Vol 1 April 7 doi:1.13/nture57 ARTICLES Meditors of vsulr remodelling o-opted for sequentil steps in lung metstsis Gorv P. Gupt 1, Don X. Nguyen 1, Anne C. Ching 1,, Pul D. Bos 1, Juliet Y. Kim 1, Cristin Ndl 1 {, Roger R. Gomis 1 {, Kti Mnov-Todorov 3 & Jon Mssgué 1, Metstsis entils numerous iologil funtions tht olletively enle nerous ells from primry site to disseminte nd overtke distnt orgns. Using geneti nd phrmologil pprohes, we show tht the epiderml growth ftor reeptor lignd epiregulin, the ylooxygense COX, nd the mtrix metlloproteinses 1 nd, when expressed in humn rest ner ells, olletively filitte the ssemly of new tumour lood vessels, the relese of tumour ells into the irultion, nd the rehing of lung pillries y irulting tumour ells to seed pulmonry metstsis. These findings revel how ggressive primry tumorigeni funtions n e mehnistilly oupled to greter lung metstti potentil, nd how suh iologil tivities my e therpeutilly trgeted with speifi drug omintions. The emergene of disseminted metstses remins the primry use of mortlity in ner ptients 1 3. Reent serhes for geneti determinnts of metstsis hve led to the identifition of gene sets, or signtures, for whih the expression in primry tumours is ssoited with high risk of metstsis nd poor disese survivl 1. It hs een proposed tht the expression of suh genes in primry tumours might diretly predispose ner ells for growth in distnt orgns 11, rising questions out the distintion etween tumorigeni genes nd genes tht medite metstsis. A ontrsting, long-held view is tht metstsis rises from rre tumour ell lones, the geneti mkeup of whih endows them with unique seletive dvntge in distnt orgn miroenvironments 1. Brest ner metstsis genes tht my orrespond to eh of these two models hve een reently identified in funtionl sreen for meditors of lung oloniztion 1. A suset of the genes identified in this mnner supports mmmry tumour growth s well s pulmonry metstsis y humn ner ells in mie, nd onstitutes lung metstsis gene signture (LMS), the expression of whih in primry tumours indites high risk of pulmonry relpse in rest ner ptients 1,13. Other genes emerging from the sme sreen, however, imprted lung metstsis virulene without ffeting primry tumour growth 1. The speifi roles tht these vrious genes my hve in metstsis hve remined unknown. Here we report tht four LMS genes olletively ontriute vsulr remodelling funtions tht n support the formtion of vsulture in mmmry tumours, the entry of tumour ells into the irultion nd the exit of tumour ells from the loodstrem into the lung prenhym. Their ility to medite distint funtions in the primry site nd in the lung metstsis setting distinguishes these genes from onogenes tht primrily support the trnsformed stte in ner ells. The present findings suggest moleulr sis for the lung metstsis prolivity of lolly ggressive primry rest tumours, nd rtionle for omintoril therpeuti interventions ginst metstsis. Geneti oopertion in mmmry tumour growth The MDA-MB-31 ell line, whih ws derived from the pleurl fluid of ptient with widely metstti rest ner 1, is heterogeneous popultion omposed of ells with diverse orgnotropi metstti potentil nd distint pro-metstti gene expression signtures 15 17. Notly, some of the LMS genes tht typify the lung metstti supopultions derived from this soure were independently identified s downstrem effetors of vsulr endothelil growth ftor (VEGF) in endothelil ells 1. This suggested to us the possiility tht, when expressed y tumour ells, these genes my onfer vsulr remodelling funtions tht re relevnt for metstti progression. These genes inlude the epiderml growth ftor reeptor (EGFR)/ pn-her lignd epiregulin (), the prostglndin-synthesizing enzyme ylooxygense (COX; lso lled PTGS), nd the mtrix-remodelling metlloproteinses MMP1 nd MMP., COX nd MMP1 re prt of the linilly vlidted LMS genes nd MMP is frequently ssoited with them 1. Unlike endothelil ells 1, the lung metstti MDA-MB-31 supopultion LM-175 (herefter lled LM) expressed these four genes in mnner tht ws neither dependent