP, matrix metallopro teinase; mAb, monoclonal antibody; PBS, phosphate-buffered saline; DT, detergent; AQ, 6 W-T. Chen, C-Y. Lin, w. L. Monsky, and Y. Yeh. A membrane protease complex, aqueous; FACS, fluorescence-activated cell sorting; HRP, horse radish peroxidase; SDS, seprase, of human malignant melanoma cells is implicated in cell surface-matrix prote sodium dodecyl sulfate. olysis, manuscript in preparation. 5702

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. INVADOPODIA-ASSOCIATEDPROTEASES over, the seprase activity was not detected in SKMEL28, a line that mAbs. The generation and characterization of anti-seprase rat mAbs, in does not express invadopodia. Furthermore, mAbs against seprase are cluding V4, D8, D28, and D43, will be described.6'7mAb C37 was directed used in combination with a cell fractionation technique (31) to show against a p90 glycoprotein that was copurified with human melanoma seprase that seprase is enriched on the ventral membrane of LOX cells in during immunoaffinity purification, and it localizes exclusively on the plasma contact with an underlying substratum. The localization of seprase and membrane. Gelatinase A on the membranes of invadopodia has been confirmed Zymography of Seprase-Antibody Complexes. LOX cells were cultured tising mAbs and confocal microscopy. LOX cells expressing high for 2 days until confluence (1 X iO@cells/iS @2flask)and extracted in 2 ml 1.5% Triton-X 114 in TBS@. The cell extract was centrifuged at 10,000 X g levels of cell surface seprase were selected using FACS analysis and at 4Â°Cfor 10 min to remove cellular debris and then phase partitioned at 37Â°C found to be more active in the local degradation assay than LOX cells for 2 mm; the detergent phase was rediluted to 1 ml in TBSP. This detergent with lower levels of cell surface seprase. Thus, seprase expression at extract (100 I.Ll)was incubated with 1, 0.1, or 0.01 g.@g/@lanti-seprasemAbs the invadopodia is an important determinant of in vitro invasive D8, D28, or D43 or anti-p90 glycoprotein mAb C37 overnight at 4Â°C.The behavior. mixture was washed by re-partitioning three times in TBSP. The resulting DT phase was solubilized in 2X Laemmli sample buffer and analyzed for proteol ytic activity by gelatin zymography (28). MATERIALS AND METHODS Immunoprecipitation of Seprase from Surface-blotinylated LOX Cells. A confluent monolayer of LOX cells was cultured overnight on loosely Cell Culture. All cell lines were cultured in a 1:1 mixture of Dulbecco's cross-linked gelatin films (2.5% gelatin and 2.5% sucrose fixed with 0.5% modified Eagle's medium and RPMI 1640supplemented with 10%calf serum, glutaraldehyde) in 100-cm2 tissue culture plates. The cells were washed four 5% Nu-serum (Collaborative Research,Inc., Bedford, MA), 2 [email protected], times with 10-mi cold PBS@@@(1mM CaCl2, 1 m@iMgCl2, and 1 mM MnCl@.) 1 unit/mI penicillin, and 10 @&g/mlstreptomycin.The human amelanotic at 4Â°Candthen incubated at 4Â°Cfor 15 min in 10 ml of 20 p@g/mlBiotin melanomacelllineLOX(35)waskindlyprovidedbyL B.Chen(Dana-Farber (Pierce, Rocklord, IL). After washing six additional times in cold TBS@@@ Cancer Institute, Harvard Medical School, Boston, MA). The melanotic mel (1 mi@sCaCI2,1 mMMgCl2,and 1 mt@iMnCl@),theventral plasma membranes anoma SKMEL28 (ATCC HTB 72), the melanotic melanoma RPMI7951 in contact with the underlying gelatin substratum and the dorsal cell mem (ATCC HTB 66), and the amelanotic melanoma A375 (ATCC CRL 1619) branes were separated as described (31). The ventral membranes attached to were purchased from the American Type Culture Collection (Rockville, MD). the substratum were solubilized directly in 2 ml 1.5% Triton X-1 14 in In wm@ Local Degradation Assay. Glutaraldehyde-cross-linkedgelatin TBSP + +. The dorsal membranes were homogenized separately with a loose films for assaying cell invasiveness were prepared by a modification of the fitting pestle and Dounce homogenizer, followed by centrifugation first at technique previously described (19). Briefly, 15-mm glass coverslips were 10,000 x g at 4Â°Cfor 15 mm to remove large particulates and then by coated with a thin layer of 2% gelatin and 2% sucrose in PBS, air dried, then centrifugation of the supernatant at 100,000 X g at 4Â°Cfor1 h. The pellet was re-hydrated with 200 pJ of deionized water at 4Â°Cfor30 mm, and fixed with dissolved in 2 ml of 1.5% Triton-X 114 in TBSP + + and phase separated to 0.5% glutaraldehyde in PBS at 4Â°Cfor 30 min. Rhodamine conjugates of obtain DT and AQ phases of the dorsal and ventral microsomal preparations. human plasma fibronectin (0.5 mg/ml) and fluorescein conjugates of Matrigel Lowry protein assays were performed. Dot blotting of equal amounts of protein (0.5 