Journal of Cell Science nrae uo growth and tumor apoptosis increased reduced degradation, gelatin resulting MMP-14, in activates and redistributes ADAM12 Article Research erdto,osre nteeivdpda a needn of independent was invadopodia, Gelatin these 2011). in observed al., matrix degradation, et and ADAM12 (Albrechtsen both (MMP)-14 containing metalloproteinase formation invadopodia the of in clusters results of ADAM12 to against ligation that antibody described (Fro monoclonal recently have a activity We 2011). catalytic al., et the Roy 2011; of engagement with without invasion) and or growth through cancer- adhesion, that cell stimulate as thought could (such processes is ADAMs related It activities, 2008). domain al., various at et these that the integrins) (Edwards as surface as domains, (such cell proteins such the transmembrane cysteine-rich exosites, other with through and interact mediated disintegrin-like functions extracellular on surface also activity, metalloproteinase cell their but on of only functions of not the rely to However, processing appear 2011). ADAMs that domain Reiss, the transmembrane clear and Saftig the become in 2005; near (Blobel, has role cleavage it undergo major years, that receptors few Edwards a past 2011; play the al., ADAMs Over et 2008). (Duffy al., cancers et many to of contribute pathogenesis and tumors the malignant in several overexpressed that are ADAMs evidence provide (a metalloproteinases) ADAMs of and functions disintegrin pathological and biological the on Studies Introduction complex protein ternary a words: that Key suggest data our conclusion, protects In ADAM12 that progression. confirmed tumor and surface of MMP-14 increased activated ADAM12- significance cell gelatin of to ADAM12, of of abolition The of leading levels on on composed implantation and increased required. apoptosis, with dependent orthotopic effect degradation against were tumors Furthermore, gelatin cells produced ADAM12 be The ADAM12. mice MMP-14-mediated tumor of nude to against BIK. in in tail antibodies cells reduction appeared and cytoplasmic MCF7 monoclonal a expressing the and BCL2L11 specific by nor by underscored activity proteins effects ADAM12 was MMP-14 pro-apoptotic apoptosis-protective of MMP-14 tumor activity the of the and catalytic activation stimulated of of ADAM12-induced the was inhibition degradation expression neither gelatin but by reduced activation, MMP-14 localization, this abrogated endogenous with to surface, Subsequent was breast cell decreased, activation. human the degradation its in at was expression in ADAM12 ADAM12 expressing resulting apoptosis and cells surface, to MMP-14 cancer cell cell contribute between breast the correlation and human to positive and tumors recruited a pathology. 293-VnR malignant was is cancer in in there of that that upregulated aspects demonstrated show are We various we cancer. in ADAM12, Here, role including cancers. key of metalloproteinases), a pathology and play the MMP-14, disintegrin and (a MMP-9 ADAMs MMP-2, Likewise, particular in (MMPs), metalloproteinases Matrix Summary 10.1242/jcs.129510 doi: 4707–4720 126, ß Science Cell of Journal 2013 July 9 Accepted ( correspondence for *Author 3 2 1 Albrechtsen Reidar on oanvlcnetfrtedvlpeto M-4treigdusi raigcancer. treating in drugs MMP-14-targeting of development the for concept novel a to point inClu Nielsen Cilius Finn h ooNrikFudto etrfrPoenRsac,Uiest fCpnae,20 oehgn Denmark Copenhagen, 2200 Copenhagen, of University Research, Denmark Denmark Protein Copenhagen, Copenhagen, 2200 for 2100 Copenhagen, Center Copenhagen, of Foundation University of Nordisk (BRIC), University Novo Centre Rigshospitalet, The Innovation Biochemistry, and Clinical Research of Biotech Department and Sciences Biomedical of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. DM2 MMP-14, ADAM12, [email protected] 2 1 a laM Wewer M. Ulla , ai Kveiborg Marie , V b nernadMP1 ttetmrcl ufc euae h ucino M-4 hsitrcinmight interaction This MMP-14. of function the regulates surface cell tumor the at MMP-14 and integrin 3 a V ) b nern eai erdto,Apoptosis degradation, Gelatin integrin, 3 1 1 n ail Fro Camilla and ot Stautz Dorte , hihe al., et ¨hlich 1 hlich ¨ oa Vikesa Jonas , ppoi Mqo ta. 02.Teatvt fMP1 is and MMP-14 CD151 cell tetraspanin of by as activity activation such including proteins The mechanisms, surface 2012). multiple al., by collagen-inducedregulated et prevent Koshikawa to 2011; (Maquoi shown al., recently apoptosis was et and Koshikawa 2010), Werb, al., 2006; and et Seiki, MMP-13, Egeblad and 2012; and al., Itoh MMP-2 et 2002; (Chan as proteins surface such cell enzymes sheds activates gelatin), MMP- collagen and 2011). fibrillar al., (i.e. components et matrix mouse Roy extracellular 2011; degrades al., 14 in et Perentes 2012; progression al., et (Fro Maquoi tumor cancer 2003; accelerates Seiki, of 2006; models and al., and et (Egeblad 2010) Jiang cancer 2009; Strongin, al., human et in Figueira upregulated 2002; Werb, is other it ADAM12-induced ADAM12, that like the suggesting mediate ADAM12, MMP-14), degradation. of gelatin (e.g. activity proteinases catalytic the uo rgeso.W on htuo nrae xrsinof expression increased upon on that impact found might this We how progression. and tumor MMP-14 of activity the influences al., Yan et 2004; Hadler-Olsen Seiki, 2010; and al., Itoh et 2011; Gonzalo 2004; al., et (Deryugina 1, M-4i lsia rnmmrn ealpoens and, metalloproteinase transmembrane classical a is MMP-14 ntepeetsuy eivsiae hte ADAM12 whether investigated we study, present the In * ˚ 2 ui .Noer B. Julie , hihe l,21;Kebr ta. 2005; al., et Kveiborg 2011; al., et ¨hlich ˜ez-Mo 1 lxne Kotzsh Alexander , ´ ta. 2008). al., et a a V V b b integrins 3 integrin 3 3 , 4707 Journal of Cell Science htAA1 n M-4pooetmraggressiveness. tumor tumor promote apoptosis increased notion MMP-14 the cell and supporting ADAM12 ADAM12 MMP-14, tumor that activated fact, reduced of levels with in