on utorine VEGF (Supplementry Fig. 1, ) nor responsive to VEGF ddition (Supplementry Fig. 1). Thus, we investigted whether the elevted expression of these genes in ner ells might repitulte trnsriptionl progrmme enoding seretory meditors of vsulr remodelling for tumorigenesis nd lung metstsis. We stly redued the expression of, COX, MMP1 nd MMP y using short hirpin RNA interferene (shrna) in LM ells 1. We lso generted ompound knokdown ells in whih up to four shrnas were expressed in given ell popultion, ttining signifint silening of ll trgeted genes nd their enoded produts (Fig. 1 nd Supplementry Fig. ). The extent of silening hieved is onsistent with multiple onstruts eing simultneously tive in most, ut not ll, ells in the knokdown popultion. Redution of, COX, MMP1 or MMP expression individully hd sttistilly signifint, yet limited, effets on tumour growth on ell inoultion into the mmmry ft pd of immunoompromised mie (Fig. 1, left grph). Nevertheless, silening of these genes in different omintions delyed tumour progression, with nerly omplete rogtion of growth hieved y silening ll four genes simultneously (Fig. 1, right grph). Eh ell line ws infeted with similr 1 Cner Biology nd Genetis Progrm, Deprtment of Mediine, 3 Moleulr Cytology Core Fility, nd Howrd Hughes Medil Institute, Memoril Slon-Kettering Cner Center, New York, New York 11, USA. {Present ddresses: Hemto-Onology Institute, Hospitl Clini de Brelon, 3 Brelon, Spin (C.N.); Onology Progrmme, Institute for Reserh in Biomedeine, Brelon Siene Prk nd University of Brelon, Brelon, Spin (R.R.G.). These uthors ontriuted eqully to this work. 7 Nture Pulishing Group 75
ARTICLES NATURE Vol 1 April 7 mrna (reltive units) Tumour volume (mm 3 ) 1 1 Prentl 7 5 3 1 Protein (reltive level) 1 9 3 sh -sh sh -sh COX MMP1 MMP Prentl COX LM- 175 LM- 175 3 5 Dys fter injetion Phospho-histone 3 Cleved spse-3 MMP1/ COX/MMP1/ /MMP1/ /COX 3 5 Dys fter injetion retrovirl titres irrespetive of the numer of different hirpin sequenes introdued. Moreover, overexpression of MMP1, MMP nd COX in the ontext of ells trgeted y ll four shrnas resulted in phenotypi resue of tumour growth to levels tht were oserved for the single knokdown (Supplementry Fig. 3, ). The omined knokdown of three lung metstsis virulene genes, IL13RA, SPARC nd VCAM1 (ref. 1), only mildly ffeted mmmry tumour growth despite strongly inhiiting lung metstsis Seretion (ng ml 1 ) 7 5 3 1 Prentl d MMP1 sh -sh LM- 175 Seretion (ng ml 1 ) 15 1 Prentl sh -sh LM- 175 COX MMP1/ COX/MMP1/ /COX /MMP1/ 1 Cleved spse-3 (ounts per field) 5 MMP Figure 1, MMP1, MMP nd COX ooperte to medite primry tumour growth., LM ells were infeted with retrovirus enoding ontrol hirpin, or with shrnas trgeting, MMP1, MMP or COX. For omintion knokdown retrovirus, multiple hirpin vetors were trnsfeted s pools into virl pkging ell lines. Infeted ells were seleted nd knokdown ws determined y quntittive (q)rt PCR, COX nlysed vi western lot, nd sereted MMP1 nd MMP mesured y ELISA. Shown re levels of eh gene produt in the prentl MDA-MB-31 ell line from whih LM ells were seleted, s well s LM ontrol, single (sh) nd qudruple knokdown (-sh) ells. n 5 3; error rs represent 95% onfidene intervl for qrt PCR nlysis nd stndrd errors of the men (s.e.m.) for ELISA., 131 ells of ontrol, single knokdowns, or the indited omintion knokdown smples were inoulted into the fourth mmmry ft pds of immunodefiient mie. Length nd width of plple tumours were mesured, nd tumour volumes lulted t the indited time points. Left: effets of single gene knokdown; right: ontrol ompred to omintion knokdown ells. n 5 ; error rs indite s.e.m.; sterisk, P,.5; doule sterisk, P,.1; triple sterisk, P,.1; lulted using two-tiled Student s t-test for tumour volumes t the lst time point, ompred to ontrol., Automted immunohistohemistry for phosphohistone 3 nd leved spse-3 detetion ws performed on tumours otined from the vrious omintion knokdown ell lines. Shown re representtive imges t n originl mgnifition of 3. d, Quntifition of leved spse-3 stining using Imge J softwre. n 5 15; error rs indite s.e.m.; single sterisk, P,.1; doule sterisk, P,.1; lulted using two-sided Wiloxon rnk-sum test, ompred to levels in ontrol tumours. 