mg/nil; Collaborative Research) were co-coupled to the surface of the from dorsal and ventral membrane preparations was labeled with streptavidin cross-linked gelatin film. Cells grown on the films were fixed with 3% horseradish peroxidase followed by ECL chemiluminescence (Amersham paraformaldehyde and 0.05% Triton X-100 in PBS. Specimens were photo Corp., Arlington Heights, IL) to show that approximately equal amounts of graphed with a Planapo 63/1.4 objective on a Zeiss Photomicroscope III (Carl biotinylated proteins were present in both the dorsal and ventral membrane Zeiss, Inc., Thornwood, NY) by transmitted illumination for differential inter preparations. Equal amounts of biotinylated protein from the AQ and DT ference contrast microscopy and then photographed by epifluorescence mi phases of the dorsal and ventral membrane preparations were brought to 200 croscopy for rhodamine and fluorescein labelings. The ultrastructure of the ,ii in TBSP + Â±and preincubated for 1 h at 4Â°Cwith Sepharose 4B (Pharmacia invadopodia was examined as described (30, 31). Ultrathin sections were Biotech, Inc., Piscataway, NJ). Seprase was izmnunoprecipitated with mAb prepared and post-stained using uranyl acetate and lead citrate according to D28-conjugated Sepharose 4B for 2 h at 4Â°Cand eluted by boiling in 2X standard procedures and photographed using a JEOL 1200 EX transmission Laemmli sample buffer containing 0.1 M dithiothreitol. The eluted proteins electron microscope operated at 60 kV. were subjected to 7.5% SDS-polyacrylamidegel electrophoresis, transferred to Rapid Procedure for IdentifyingSepraseand GelntinaseA. Tumorcells nitrocellulose, and immunoblotted using streptavidin-HRP as described were cultured in the serum-free media (14 millS cm2 flask) for 2 days until previously (31). confluence (1 x i07 cells/75 cm2 flask). Medium from each flask was Confocal Microscopy. Confocal microscopy was used to demonstrate that collected and centrifuged to remove cellular debris and stored at â€”84Â°Cfor seprase and Gelatinase A are localized on invadopodia. LOX cells were analysis. The cell layer was washed three times with cold Tris-buffered saline cultured overnight on the fluorescent-fibronectin-gelatin films, fixed, post Ã§rBS;150mMNaCI/50mMTris-HCI,pH 7.4). Cold 13% TritonX-114in permeabilized in 0.5% Triton X-100, and immunolabeled as described above TBSP(2 ml TBSand5 nmsEDTA)wasaddedto thecell layerandincubated with rat anti-seprase mAb D28 or rabbit polyclonal anti-Gelatinase A antibody at 4Â°Cfor 15 min. The Triton X-114 extract was scraped with a Nunc cell Ab45 (Ref. 24; provided by William 0. Stetler-Stevenson, National Cancer scraper and collected from each flask (â€”2.4ml)and equally aliquoted into two Institute, NIH, Bethesda, MD), followed by Texas Red-goat-anti-rat or don 1.5-mI microfuge tubes. The total Triton X-114 extracts of cells were centri key-anti-rabbit (Jackson Immuno Research Laboratories, West Grove, PA), fuged at 10,000 x g at 4Â°Cfor 2 min to remove insoluble particulates. The respectively. Rat mAb ES238, specific for the chicken fibronectin receptor, supernatant was phase partitioned into DT and AQ phases in a 37Â°C water bath was used for a negative control for rat mAb D28. Preimmune rabbit serum was for2 mm,untilcloudyin appearance,andthencentrifugedatroomtempera not available as a negative control for Ab45; therefore, secondary antibody hire at 5,000 X g for 1 min (36). The DT fraction was diluted with cold â€˜@ra5@ alone was used for negative control. Confocal images of the invading LOX to 1 nil. When isolating seprase activity, DT, AQ, and medium fractions (1 ml cells and the substratum were obtained using an MRC-600 laser scanning each) were incubated with 20 @.dofpacked amino phenyl mercuric acetate confocal system (Bio-Rad, Hercules, CA) equipped with a Nikon inverted Sepharose beads (37) end-over-end at 4Â°Cfor 30 miii, washed 3 times with microscope using a X 40 Nikon oil objective with a variable numerical cold TBSP, and eluted with 20 ,tl of 2X Laemmli sample buffer (38) con aperture set at NA 1.1. A series of [email protected] of the substratum taming 0.1 Mdithiothreitol at 25Â°Cfor15 mm. This procedure was also used and labeled cells was obtained. Pixel intensities of invadopodial membranes to obtain Gelatinase A activity with the exceptions that: (a) TBS containing 2 and dorsal membranes were compared using software provided with the mM N-ethylmaleimide instead of EDTA was used throughout the procedure; instrument. The significance of the differences between means was determined and (b) gelatin-Sepharose was used, and the protease was eluted with 2X using the Student t test. Lacmmli sample buffer, without dithiothreitol. After centrifugation at