increased and correlated that, breast and by human found of progression model reduced and mouse a the significantly in cancer tested ADAM12 increased activity we be of Finally, effect proteolytic ADAM12. could against MMP-14 antibodies ADAM12 monoclonal the by Interestingly, of enhanced of presence surface. the reduction on cell and dependent to appeared and strongly degradation degradation be surface gelatin gelatin The cell apoptosis. in the collagen-induced to resulting redistributed activated, was MMP-14 ADAM12, 4708 ora fCl cec 2 (20) 126 Science Cell of Journal a V b nerna the at integrin 3 Results yolsi aladi hrfr edl ietdt h cell the to directed readily therefore is 1B). Fig. and panels; upper tail 1A, cytoplasmic ADAM12 (Fig. with MMP- surface transfection exhibited cell upon cells However, the few at very in staining whereas nucleus cells, 14 the 293-VnR the to of in close 90% localization nearly MMP-14 dot-like a endogenous demonstrated cells of that line Immunostaining cell HEK293 2001). a of advantage expresses took stably we surface, MMP-14 cell of the recruitment to affects ADAM12 whether investigate To stimulated is surface ADAM12 cell by tumor the to recruitment MMP-14 a V b nern ald23VR(ajye al., et (Sanjay 293-VnR called integrin, 3 (F,G,I). Student’s oa cells. total muotie n one sdsrbdi C. in described Means as counted and immunostained cells MDA-MB-231 siRNA-treated qPCR to detect subjected to then hours, and 48 extracted, for was siRNA mRNA control or with siRNA treated ADAM12 were cells (blue). MDA-MD-231 DAPI (H) and (red) ADAM12 MMP-14 surface (green), cell for immunostained cells MDA-MB-231 non-permeabilized cells. of total Cytospin of (G) percentage means the The as C. expressed in described and as MMP-14 counted surface cell for that immunostained Cells cells. non- MCF7 of permeabilized experiments western Cytospin by (F) ADAM12 blotting. and MMP-14 for analyzed MCF7- MCF7, A12 Total wild-type (E) from lysates. lysates cell total cell in shown also is protein rcpttswr nlzdfrMP1 and MMP-14 ADAM12 for analyzed were precipitates Streptavidin assay. biotinylation surface experiment, a as cells bars) mean (green GFP and bars) of (red distribution MMP-14 the shows graph The cells. non-permeabilized 293-VnR in MMP-14 surface experiments Cytospin detected (C) cells. total of percentage nuclear means juxta The the region. to MMP-14 of localization xrsin B el eesanda ecie in described as and stained A were Cells protein (B) visualize expression. to used was Confocal microscopy (blue). nucleus the and (red), (green) MMP-14 ADAM12 for stained and permeabilized i.1 erimn fMP1 otecell the to MMP-14 of Recruitment 1. Fig. n el eetasetdwt ihrvector ADAM12 either or with control transfected were cells ADAM12. VnR by stimulated is surface D 6 y,MCF7-A12 cyt, 6 . ..pretg ftettlcls o each For cells. total the of percentage s.d. ..aeepesda h ecnaeof percentage the as expressed are s.d. 0 el e xeietwr one for counted were experiment per cells 500 ADAM12 D t ts.Saebr:6 bars: Scale -test. * y-F ywsenbotn.MMP-14 blotting. western by cyt-GFP P . , 00clswr one.()Cell (D) counted. were cells 1000 .5 ** 0.05, 6 and D ..aeepesda the as expressed are s.d. D y-F.()Clswere Cells (A) cyt-GFP. y+o el were cells cyt+dox MMP14 P D , y,wihlcsthe lacks which cyt, .1 *** 0.01, m A;12 (A); m I yopnof Cytospin (I) . P AD 293- (A–D) 6 , ..are s.d. 0.001; m m Journal of Cell Science nlzd el rnfce ihGPtge ADAM12 is GFP-tagged localization with surface transfected cytospin cell in Cells on specifically assessed analyzed. ADAM12 immunostaining where subsequently of MMP-14 effect was experiments, MMP-14 dot-like The localization 1B). with MMP-14 exhibited (Fig. and cells panels), significantly cells lower of decreased 1A, 2000), (Fig. number surface the al., cell the et at immunostaining (Hougaard surface el ufc a nlzduigteDoiktcnqe(briefly technique Duolink the the using at analyzed MMP-14 was and surface ADAM12 cells between relationship spatial The cell degradation tumor gelatin the induces at surface colocalization MMP-14 and surface. ADAM12 cell tumor the to MMP-14 of regulate recruitment might ADAM12 the that confirm further of data These level 1H). (Fig. the Notably, S1C). MMP14 Fig. FACS material by confirmed (supplementary were than data analysis These siRNA 1I). ADAM12 (Fig. significantly cells with treated was treated control staining cells MDA-MB-231 surface in cell lower surface MMP-14 number exhibiting cell the cells and the of permeabilization, at membrane without immunolocalization analyzed in MMP-14 was MDA- reduction 1H) in significant (Fig. ADAM12 a levels of caused knockdown cells siRNA MB-231 1G). (Fig. surface the cell at furthermore ADAM12 to juxtaposition and in surface MMP-14 experiments, of cell presence the cytospin had showed line by ADAM12 this ADAM12 that express of confirmed to expression We 2010). shown al., previously MDA- et line been (Solomon cell MMP-14. cancer has breast of which invasive the MB-231, distribution used we cellular purpose, this the For influence would ADAM12 S1B). Fig. material (supplementary analysis FACS by by increased MCF7-A12 and was staining MCF7-A12 in 10-fold surface than more cell of MMP-14 number exhibiting the was cells were and surface permeabilization, we cell membrane the 2012), without at analyzed al., localization experimental MMP-14 et three 1E). all Maquoi under (Fig. MMP-14 conditions 2009; of levels al., equal (MCF7- detect et to able Figueira expression 2000; al., ADAM12 the et off to doxycycline switched A12 adding medium and induced 1E), growth we (Fig. ADAM12 hence, expressed 1E); MCF7-A12 (Fig. (MCF7-A12 ADAM12 ADAM12 system. of no expression the or and little low-invasive the express using lines, cells to MCF7 cell Wild-type MCF7. sought cancer line cell breast-cancer non-metastatic in first surface we cell the setting, to cancer translocation MMP-14 a stimulates also ADAM12 whether in determine MMP-14 of and recruitment ADAM12 cells. 