5,000 x g for 30 a, the sample buffer eluates were electrophoresed immedi 7 C-Y. Un, Y. Yeh, and W-T. Chen. Identification of novel seprase-related protease ately on a gelatin zymogram as described (28). complexes on the cell surface of human placental endothelia, manuscript in preparation. 5703

FACS Analysis. Subconfluentmonolayersof LOX melanomacells were TableisnmobilizedCa@b6h 1 Percentageof human melanoma cells forming invado on suspended using PBS and were immunolabeled on ice. Rat anti-seprase mAbs ECMflin?podia @ D28 and V4, rat anti-p90 mAb C37, as well as rat anti-avian integrin ES238, 18h24hLOX21.3 were used to label suspended cells on ice, followed by labeling with fluores 4.2%RPMI795113.3 Â±33%C 36.9 Â±6.8%41.3 Â± cein-conjugated secondary goat anti-rat antibody (Rockland, Gilbertsville, 2.9%A3750 Â±2.1% 28.5 Â±3.5%33.6 Â± PA). MAb ES238 was used as a negativecontrolsince it is specific for avian 2.3%SKMEL280 3.1 Â±1.9%9.4 Â± integnn P1 (39). A Becton Dickson FA@SStarwas used for sterile sorting and 00 measuring antigen levels. Cell populations representing top and bottom levels a In all cases, fluorescein conjugates of fibronectin-coated, cross-linked gelatin films were used to culturedifferentcell lines as indicated.Cell invasivenesswas evaluatedby of antigen expression were deposited onto fluorescent, fibronectin-coated the appearance of invadopodia and surface indentations on the film. gelatin films at about 5 X 10@cells/iS-mm coverslip for 18 h. b At 6 h, significantly more LOX cells form invadopodia than RPM17951 cells (p < 0.025). At 6, 18, and 24 h, significantly more LOX cells form invadopodia than A375 cells (p < 0.005 in each case). At 6, 18, and 24 h, significantly more RPM17951cells form RESULTS invadopodia than A375 cells (p < 0.005 in each case). At 18 and 24 h, significantly more A375 cells from invadopodia than SKMEL cells (p < 0.025 and p < 0.005, respectively). Local Degradation of Matrix by Invadopodia. Tumor cells such C Percentage of cells invading the film shown was mean Â± SD from three independent as LOX and RPM17951 growing on a thick, weakly cross-linked experiments in which 200 random cell counts were made. gelatin film were prepared as described in â€œMaterialsandMethods,â€• and elaborate specialized membrane structures at the ventral surface of the cells, termed invadopodia, are shown in Fig. 1, a and b, were co-coupled on a cross-linked gelatin film at localized, specific arrowheads. Transmission electron microscopy is also used to visu points of invadopodial contact between the cells and substratum alize the invadopodia of LOX cells (Fig. 1, d and e). Invadopodia within 6 h after seeding these cells. The dark spots were largely found extend into the gelatin bead, where ECM fibers appear to be removed underlying cells, as would be expected for invadopodium-mediated (Fig. 1, d and e). LOX and RPMI7951 cells extend invadopodia into matrix degradation. Other dark spots are evidence of prior invadopo the underlying substrate within 6 h of seeding the cells (Table 1). dial degradation when migrating cells are no longer associated with After 1 day, more invadopodia are seen under each cell (Table 1; Fig. them (Figs. 2, g and h). After 1 day, 60% of the LOX cells and 47.4% 1, a and d, arrows), and large indentations in the gelatin substratum, of the RPMI7951 cells formed invadopodia and locally removed the indicating matrix degradation, are more frequent (Fig. 1, a and b, underlying fibronectin and Matrigel substrata (data not shown; see arrowheads). However, SKMEL28 cells do not form invadopodia and also Table 1). do not invade the gelatin film (Fig. lc; Table 1). Significantly, more Membrane Association of Seprase and Gelatinase A. To exam LOX cells form invadopodia than RPM17951 cells at 6 h (Table 1). inc whether seprase is associated with the membrane of invasive Furthermore, more LOX and RPM17951 cells form invadopodia than human melanoma cells, we have developed a sensitive approach that A375 cells, and A375 cells form more invadopodia than the nonin allows the rapid identification of seprase from Triton X-114 extracts vasive SKMEL28 cells (Table 1). In addition, these invadopodia are of cultured cells. The detergent phase of this extract contains hydro sites of active matrix proteolysis. LOX and RPM17951 degrade fi phobic proteins; thus, membrane-associated molecules are enriched. bronectin (Fig. 2, c and d) and Matrigel (Fig. 2, e andf) substrates that Cell monolayers were extracted at 4Â°Cin Triton X-114, and the

Fig. 1. Visualization of invadopodia from LOX A, â€¢1@ and RPMI7951 invading their underlying gelatin films. Differential interference contrast images of ;t cell culturesshow invadopodia(arrows)andinden tations on the cross-linked gelatin film (arrow heads) formed under both LOX at 1 day (a) and RPM17951 cells at 1 day (b) but not noninvasive SKMEL28 cells at 1 day (c). Bar, 25 pm. Trans mission electron microscopy is also used to visu a b c alize invadopodia (arrow) of LOX cells invading intoa gelatinbead(d ande). Bar, 1 p.m.The same cell is shown at differentmagnifications.Ventral membrane protrusions (arrow) penetrating the bead and endocytotic vesicles containing gelatin substratum (asterisks) are shown. Vesicles are of ten associatedwith invadopodia(arrowheads). @ . .@ ,â€˜,,@, :.. @ <@.. . .. @â€˜., @ .. â€˜I â€˜._4@., . . -. @ @â€˜. , d,,@, ,

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@ . .@â€˜â€˜;,f-@ . @ @4. :â€˜.@â€˜t@.. . â€˜ Fig. 2. Direct fluorescent visualization of the localdegradationofrhodamine-fibronectinandflu :â€˜ orescein/Matrigel-coated gelatin films under the ventral surface of LOX and RPM17951cells. The :@ :3@@ :@,,I @ cells were grown for 6 h on these films. Differential ,@ @. @_1,@ interference contrast microscopy is first used to @@@@ visualize the LOX cells (a) and RPMI7951 cells :. â€˜â€˜ (b). The same fields are then observedwith fiuo ,@, @ rescence microscopy using different filters to visu . -. â€˜, alize the degradation of the rhodamine-fibronectin by LOXcells(c) and RPM17951cells(d) or the degradatiOn of fluorescein-Matrigel by LOX (e) e g and RPM17951cells(1). Compositeimagesof @ differential interference contrast and rhodamine .@: . , florescence microscopy (g is a composite of a and c; h is a composite of b and d) show active degra @_. .;â€˜, - dation of the ECM film under both LOX and RPM17951cells, respectively. Black spots in fluo rescent images indicate areas under both LOX and RPMI7951 cells where fibronectin-Matrigel-gela tin substrates have been degraded (c and e; d and f). Bar, 25 i@m. @1c1@@t: h.