293-VnR stimulates of surface ADAM12 cell the that to data finding MMP-14 these together, the Taken 1D). support (Fig. ADAM12 endogenous cells overexpressing control of when with levels lysate compared expression cell the total in in MMP-14 changes We observe S1A). in Fig. not material detected did supplementary 1D; be (Fig. we cells could transfected MMP-14 analysis, surface (FACS) ADAM12 cell sorting that assay vector- cell biotinylation demonstrated a control fluorescence-activated in with Finally, and 1C). compared (Fig. staining cells GFP-expressing surface cell MMP-14 an exhibited oepoetefntoa eeac fcmie xrsinof expression combined of relevance functional the explore To et eivsiae hte noeosyexpressed endogenously whether investigated we Next, D y+o) ncnrs oerirpbiain (Deryugina publications earlier to contrast In cyt+dox). RAwsntafce ykokono ADAM12 of knockdown by affected not was mRNA D y-rnfce 9-n el,btnti control- in not but cells, 293-VnR cyt-transfected , D 0fl nraei h ubro el with cells of number the in increase 10-fold y+o el Fg F.Teedt a confirmed was data These 1F). (Fig. cells cyt+dox D y el rwn ihu doxycycline without growing cells cyt D y el scmae ihMCF7 with compared as cells cyt D y)uigaTet-Off a using cyt) ADAM12 mRNA D D cyt cyt h edfrAA1 n M-4t rnlct otecell the to translocate to 2C). (Fig. MMP-14 hours and 20 in ADAM12 expressing after hours for times 4 need cultures five after The about In increased detectable transfection but was culture, the S2A). degradation for gelatin Fig. used ADAM12, were material plasmid (supplementary ADAM12 increasing 2B). with when of (Fig. expressing substantially degradation transfected amounts increased not gelatin degradation cells no gelatin cells or or The low whereas showed cell control) gelatin, non-transfected vector degrade (i.e. to ADAM12 able ADAM12 cells. were behind the leaving underneath fluorescence, areas gelatin black was of degradation disappearance cells as Gelatin 293-VnR defined conditions. of activity experimental degradation various matrix under the assay test degradation to gelatin used A was MMP-14. biological of altered characteristic in resulted activities MMP-14 of localization subcellular in as 2A). (Fig. or, surface expressed exogenously endogenously cell was cells, surface the ADAM12 MDA-MB-231 cell at the whether at dots of colocalized MMP-14 independently red and and bright ADAM12 ADAM12 2A). as (Fig. of visualized lines colocalization was cell on MMP-14 and performed three were cells, the experiments Duolink in non-permeabilized The 1. Methods) Fig. and in Materials examined the in described ooaiea h uo elsraeadta DM2i an is cell cancer ADAM12 breast that in and degradation surface gelatin lines. cell of regulator tumor important decreased the endogenous at significantly colocalize of cells knockdown 2F,G). MDA-MB-231 (Fig. degradation siRNA gelatin in Here, that 2009). al., are ADAM12 et demonstrated cells (Yamaguchi se MDA-MB-231 per we 2D,E). gelatin (Fig. degrade and to timeframe known MCF7 this wild-type in degrade the gelatin to whereas ability hours, MCF7-A12 their 48 influenced after 1, degradation Fig. MCF7-A12 in cell carcinoma gelatin. shown breast as in MMP-14 lines, of localization subcellular S2B). the Fig. material (supplementary al., cells surface cell than et the degradation ADAM12 to (Hougaard of gelatin expression efficient intracellularly ectopic less with retained showed mostly and 2000), is ADAM12- full-length by which tested with further L, cells was 293-VnR occur transfecting to degradation transient gelatin the for surface aayi ciiyo DM2isl a eurdfrthe cells for 293-VnR required transfected was the we the itself for ADAM12 degradation, with whether ADAM12 responsible gelatin test of was observed To activity proteinase degradation. catalytic which gelatin asked ADAM12-induced next We by caused MMP-14 is degradation gelatin ADAM12-induced eai erdto a iia fe rnfcinwt wild-type with transfection after ADAM12 similar was degradation Gelatin infcn euto ngltndgaain(i.3B). (Fig. degradation gelatin in reduction 1994; caused al., TAPI-2 significant and et GM6001 Gijbels Both a 1997; 2007). on Rasmussen, al., and grown inhibitors et Moss broad (Black cells both are metalloproteinases treated which of TAPI-2, we or results GM6001 activity, degradation with degradation gelatin gelatin gelatin metalloproteinase the the that a confirm for to from order responsible In 3A). itself (Fig. not is ADAM12 et eakdwehrteAA1-eitdcag nthe in change ADAM12-mediated the whether asked we Next, ae oehr hs aasgetta DM2adMMP-14 and ADAM12 that suggest data these together, Taken in change ADAM12-dependent the whether analyzed next We D D y n ADAM12 and cyt y+o el hwdltl ednyt degrade to tendency little showed cells cyt+dox D y otiigactltcst uain(E351Q). mutation site catalytic a containing cyt DM2atvtsMP1 4709 MMP-14 activates ADAM12 D y el xiie infcn gelatin significant exhibited cells cyt D yE5Q niaigthat indicating cytE351Q, D y,wihraiylocates readily which cyt, D y-rnfce cells cyt-transfected Journal of Cell Science eetasetddrcl ihMP1,teatv mtr)form (mature) active with the transfected MMP-14, with were directly transfected cells were the when observed ADAM12 form was mature no MMP-2 and detected cells of be 293-VnR could untransfected MMP-2 Only from of medium panel). proform in upper inactive the 3D, of (Fig. and presence zymography presence the by the were MMP-2 for cells of examined activity and 293-VnR degradation transfected gelatin for from tested supernatants serum-free culture First, and was degradation. cell MMP-2 gelatin whether gelatin observed test the to degrades us for led responsible This and 2008). al., activated et (Itoh collagen becomes MMP-2 under surface, degradation gelatin to conditions. contributor experimental MMP-14 example, major these (for either the metalloproteinase that is MMP-14-activated MMP-2) indicating an compared 3C), or (Fig. knockdown itself was siRNA MMP-14 degradation control Gelatin upon with ADAM12 3C). decreased (Fig. in significantly we cells MMP-14 involved, 293-VnR of is transfected knockdown MMP-14 siRNA whether performed determine to Subsequently, 4710 ti eletbihdta pnMP1 erimn otecell the to recruitment MMP-14 upon that established well is It ora fCl cec 2 (20) 126 Science Cell of Journal D y ra mt etrcnrl ncnrs,we cells when contrast, In control. vector empty an or cyt D cyt- htMP2i o h rtiaeta srsosbefrthe for responsible is that proteinase the degradation. not gelatin ADAM12-induced indicate is together data MMP-2 these Thus, that 3E). 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ADAM12 or control siRNA with treated of cells hours) MDA-MB-231 (20 assay (F) gelatinase 100%). solid-phase at (set value MCF7-A12 degradation mean to relative expressed gelatin-degradation ( of percentage Mean (E) A12 fe n 0hus ra ihu green ( without measured Areas were hours. fluorescence 20 and degradation 4 gelatin after of Percentage nucleus (C) the (blue). and (red) expression ADAM12 ADAM12 data percentage ( Mean calculated. degradation was gelatin cell the per and B in experiments el rnfce ihvco oto or control ADAM12 vector with transfected 293-VnR cells using assay (B) gelatinase surface. solid-phase cell the at indicate colocalization spots Red (6E6) MMP-14. ADAM12 and to antibodies with Duolink reagents to subjected were panels) (right cells MDA-MB-231 non-permeabilized of cytospin A12 non- MCF7-A12 panels), permeabilized (left vector control or 9-n el rnfce ihADAM12 with transfected cells 293-VnR induces degradation. surface gelatin cell the at MMP-14 colocalization and ADAM12 2. Fig. 6 6 m P .. o h C7clssoni D in shown cells MCF7 the for s.d.) .. r xrse eaiet h mean the to relative expressed are s.d.) D A ih aes;40 panels); right (A, m , D D D y-rnfce utr ihu the without culture cyt-transfected y+o) 10 cyt+dox); y n MCF7-A12 and cyt y+o el mdl aes,and panels), (middle cells cyt+dox .1 *** 0.01, a D D D V D y) 100 cyt), y 0hu percentage hour 20 cyt y.Cl utrswr etdfor tested were cultures Cell cyt. y) 20 cyt); b m D ´ nern(Borrirukwanit integrin 3 P vze l,20) nthe On 2002). al., et lvez A etadmdl panels), middle and left (A, m y+o losoe no showed also cyt+dox , .0,Student’s 0.001, m (F). m m m A Non-permeabilized (A) B etrcnrl D, control; vector (B, m D C7 MCF7- MCF7, (D, m D D y+o cells. cyt+dox m y n MCF7- and cyt (B, m m m 2 * from ) P 6 t , -test. .. for s.d.) nsitu In nsitu In nsitu In 0.05, D cyt D cyt H Journal of Cell Science ooaiaina h elsraewsosre between observed was surface cell the and ADAM12 at ADAM12 with colocalization between transfected 293-VnR non-permeabilized colocalization on cells technique the Duolink the visualize employed and to MMP-14 addition, cellADAM12, the to In complex triple protein surface. the MMP-14 of transport and correct for ADAM12 player however, lysate; between the interaction in occurs indirect still or a that we suggests direct This S3C). MMP- Fig. ADAM12, material and (supplementary proteins ADAM12 expressing 14 between coimmunprecipitation HEK293 a in observed cell detect to MMP-14 unable were surface we and ADAM12 though even Interestingly, using MMP-14 4C). ADAM12. (Fig. both studies against with (mAbs) immunoprecipitation coimmunoprecipitated antibodies performed monoclonal we complex, with associates membrane-anchored ADAM12 whether analyze to Therefore, 4B). for axis. (Fig. role MMP-14 LM609 a LM609 of suggest presence results inhibitory the These in the decreased significantly added was we against cells, antibody the 293-VnR test To transfected 4A). (Fig. degradation of gelatin role no found and non-permeabilized ADAM12 S3B) in Fig. MMP-14 expressing of localization HEK293 as surface extent detect same unable to were parental the We S3A). to Fig. with material (supplementary MMP-14 cells exhibit 293-VnR experiments express not but do undertook expression, cells 293-VnR integrin we unlike which data cells, HEK293 these of basis a V b nerno eai erdto nADAM12 in degradation gelatin on integrin 3 a V a V b nernadbetween and integrin 3 b nernt h el.Gltndegradation Gelatin cells. the to integrin 3 a a V V b b a nernadMP1 nalarger a in MMP-14 and integrin 3 nerna h elsrae we surface, cell the at integrin 3 V a D b V D nernsest etekey- the be to seems integrin 3 y splmnaymaterial (supplementary cyt b y.A hw nFg 4D, Fig. in shown As cyt. nerni h ADAM12– the in integrin 3 a V b nernand integrin 3 a V b integrin 3 a D V cyt- b 3 ufc Abehsne l,21) a oefc ngelatin 8F8) and on (7B8 ADAM12 effect ADAM12 against with no mAbs transfected other had two cells Interestingly, 2011), 293-VnR al., cell in the degradation et at ADAM12 (Albrechtsen cluster to 6E6, surface shown mAb been function- The previously assay. for has degradation tested which gelatin were the these in of activity series blocking a to and (mAbs) ADAM12, antibodies human monoclonal developed previously have We degradation prodomain gelatin ADAM12 inhibit the against antibodies Monoclonal oehr hs aasrnl implicate Taken MMP-14. of strongly 4G). activation (Fig. ADAM12-induced data cultures MCF7-A12 these IgG-treated LM609-treated together, with of compared reduction in 80% cultures an degradation observed We gelatin antibody. LM609 inhibitory D-B21clsepesmdrt eesof that showing levels reports 2011) moderate al., previous express et confirm cells Taherian MDA-MB-231 we 2007; of al., addition, levels et In of surface (Borrirukwanit 4E,F). cell overexpression (Fig. increases that cells integrin MCF7 showed in FACS immunostaining both ADAM12 however, 2011); and 2000; al., al., et analysis et Taherian (Deryugina 2009; cells al., MCF7 et of Figueira surface cell the at integrin molecules. three the of between portion interaction the that and reveal MMP-14 ADAM12, data these ADAM12 examined, used cells we the Because 4D). (Fig. MMP-14 Fg E.Teeoe oaayewehrADAM12-dependent MCF7-A12 to whether treated related was analyze MMP-14 of to