detergent extracts were quickly phase separated at 37Â°Cinto the AQ approximately Mr 300,000, a much higher molecular weight than the and DT phases. Seprase activities in the DT phase were then identified uncomplexed M@ 170,000 seprase activity (Fig. 4A, under D8, D43, using organomercurial Sepharose chromatography, followed by gel and D28). However, the control anti-p90 glycoprotein mAb C37 did atin zymography. LOX and RPM17951 cells contain gelatinase activ not form a complex with seprase, and the protease activity was seen ities exclusively in the DT phase, which appear as a Mr 170,000/ at Mr 170,000 (Fig. 4A). Immunoblotting analysis of the mAb-seprase 150,000 doublet (Fig. 3A, Lane D under LOX and RPM!). A375 cells complex confirmed that this high molecular weight complex consists exhibit a lower level of the M1 170,000 gelatinase activity but no of seprase and the bound mAb (data not shown), indicating that mAbs detectableM@150,000activityin theDT phase(Fig.3A,LaneD under D8, D28, and D43 bind to active seprase. mAb D28 recognized the A375). SKMEL28 cells exhibit no detectable gelatinase activity in the melanoma seprase at Mr 170,000 (Fig. 4B, Lane N) and the Mr 97,000 DT (Fig. 3A, Lane D underSKMEL).None of the aqueousfractions seprasesubunitwhen theproteasecomplex was dissociatedby heating from these four cell lines (Fig. 3A, Lane A under each cell line) to 100Â°Cfor 3 mm (Fig. 4B, Lane B). The protease complex immu contained this protease activity. noprecipitated by anti-seprase mAbs was recognized by anti-seprase To further substantiate the observation (24) that Gelatinase A mAbs but not by anti-p90 glycoprotein mAb C37 (Fig. 4E). On the associates with the membranes of invasive cells, we used a similar other hand, mAb C37 immunoblotted a Mr 90,000 polypeptide iso approach to that used for isolating seprase activity, and Gelatinase A lated by mAb C37 (Fig. 4E, Lane 4) but not the protease complex was enriched using gelatin-Sepharose (Fig. 3B). RPM17951 cells have immunoprecipitated with D28 anti-seprase mAb (Fig. 4E, Lane 3). high levels of Gelatinase A and B (Mr 72,000 and 92,000, respec To examine whether seprase is specifically expressed at the ventral tively) activities in the AQ phase (Fig. 3B, LaneA underRPM!), but cell surface of cells invading the ECM, LOX cells were grown to LOX cells have very low levels of Mr 72,000, with latent Gelatinase confluency on gelatin-coated dishes, surface biotinylated, and then A activity in the AQ phase (Fig. 3B, LaneA underLOX). Strikingly, mechanically sheared to separate dorsal from ventral membranes (31). both LOX and RPM17951 cells contain the Mr 62,000 Gelatinase A Membrane preparations of each were solubilized in Triton X-114. The activityin the DT phase,which may representanactivatedformof the DT phases of these dorsal and ventral membrane preparations were Mr 72,000 Gelatinase A (Fig. 3B, Lane D under LOX and RPM!). The immunoprecipitated with mAb D28, resolved by SDS-polyacrylamide DT phase of RPMI7951 Triton X-114 extracts also contains the Mr gel electrophoresis, transferred to nitrocellulose, and blotted with 92,000 Gelatinase B activity (Fig. 3B, Lane D under RPM!). A375 streptavidin-HRP. To elute the antigen from mAb D28-Sepharose and SKMEL28 cells contain the Mr 72,000 latent Gelatinase A activ beads, the sample was boiled, and the Mr 97,000 seprase subunit was ity in both the AQ and DT phases (Fig. 3B, Lanes A and D under A375 obtained (see Fig. 4B). Seprase was enriched in the ventral membrane and SKMEL). These gelatinolytic activities are MMPs because they preparation (Fig. 4C, Lane D under Ventral) compared to the amount were inhibited by EDTA but not N-ethylmaleimide or phenylmethyl present on dorsal membranes (Fig. 4C, Lane D under Dorsal). Fur sulfonyl fluoride (data not shown). The biochemical results, together thermore, no seprase was detected in the AQ phase obtained from the with the cell degradationassay,indicateda correlationbetweenhighly dorsal or ventral membrane Triton X-1 14 extracts (Fig. 4C, Lanes A invasive cells and the presence of seprase and active Gelatinase A in under Dorsal and Ventral, respectively). To assure that the immuno the DT phases of extracts from the invasive cells. precipitation was from equal starting amounts of dorsal and ventral Localization of Seprase and Gelatinase A on Invadopodia. The biotinylated proteins, the protein concentrations of the detergent ex possibility that seprase is located on the invadopodial surface was tracts were determined by the Lowry method, then dot blots with investigated using a panel of anti-seprase mAbs. Three experiments streptavidin-HRP on 2 @tg/mlbiotinylated protein were performed. were used to show the specific reactivity of anti-seprase mAbs to the We found that the total amount of biotinylated proteins in the DT melanoma antigen: antibody-protease complex formation (Fig. 4A); phases of dorsal and ventral membrane microsome preparations were immunoblotting (Fig. 4B); and immunoprecipitation (Fig. 4E). When equal (Fig. 4D, Lanes D and V, respectively). The amount of bioti the seprasefromLOX cell Triton X-114 extractswascomplexedwith nylated proteins in the AQ phases were equal as well (data not anti-seprase mAbs D8, D28, or D43, proteolytic activity shifted to shown). 5705

calize with holes in the substratum, showing that these invadopodia invade the underlying matrix. Seprase is specifically enriched on A invadopodia penetrating the fluorescein/fibronectin-coated gelatin LOX RPM A375 SKMEL film (Fig. 5, b and d). Further processing of the image shows that A D A DA DAD seprase labeling is highest at the perimeter of the invadopodia, sug

D@ C37 1 .1 .01 1 .1 .01 1 .1 .01 1 .1 .01 @r-Complexed

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@. ..@, D E i 2 3 4 DBV kDa .