activation Therefore, 4E). (Fig. eea eot aedmntae o rn eesof levels no or low demonstrated have reports Several oto uprpnl.Teiae ntelwrpanel lower the in images The panel). (upper control with ADAM12 co-transfected and or MMP-14 MMP-14, ADAM12 or with control, transfected vector cells 293-VnR 293-VnR or untransfected cells MMP-14. for for zymography analyzed MMP-2 lysates (D) western cell Inset, total 100%). of at blots (set value degradation control a uniidb esrn h erddae in area degraded the measuring degradation by Gelatin quantified cultures. was respective the staining in nuclear (DAPI) and degradation gelatin illustrate aa( data eaiaeasyo 9-n el otasetdwith co-transfected cells ADAM12 293-VnR of assay gelatinase (C) cells. ADAM12-positive of ubro eai-erdn DM2pstv cells ( mean ADAM12-positive The gelatin-degrading (DAPI). of nucleus number for the stained for were and Cells ADAM12 overnight. (control) only vehicle (10 GM6001 with treated ADAM12 with transfected situ ADAM12 eai erdto ( degradation gelatin ADAM12 eaiaeasyuig23VRclstasetdwith transfected cells ADAM12 293-VnR using assay gelatinase MMP-14. is by degradation mediated gelatin ADAM12-induced 3. Fig. n orltdt h ubro el.Saebr 10 MCF7-A12 MCF7-A12 bar: for Scale zymography cells. MMP-2 of (E) number the to correlated and 6 D DM2atvtsMP1 4711 MMP-14 activates ADAM12 .. speetda ecnaeo h oa number total the of percentage a as presented is s.d.) oi-hs eaiaeasyo 9-n cells 293-VnR of assay gelatinase solid-phase y elclue ihthe with cultures cell cyt 6 .. r xrse eaiet h ensiRNA mean the to relative expressed are s.d.) a D D D D V D y n oto rMP1 iN.Mean siRNA. MMP-14 or control and cyt y erdto au sta 0%.(B) 100%). at (set value degradation cyt y-31.Dt r xrse stemean the as expressed are Data cyt-E351Q. y risctltciatv form inactive catalytic its or cyt b y+o.*** cyt+dox. nernrltst h extracellular the to relates integrin 3 6 .. eaiet h mean the to relative s.d.) a D (A) V y rMP1 n vector and MMP-14 or cyt P m D b , ) AI2(10 TAPI-2 M), y rcnrlvco and vector control or cyt nsitu In nernfnto,we function, integrin 3 a .0,Student’s 0.001, V nsitu In b nerni the in integrin 3 solid-phase D y otransfect to cyt a a solid-phase V V D b y and cyt b integrin 3 D integrin 3 m y cell cyt )or M) t -test. a a D D V V m cyt. cyt, m m b b In m. 2 3 3 Journal of Cell Science xrse eaiet enMCF7-A12 mean to relative expressed ecnaeo eai erdto ( degradation gelatin of percentage MCF7-A12 n eaiecnrlmueadrbi g.()Ma ecnaecl ufc tiigof staining surface MCF7-A12 cell MCF7, percentage Mean (E) IgG. rabbit and mouse control negative and el( cell g rmb gis DM2(G r88.Peiiae n nu ape eeaaye ywsenbotn ihteidctdatbde.()Non-perm (D) antibodies. indicated the with blotting western by analyzed were samples ADAM12 input with and transfected Precipitates cells 8F8). 293-VnR or (7G3 ADAM12 against mAbs or IgG DM2o h blt fMCF7-A12 of ability the on ADAM12 gelatin S4B). on Fig. these material effect (supplementary MMP-14 inhibitory ADAM12 Importantly, with of transfected any were instead shown). that exert catalytic cells 293-VnR not the not in on degradation did (data 7B8 also and ADAM12 8F8 antibodies of of effect inhibitory activity that no demonstrated we was prodomain assay, there shedding the ectodomain cell-based in a Using as epitope S4A). 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Journal of Cell Science tde ihAA1- n DM2L(o ta. 2011), al., et (Roy ADAM12 ADAM12-L of expression and increased ADAM12-S with studies MCF7-A12 in MCF7-A12 ADAM12 of expression oa f73hmnbes uo ape.We l tumors, all When samples. tumor a breast for Methods, human and 733 Materials of in breast described total four from as human datasets cohorts, profile in different expression tumor expression gene for combined correlation MMP-14 We advantage the tumors. and and investigate an ADAM12 to ADAM12 be mouse aimed between both would we and of Therefore, tumors cultures progression. levels breast cell increased in from MMP-14 that obtained suggest results, studies, present cancer Our breast human MMP-2 in and MMP-14 expression with correlates ADAM12 of Expression rgeso smdae hog erae ppoi,possibly MMP-14. of apoptosis, activity decreased increased an through through mediated is progression MCF7-A12 MCF7-A12 in These MMP-14 control of with level fragment increased compared kDa significantly 43 a the active observed of of 2006), we quantification of Itoh, by intensities, determined 2001; band indicative As al., the 7E). et is (Fig. Ellerbroek blotting which western 2008; by al., MMP-14, et (Cho autodigested of partially MMP-14 the fragment of MCF7- kDa MMP- level on the 43 in ADAM12 analyzed of we cells influence activation, the 14 tumor evaluate To 7D). apoptotic (Fig. mice of significant number a revealed A12 the staining staining Ki67 in ApopTag by contrast, decrease determined In as 7C). proliferation, (Fig. cell tumor affect D y-ncltdmc oprdwt MCF7-A12 with compared mice cyt-inoculated nvivo in D y+o uos(i.7) eiyn previous Verifying 7B). (Fig. tumors cyt+dox aasgetta h feto DM2o tumor on ADAM12 of effect the that suggest data D y nMF uosddnot did tumors MCF7 in cyt D y+o uos(i.7F). (Fig. tumors cyt+dox D y uosbtntin not but tumors cyt D y tumors cyt D cyt+dox opnnso h toa(gba n eb 2002; Werb, and (Egeblad structural stroma main basement degrade the the to collagens, able of are and MMP-14) components components (e.g. few process, membrane a this only In 2011). (Lo but proteins action Weinberg, in matrix are and extracellular enzymes (Hanahan multiple of types variety cell a and of stroma, grow composed surrounding the to is into ability islets which tumor their from by directly invade characterized and are be cells that tumor might Malignant suggest activity, data Discussion MMP-14 These tumors. of breast 8B). human ER-positive modulator (Fig. in in relevant a biologically TNBC tumors, as all in ADAM12, in and found This MMP-2. tumors was an and found correlation ADAM12 we between positive lacks Interestingly, correlation growth that 8A). stronger (Fig. epidermal cancer even and (ERBB2) breast receptor receptor of progesterone factor triple-negative subtype ER, of in a expression in and (TNBC), retained tumors tumors was breast MMP-14 (ER)-positive correlation and receptor positive ADAM12 This estrogen between 8A). 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A12 of analysis MCF7-A12 blot Western (E) tissue. of ( number cells mean ApopTag-positive the estimate was to C for used that to method A counting (D) similar staining. Ki67 for immunostained A12 fec MCF7-A12 each areas five of from images of counting for nuclei program software Metamorf the cell ( Mean proliferation (C) ADAM12. tumor for of extracts analysis blot Western (B) ersn entmrms ( mass tumor mean represent A12 n (MCF7-A12 drinking water their in mice doxycycline Some received mice. female of glands mammary the in implanted orthotopically vivo apoptosis tumor inhibits and tumor growth accelerates ADAM12 7. Fig. 5 P ) hra te ie(MCF7- mice other whereas 8), , ´ . D D D e-tnadMtiin 2007), Matrisian, and pez-Otin MCF7-A12 y+o uo xrcsfrMMP- for extracts tumor cyt+dox tissue tumor cyt+dox y tumors, cyt .1 *** 0.01, D y n MCF7- and cyt 6 P .. a acltdusing calculated was s.d.) , D n D y uo el were cells tumor cyt .0,Student’s 0.001, 5 D y+o tumors, cyt+dox * )ddnt A.Data (A). not. did 8) y n MCF7- and cyt P , 6 0.05, .. ntumor in s.d.) 6 s.d.). t -test. in Journal of Cell Science hc niisMP1 u o DM2 significantly ADAM12, not but MMP-14 key GM6001, a inhibitor inhibits is metalloproteinase MMP-14 The which process. an that this of notion in cell knockdown reporting the driver siRNA-mediated tumor supporting by as further reduced rescued MMP-14, The be well cells. could BCL2L11 apoptosis as in tumor and MMP-14 ADAM12-expressing BIK apoptosis, both and in of ADAM12 expression cell decreased between accompanying link tumor by direct the finding reducing a their of extend to expression now data pointing the Our decreased BIK. protein MMP-14 pro-apoptotic matrix, confer cleaving collagen by The that, to the a mice. was in mechanism in implantation molecular shown embedded orthotopic underlying after suggested when as was cells well as cancer gel, al., MMP-14 collagen breast et to specifically, resistance (Maquoi apoptotic apoptosis tumor More cell primary new tumor accelerate 2012). a to circumventing capacity However, by the 2011). growth Noe has Agnes al., also 2011; of MMP-14 et al., laboratory that et the Zarrabi (Perentes from 2008; se report exciting per al., growth et tumor on Szabova with effect affect matrix, no mainly extracellular or degrading to little all by thought dissemination in TNBC was cell in present MMP-14 tumor and tumors recently, was ER-positive Until in which samples. as well cancer, as breast analyzed MMP-14 samples human and the ADAM12 for in here between report correlation expression 2011; We positive al., cancer 2011). a et al., both time Roy et first of 2011; Zarrabi and al., 2008; et al., 1997), models et Perentes Szabova 2006; al., al., mouse et et Peduto Ueno 2012; in 2004; progression al., (Fro et tumor Roy (Figueira accelerate 2005; carcinoma al., breast Fro et including 2009; many in al., cancer, upregulated et human highly are of both types is, that proteinases; cancer ecie,wt hsitrcinrsligi naprn striking been apparent behavior has an cell tumor in MMP-14 on resulting effect and interaction this ADAM12 with described, between interaction direct 4716 niiuly M-4adAA1 r osdrdimportant considered are ADAM12 and MMP-14 Individually, hihe l,21;Kebr ta. 05 auie al., et Maquoi 2005; al., et Kveiborg 2011; al., et ¨hlich ora fCl cec 2 (20) 126 Science Cell of Journal hihe l,21;Iae l,19;Lendeckel 1999; al., et Iba 2011; al., et ¨hlich nvitro in and nvivo in ldemonstrated ¨l . ta. 02 ozl ta. 00.I atclr h interaction the particular, and In Ga 2010). 2001; MMP-14 al., al., et between et Gonzalo (Ellerbroek 2002; MMP-14 al., important et of an play activation to for seems role integrins surface cell with interaction extracts, form. active tissue an tumor 2008; in present and was al., MMP-14 cell kDa that tumor demonstrating et 43 further a both the (Cho detected in we fragment domain Here, generate 2006). MMP-14 kDa Itoh, catalytic 18 2001; to al., soluble the et Ellerbroek a of cleaved and fragment in kDa autocatalytically 43 inactive of MMP- membrane of is active fragment the membrane-anchored which Processing plasma in proteinases event 2003). an is the 14 (Seiki surface cell findings the at at on MMP-14 earlier relies but localization with level, accordance protein appropriate or increased mRNA an the to well- its noted due at be not MMP-14 should was Itoh otherwise of It activity MMP-14-mediated amount 2010). 1998; MMP-14 Strongin, in an al., 2001; an increase al., et the that et MMP-2, (d’Ortho of Itoh 2011; MMP-14 al., effect gelatinase active et of indirect the effect described an of rather being gelatin activation degrades it directly activity. MMP-14 catalytic MMP-14 than that own indicate its for data of Our regulator independently degradation key gelatin important an cases. clinical in as activation also function ADAM12 that express hypothesize can simultaneously we MMP-14, humans and in ADAM12 both tumors high a malignant Because of cells. increased tumor number of by showed mechanistic capacity apparently that apoptotic growth the 2011) previous tumor diminishing important al., accelerated et ADAM12 in of Roy an expression 2005; al., observations provide et (Kveiborg to data on axis explanation ADAM12–MMP-14 These the indicates of which effect apoptosis. the apoptosis, inhibit can cell they tumor that to increased antibodies also monoclonal that ADAM12 report we Importantly, ADAM12-expressing cells. tumor collagen-embedded in apoptosis increased M-4atvt srgltda eea ifrn ees and levels, different several at regulated is activity MMP-14 induces ADAM12 that revealed we study, present the In a V b nernhsbe implicated been has integrin 3 n MMP-2. and ADAM12 between Correlation (B) MMP-14. and ADAM12 between Correlation (A) correlation. 