â€¢@95

Fig. 3. Rapid identification of seprase and Gelatinase A activities in the detergent extracts of human melanoma cells. A, identification of seprase activities in the detergent phase (D) and aqueous phase (A) ofTriton X-114 extracts derived from LOX, RPM17951 (RPM!), A375 (A375), and SKMEL28 (SKMEL) human melanoma cells using amino phenyl mercuric acetate-Sepharose. Approximately equal amounts of proteins derived Fig. 4. Cell surface localization of seprase on LOX melanoma cells. A, gelatin zymo from the DT and AQ phases of the four cells, respectively (see â€œMaterialsandMethodsâ€•), gram of a SDS gel-shift experiment using anti-seprase mAbs. The DT phases of LOX cell were analyzed. Seprase appears as a M, 170,000/150,000 doublet in the DT phase (D) of Triton X-114 extracts were incubated 2 h with anti-seprase mAbs D8, D28, and D43, or LOXandRPM17951(RPM!)anda Mr170,000singletin thatof A375(A375.D).The anti-p90 mAb C37 at either 1, 0.1, or 0.01 mg/ml concentrations. Seprase activities were Mr 170,000 activity is not seenin the aqueousphase(A) of thesecell extracts.Further detected at a much higher molecular weight, approximately Mr300,000 (300 Wa; marked more, no Mr 170,000 activity is detected in the DT phase (D) or AQ phase (A) of Complexed), than Mr 170,000 (170 Wa; marked Uncomplexed) after incubating with SKMEL28 (SKMEL) cell extracts. Ordinate, molecular weights in kilodaltons (Wa). B, anti-seprase mAbs D8, D28, and D43. These results are interpreted as formation of identification of MMP activities, using gelatin Sepharose, in the AQ phase (A) and DT antibody-protease complexes that were not separated in the SDS buffer. B, immunoblot phase (D) of Triton X-1 14 cell extracts derived from LOX, RPM17951 (RPM!), A375 analysis of seprase. The DT phase of LOX cell Triton X-114 extracts, in reduced and (A375), and SKMEL28 (SKMEL). The Mr 72,000 (72 Wa) Gelatinase A activity appears nonboiled conditions (Lane N), as well as reduced and boiled conditions (Lane B), was in the AQ phase (A) and the DT phase (D) of RPM17951(RPM!), A375 (A375), and blotted and stained by anti-seprase mAb D28. Seprase appears as a M, 170,000 (170 Wa) SKMEL28 (SKMEL) extracts, whereas the M@62,000 (62 Wa) active Gelatinase A band when not boiled and a M, 97,000 (97 Wa) band when boiled. C, immunoblot activity is shown in the DT phase (D) of LOX and RPM17951(RPM!) extracts. Ordinate, localization of seprase in the ventral cell membrane. The DT phase (D) and AQ phase (A) molecular weights in kilodaltons (Wa). of dorsalandventralmembranesofLOX melanomacellswereobtainedafterthe living cell surface was biotinylated at 4Â°C.Thecell extracts were immunoprecipitated using mAb D28. The biotinylated seprase was then blotted and stained with streptavidin-HRP. D, dot blot analysisof 2 g.@g/mlbiotinylatedproteinsfrom the dorsalmembraneDT phase (D) and the ventral membraneDT phase(V) using labeled streptavidin.Bovine serum To examine the distribution of seprase and Gelatinase A on the albumin (2 p@g/mI)was used as a control for nonspecific binding of labeled streptavidin surface of invading cells, we used confocal microscopy. LOX cells (B). E, immunoblot analysis of D28 and C37 antigens. These antigens were immunopre invading fluorescein/fibronectin-coated gelatin films were labeled cipitated from the DT phase of LOX cell Triton X-114 extracts using mAb D28-coupled Sepharose (Lanes 1 and 3) or mAb C37-coupled Sepharose (Lanes 2 and 4), eluted in with anti-seprase mAb D28 (Fig. 5) or anti-Gelatinase A mAb Ab45 reduced and boiled conditions, then immunoblotted with either mAb D28 (Lanes 1 and 2) (Fig. 6). Figs. 5 and 6 show that brightly labeled invadopodia cob or mAb C37 (Lanes 3 and 4). Ordinate, relative molecular weights in kilodaltons (Wa). 5706