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Fig. nvivo in n 5 3)and 534) studies n 5 ´ lvez 145) Journal of Cell Science navreyo rcse novdi uo progression tumor in interact Deryugina to (i.e. shown 2002; been integrins involved has several al., ADAM12 with et MMP-14, processes Like Deryugina 2004). al., 2007; of et al., et variety (Borrirukwanit a in ppoi yehnigcl ufc eesadsubsequent cell and tumor levels and surface cell degradation enhancing gelatin by apoptosis regulates ADAM12 ta. 05;Za ta. 04.W eetyfudthat found recently We 2004). al., Thodeti et 2005a; Zhao al., myotube et 2005b; and Thodeti al., 2005; migration al., et cell et cytoskeleton, (Lafuste formation actin the affecting eessmlrt 9-n el u ontexpress at not do MMP-14 but degradation, express cells which 293-VnR gelatin to cells, similar subsequent HEK293 levels parental and the MMP-14 whereas of that express localization 293- demonstrated and which ADAM12-producing MMP-14 cells, Moreover, we surface. both VnR cell study, with the at colocalized present integrin ADAM12 and the that MMP-14 and both In immunoprecipitated 2011). ADAM12 al., MMP-14-positive et required of clusters invadopodia-like formation ADAM12-antibody-induced erdto fe ramn ihihbtr nioisagainst antibodies gelatin decreased inhibitory of with finding a treatment the the after support by degradation provided to was evidence degradation Further relocate RACK1 of 2008). with to involvement interactions al., shown through et been surface (Bourd-Boittin cell previously the has to ADAM12 integrin of and Interestingly, surface expression. activation cell ADAM12 upon the even to gelatin, MMP-14 degrading redistributing of incapable were a bet omuorcptt DM2tgte ihMMP- with and together 14 ADAM12 coimmunoprecipitate to able was ADAM12, of consisting and complex functional MMP-14 protein the ternary al., disrupt the ADAM12 of of et against activities 8F8) antibodies and (Wewer monoclonal (7B8 the prodomain bonds that the indicated noncovalent data the Our with through 2006). associated remains molecule Interestingly, prodomain domain. mature the site catalytic cleavage, furin the the furin at and after cleavage prodomain al., after the until et ADAM12 form between the inactive (Gilpin keep an primarily tail in to molecule thought cytoplasmic is of prodomain a a The 1998). consists domain, and a domain, disintegrin-like domain factor-like it a growth transmembrane epidermal domain, N-terminus, an domain, catalytic cysteine-rich the a prodomain, From a protein. multidomain effects profound for behavior. responsible ternary cell together tumor a on three of all part with a surface, becomes with thus complex and initiates protein MMP-14 integrin, of the activation of clustering an a through probably ADAM12, A12 n/ratrisrfnditrcinwt h MMP-14– the with interaction molecule refined ADAM12 its the of alter conformation and/or the change somehow h yolsi alapae ob eurdfrgelatin for surface, required cell signal the and key be at a MMP-14 provides that, for prodomain, to propose its through us probably appeared let ADAM12, data nor tail These ADAM12, ADAM12 degradation. draw of of cytoplasmic domains activity cannot functional other catalytic We the the the surface. the neither of cell interestingly, role of but, the the on at disruption conclusions complex any in the of resulting activities complex, integrin V ncnlso,tedt rsne eedmntaethat demonstrate here presented data the conclusion, In h ebaeacoe omo ua DM2Li a is ADAM12-L human of form membrane-anchored The b D nerni ohAA1-rdcn 9-n n MCF7- and 293-VnR ADAM12-producing both in integrin 3 y el.O h ai fteefnig,w ugs that suggest we findings, these of basis the On cells. cyt a V b nern ehpteieta As78ad8F8 and 7B8 mAbs that hypothesize we integrin, 3 a V a b a V nerna h elsrae lhuh8F8 Although surface. cell the at integrin 3 V b b nerni DM2idcdgelatin ADAM12-induced in integrin 3 nernt rmt uo progression. tumor promote to integrin 3 a V a b 9 a nernadMP1 ttecell the at MMP-14 and integrin 3 b V 1, b nern nue elsurface cell induced integrin, 3 a 7 b a 1, V a b 5 nern(Albrechtsen integrin 3 b ,and 1, a a V V b b b integrin, 3 ) thereby 3), integrin, 3 a a V V b b 3- 3 M-4(ba,Cmrde A S n iepnBocec,Sate WA, Seattle, Bioscience, against LifeSpan and and antibodies USA Ta USA) (Clontech polyclonal Biosite, MA, Nordic Cambridge, rabbit USA; (Abcam, GFP and CA, MMP-14 a Biotechnology, USA), BIK Cruz against MA, (Santa against Ballerica, BCL2L11 antibody (Calbiochem, antibodies actin polyclonal and goat monoclonal USA), MA, USA), Ballerica, mouse CA, Millipore, View, in include used Mountain antibodies Laboratories, Other 2004). study as al., Furthermore, et 2004). (Sundberg were the and described al., (7B8 as et rb122) ADAM12 generated Sundberg against were 2005; antibodies 7G3) rabbit monoclonal al., mouse et described and Kveiborg previously 1999; not 6E6 al., Fro et 2011; Iba 8F8, al., 1998; et (mouse (Albrechtsen described ADAM12 previously against Antibodies chemicals and Antibodies Methods and Materials 4sann el eecutduigteMtMrhsfwr ihmulti with software MetaMorph the using MMP- and program. counted scoring ADAM12- cell USA). were wavelength 60133, cells IL without centrifuge Inc., stained cytospin al. 14-staining and a Scientific et in trypsinized Fisher Kawaguchi spun were Thermo and by cells (Sandon, paraformaldehyde described the 4% secondary that in brief, fixed to and In and permeabilization, method 2003). MMP- similar al., and serum), primary ADAM12 et a for (Kawaguchi using goat staining before performed surface was cell normal 100) the 14 experiments, cytospin Triton-X 1% In added. (0.5% and 4 and not BSA antibodies or (1% and permeabilized MMP-14 blocked 2011). al., of et paraformaldehyde, (Albrechtsen visualization previously described as For was ADAM12 of Visualization staining Immunofluorescence siRNA Sigma-Aldrich. siGENOME OPTI-MEM from using as was control performed Dharmacon obtained RNAs negative was Scientific universal were interfering transfection siRNA Thermo Small ADAM12 and from medium. USA), and reagents growth MMP-14 the SMARTpool against to 100 added (siRNAs) MCF7-A12 was and the BD (Sigma-Aldrich) in Science) (Clontech expression Applied Tet-Off ADAM12 (Roche MCF7- MCF7 stable B The into 2002). al., ADAM12 transduced et 2000). A12 (Rønnov-Jessen previously stably al., described et as media was (Kang Sciences) culture described vector some previously in as pRevTRE used used was was FBS and previously Fro Gelatinase-depleted as Denmark). 