7 noninvadopodial ventral membrane (Fig. 5e). LOX cells were stained with anti-chicken fibronectin receptor, a rat mAb ES238 that does not .,, ,@ react with human fibronectin receptor, and this measurement was used Â¶1@0bj,â€”as a background control. This control labeling was significantly lower than the labeling at the dorsal protrusions and at the noninvadopodial C ventral membrane (p < 0.05). Similarly, the distribution of Gebatinase A on the plasma membrane of LOX cells was analyzed by confocal 250 @:1 microscopy. Fig. 6 shows that Gelatinase A-labeled invadopodia

200

. @50 Ptxel @ Intensity

8Distarn.@e2 3456 7 :@a@b dT (microns) 180

160 140 140 120 Pixel 100 120 VentralMembraneN DorsalMembrane0 Intensity 80 60 100 40

20 80 Pixel 0 @ Intensity60 0 2 3 4 5 6 7 8 9 10 Distance (microns) d 40 120 20 100 n VentralMâ€¢mbrane 0 j L . Dorsal Membrane 0 Invadopodia e 80 Fig. 5. COnIOCa1microscopyof seprase distribution on the invadopodia of LOX cells. A LOX cell, invading a thick fluorescein/fibronectin-coatedgelatinfilm (thickerthan Pixel 60 what is shown in Fig. 6), was labeled with anti-seprase mAb D28. a, dark, fluorescent Intensity negative holes are seen in the substratum underlying an invasive cell (arrow y). b, one large, brightly stained invadopodium colocalizes with these holes (arrow x). The image of the invadopodium(b) is 2.0 p.mventralto the imageof the substratum(a). Bar, 10 ,sm. 40 C,ahighermagnificationviewoftheinvadopodia[seenatxof(b)Jprocessedtoremove background intensity and colorized according to pixel intensity using confocal microscopy software (Bio-Rad, Hercules, CA) for increased detection of variations in staining mIen 20 sities. Bar, 2 g.un.d@within the area oflocal degradation, the pixel intensity across the area of the substratumbeing invaded (a, arrow y) is inversely correlatedwith the pixel intensity across the same length of the invadopodia stained by mAb D28 (b, arrow x). 0 Areas of low pixel intensity on the degraded film are seen at the same areas of high pixel intensity on the cell. e, the average pixel intensity of ten 4-@m2areas of representative invadopodiaas comparedwith 4-p@m2areasof ten dorsalmembraneprotrusionsandthat e of ventral membranes. The background staining pixel intensity has been subtracted from Fig. 6. COnfOCaImicroscopyof Gelatinase A distribution on the invadopodia of LOX these of the invadopodia, ventral and dorsal membranes. cells. A LOX cell, invading a thin fluorescein/fibronectin-coated gelatin film, was labeled with anti-Gelatinase A rabbit polyclonal antibody Ab45. a, dark, fluorescent-negative holesare seen in the substratumunderlyingtheinvasivecell (arrowy). b, one large gesting an association of seprase with the plasma membrane (Fig. Sc). brightly stained invadopodium colocalizes with these holes (arrow x). The image of the cell (b) is 2.0 @mventral to the image of the substratum (a). Bar, 10 p.m. c, a higher Statistical analysis of seprase distribution on the plasma membrane magnification view of the invadopodia [seen at x of (b)l is processed to remove back shows that the average pixel intensity of 4 @m2areas from 10 ground intensity and colorized according to pixel intensity using confocal microscopy representative invadopodia (131.8 Â±7.9) was significantly higher software (Bio-Rad, Hercules, CA) for higher resolution of the staining. Bar, 2 p.m. d, the area of local degradation, the pixel intensity across the area of the substratum being than the average pixel intensity at the dorsal membrane protrusions invaded (a, arrow y), is colocalized with the pixel intensity across the same length of the (23.1 Â± 4.7; p < 0.0005) and the surrounding, noninvadopodial invadopodia stained by Ab45 (b, arrow x). Areas of low pixel intensity on the degraded film are seen at the same areas of high pixel intensity on the cell. e, the average pixel ventral membranes (19.1 Â±3.5; p < 0.0005) (Fig. 5e). Furthermore, intensity of ten 4-g.tm2areasof representative invadopodia as compared with 4-@un2areas the dorsal protrusion labeling was not significantly greater than the of ten dorsal membrane protrusions and that of ventral membranes. 5707