2011; cultured Hvidovre, FuGENE al., Sweden), using Boras, MDA-MB-231 et transfected and AB, (Albrechtsen MCF7 Stantards HEK293, (LGC described 2001). 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Green it)wsmaue vrtm.Mc eekle sso soemuedisplayed mouse the one as of soon and as (length rest killed size cm were the Mice Tumor 1.2 time. a water. over during drinking measured their was and width) in injection (Sigma-Aldrich) doxycycline MCF7-A12 cell with ml injected mice tumor Some 0.667 water. to drinking given were prior mice week experiment, One mouse. speiul ecie Ttie l,2008). al., performed et was grown zymography (Tatti and Gelatin collagen described embeded 2012). for 3D previously were al., the as et cells stained (Maquoi making and previously cells for protocol described used manufacturer’s as of the was to number solution (Advanced and according USA) PureCol the gels CA, times. software three as Diego, type least MetaMorph same San at well and BioMatrix, independently counted of as were repeated cells was use 1000 cells experiment than of by more of experiments fluorescence) each number For ADAM12. gelatin the the to correlated in holes black o ooaiaina h elsrae Duolink surface, cell the at colocalization For H eaiae Dclae sasadgltnzymography gelatin and assays collagen 3D gelatinase, uoiei assay tumorigenic 8 y G116 rmMlclrPoe Lf ehoois Naerum, Technologies, (Life Probes Molecular from (G-13186) dye 488 2 H uo,adtsu a isce sdsrbdpeiul (Fro previously described as dissected was tissue and tumor, eoiaeIO ppoi eeto i Mlioe a sdon used was (Millipore) kit detection apoptosis ISOL peroxidase ora fCl cec 2 (20) 126 Science Cell of Journal nsitu in H irsoyatmto n mg nlsssfwr a used was software analysis image and automation microscopy rxmt iainasy(L)tcnqe hr w primary two where technique, (PLA) assay ligation proximity 6 D 14Pa-pcrmtwtrimrinobjective immersion water Plan-Apochromat /1.4 y el eeijce rhtpclyit the into orthotopically injected were cells cyt m /lestradiol-17 g/ml a hihe l,21;Sat ta. 2012). al., et Stautz 2011; al., et ¨hlich V b nern pce-pcfcsecondary Species-specific integrin) 3 H egnsfo ln (Uppsala, Olink from reagents scid D y el lorcie mg/ 2 received also cells cyt 6 m b I12rg 10 /l ope oOregon to coupled g/ml) SgaAdih ntheir in (Sigma-Aldrich) 6 tm1Wjl n 3 and hihe l,2011; al., et ¨hlich m m SJmc (The mice /SzJ 6 2 10 osre as (observed hihe al., et ¨hlich 6 el per cells eygn,E . ankv .I,Y,Q,Bcu .C,Rznv .V n Strongin, and V. D. Rozanov, C., P. Baciu, Q., Yu, I., B. Ratnikov, I., E. Deryugina, eygn,E . ankv .I,Psnv,T . oao,D .adSrni,A Y. A. Strongin, and V. D. Rozanov, I., T. Postnova, I., B. Y. Ratnikov, A. I., Strongin, E. and Deryugina, W. J. Smith, K., Jungwirth, A., M. Bourdon, I., E. Deryugina, orltdt h xrsino M-4(fyerxpoe222__t and was 202828_s_at) 213790_at) probe using (Affymetrix probe performed were Prism. MMP-14 correlations (Affymetrix GraphPad Pearson of 201069_at). ADAM12 probe expression (Affymetrix of MMP-2 the level to expression correlated the where ’rh,M . tno,H,Bte,M,Akno,S . upy .adHembry, and G. Murphy, J., S. Atkinson, M., Butler, H., Stanton, P., M. d’Ortho, h,J . snosi . ho . i,S,Tt,M,Cl,K,Aokme,A., Aboukameel, K., Cole, M., Toth, S., Kim, H., Zhao, P., Osenkowski, A., J. Cho, P. C. Blobel, eindtesuy otiue oeprmnsadwoethe wrote and C.F. experiments submission. to to contributed prior study, manuscript of interpretation the the and with M.K. wrote/edited designed contributed material. study, and and A.K. the patient data design research, and the to J.B.N. from helped main U.W. D.S., obtained gene the the profiles manuscript. analyzed F.C.N. expression the and performed J.V. of experiments. additional study, performed writing to the contributed designed R.A. contributions Author manuscript. the manuscript, editing the for of reading Raab FACS critical with Linda help for and her Carlsson for Robert Fossum B. Nils Kweder Anna analysis, Ramay thank technicians We laboratory Valentin. of Brit help and the for grateful are We the using analyzed Acknowledgements association was The inhibition test. exact antibody (ANOVA). Fisher’s variance and of or degradation analysis groups Statistical gelatin independent experiments. Student’s between the two independent with Software three comparing GraphPad least or for Excel at using performed in was performed analysis were assays All analysis Statistical orrkai,K,Lfer .A,Mrui .A,Bik . rc,J . Fridman, T., J. Price, T., Blick, A., F. Mercuri, A., M. Lafleur, K., Borrirukwanit, Wolfson, L., J. Slack, J., J. Peschon, J., C. Kozlosky, T., C. M. Rauch, A., U. R. Wewer, Black, and M. Kveiborg, A., Sanjay, D., Stautz, R., Albrechtsen, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.129510/-/DC1 online [grant available material Society Supplementary Nilsson’s Cancer Arvid Danish the the and by R40-A1750-11-S2]; Foundation. supported DP08019, was numbers work The Funding manuscript. hn .M,Wn,H . i,G,Lu . a,R,Co . et,K,Tryggvason, K., Lehti, Y., Cao, R., Cao, The B., Liu, and G., Jin, A. L., H. Wong, L’Helgoualc’h, M., K. D., Chan, Bonnier, H., Pabic, Le K., Bourd-Boittin, M nFrnNgtv oo acnm ooCells. LoVo Carcinoma Colon Furin-Negative in MMP Y. 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