LOX Cell Invasion IHIgh@% DLow@% 80 C 70 60 50 Fig. 7. Degradative activities of LOX cells cx Invasive Cells pressing high and low levels of seprase as deter mined by FACS. A, cell populations representing (%) the highest and lowest 1%of anti-seprase antibody binding were sorted, and their degradationof fi bronectin substrata were determined by counting 20 the number of cells associated with sites of deg radation. Five sets of coverslips were used for three separate experiments each, and 200 cells 0 @,-=. were counted on each coverslip. The values are D28 V4 mean Â±SD. Cells containing the top 1% expres sion of V4 and D28 antigens were significantly Monoclonal Antibody Selection more degradative than populations representing the bottom 1% antibody binding (p < 0.005). B, @ * <0.005 cell populations representing the highest and low est 10% of antibody binding were sorted, and their degradative activities were determined by count ing sites of degradation by the cell per 100 cells. B Cells expressing high levels of seprase were sig ni.ficantly more degradative (p < 0.005) than those expressing low levels. However, cells sorted based on expression of the control anti-p90 mAb C37 LOX Cell Invasion had similar invasive potential, regardless of the level of p90 expression. All staining was done on . HIgh @0% nonpermeabilized living cells at 4Â°C;thus, D28, 00 0 Low @0% V4, andC37 antigensexpressedon thecell surface 90 were labeled. 80 70 Invasive Cells 60 50 (%) 40 30 20 10 0 D28 C37 Monoclonal Antibody Selection

* P <0.005

colocalize with holes in the fibronectin-coated substratum. Gelatinase surface. We then used the in vitro local degradation assay to assess the A labeling is also highest at the perimeter ofthe invadopodia (Fig. 6c), degradative activity of the different cell subpopulations. As shown in supporting our previous finding that exogeneously added Gelatinase Fig. 7, the cell populations representing the top 1 or 10% of seprase A binds to invadopodia (24). Statistical analysis (Fig. 6e) shows that expression on the cell surface were significantly more invasive than the average pixel intensity of ten 4 p@m2areas at representative those representing the bottom 1 or 10% of seprase expression; 68% of invadopodial protrusions (108.9 Â±6.4) is significantly higher than the cell population representing the top 1% of anti-seprase mAb D28 that at the dorsal membranes (36.8 Â±3.9; p'

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. INVADOPODIA-ASSOCtATED PROTEASES same degradative activity, regardless of the level of mAb C37 binding cell membranes have a plasma membrane-associated binding protein (p90 expression; Fig. 7B). for Gelatinase A, and binding of the Gelatinase A to cell membranes with subsequent activation may occur during the formation of inva dopodia and the initiation of active ECM degradation (14, 24, 47â€”49). DISCUSSION RPM! 7951 cells appear to secrete more Gelatinase A than LOX cells We have investigated the ability of four human melanoma cell lines (Fig. 3), but they are less invasive (Fig. 2). A possible explanation for to locally degradeandinvadeinto the ECM.We have basedthis study this is the Gelatinase A localization at LOX invadopodia (Fig. 5). We on two morphological criteria to determine the initial stage of cell were not able to use the cell fractionation experiments described here invasion. The first is the ability of the tumor cells to form invadopodia to conclusivelystudyinvadopodia-associatedMMPactivity. Much of and create surface indentations into cross-linked gelatin films that can this activity adheres to the underlying gelatin via the gelatin-binding be visualized by differential interference contrast microscopy and domain of the enzyme and is not extracted in the Triton X-114- transmission electron microscopy (Fig. 1; Table 1). The second is the containing buffer. Although it can then be extracted with SDS, the ability of tumor cells to locally degrade fibronectin and Matrigel ability to separate secretions from invadopodia-associated activity is coatedonlooselycross-linkedgelatinfilmsasvisualizedbyfluores lost. cence microscopy (Fig. 2). Invadopodial structure and function have been descn'bed in Rous sarcoma virus-transformed chicken embryonic ACKNOWLEDGMENTS fibroblasts (29, 31). However, in that system, it was possible that these membrane protrusions were the result of the viral transformation. We We are most gratefulto Chong-ChouLee, Leslie Goldstein,andJohannes have shown here that human melanoma cell lines also elaborate Dodt for critical reviews of this work, and Yunyun Yeh, Maozheng Dai, and invadopodia and that ECM substrates are degraded at these specific Jing-Yi Zhou for technical assistance. membrane protrusions. Membrane protrusions have been described on other neoplastic cells (40, 41). These membraneprotrusionsmay be REFERENCES functionally related to the ECM-degrading invadopodia described 1. Nicolson,0. L Cancermetastasis:tumorcell andhostorganpropertiesimportantin here. However, invadopodia are initially defined by their association metastasis to specific sites.. Biochim. Biophys. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1994 American Association for Cancer Research. A Potential Marker Protease of Invasiveness, Separase, Is Localized on Invadopodia of Human Malignant Melanoma Cells

Wayne L. Monsky, Chen-Yong Lin, Atsuko Aoyama, et al.

Cancer Res 1994;54:5702-5710.

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