7 (Supplementry Fig. 3d f). Thus, not ll omintions of lung metstsis meditors stimulte primry tumorigenesis. Overll, these results unover speifi geneti intertions etween, MMP1, MMP nd COX tht olletively filitte elerted mmmry tumour growth. Quntified phosphorylted histone H3 levels indited tht none of the single or omined knokdowns of these genes signifintly ltered the prolifertion rte of the tumour ells (Fig. 1 nd Supplementry Fig. ). In ontrst, n inresed rte of poptosis ws evident in tumours with omintoril knokdown, s mesured y leved spse-3 stining (Fig. 1, d). Dul inhiition of nd COX resulted in synergisti rise in the rte of primry tumour poptosis (Supplementry Fig. ). Although further inhiition of MMP1 nd MMP did not reh the sttistil threshold for synergy, redution of these genes resulted in supr-dditive effet ove the level of poptosis oserved in the /COX knokdown (Supplementry Fig. ). Role in mmmry tumour ngiogenesis Inresed rtes of tumour ell deth might e seondry to defets in ngiogenesis. Indeed, histologil stining for endothelil ell mrker CD31 reveled profound defets in the vsulr morphology of tumours tht hd redued expression of ll four of these lung metstsis genes (Fig. ). Digitl imging nlysis nd quntifition of vessel struture demonstrted tht lthough the verge numer of disrete vessel units ws not onsiderly ltered, the length, numer of lumens nd extent of rnhing of the existing vsulture were signifintly redued in the omined knokdown tumours (Fig. ). These morphologil hnges were lso visulized using CD31 immunofluoresene nd onfol mirosopy to imge the tumour vsulture (Supplementry Fig. 5). Co-stining for CD31 nd NG, smooth musle periyte mrker, did not revel ny mjor differenes in periyte reruitment (Supplementry Fig. 5). Nevertheless, vessels in the qudruple knokdown tumours exhiited diminished effusion of intrvenously injeted dextrn, onsistent with ttenuted vsulr permeility (Fig. ). Of note, these defets in primry tumour vessel morphology nd funtion ourred in the sene of differenes in VEGF levels etween ontrol nd knokdown tumour ells (Supplementry Fig. 1). This suggested tht the forementioned genes promote the formtion of the dilted, tortuous nd No. of vessel units (>1 µm) 7 Nture Pulishing Group 75.7 ± 11.. ±. Averge length (µm) 1. ±. 37. ±. No. of lumen per field 3.5 ± 7. 3.7 ±. No. of rnh points per vessel 3. ±.75 1.±. Figure, MMP1, MMP nd COX medite tumour ngiogeni progression., Automted nti-cd31 immunohistohemistry ws performed on ontrol nd -trgeted tumours. Representtive imges were otined t 31 mgnifition., Tumour setions stined with nti-cd31 ntiody were used for morphometri vessel nlysis. Vessel units (defined s $1 mm in length), vessel length, numer of lumen nd verge numer of rnh points were quntified s desried in Methods. n 5 15; error rs indite s.e.m.; doule sterisk, P,.1; triple sterisk, P,.1; lulted using two-tiled Student s t-test., Mie ering tumours were injeted with rhodmine-onjugted dextrn. Tumours were extrted nd setions exmined for vessel permeility t 31 mgnifition. Sle rs, 5 mm.
NATURE Vol 1 April 7 ARTICLES leky lood vessels tht typify the neovsulture of ggressive primry tumours 19. Tumour ell extrvstion from lung pillries To ssess the importne of these genes s meditors of pulmonry oloniztion, the knokdown lines were injeted intrvenously into mie, nd lung metstti progression ws monitored y ioluminesene (Fig. 3) nd histologil exmintion (Supplementry Fig. ). Independent silening of these genes hd little impt in this lung oloniztion ssy. As in the mmmry tumorigenesis ssys, inhiition of lung oloniztion ws ttined when these genes were silened in omintion, with knokdown of ll four genes yielding the most slient defet. Metstti olonies were eventully oserved in the omintion knokdown smples, ut this ws ttriutle to Normlized photon flux 1, 1 1 MMP1 MMP COX 1 1 3 1 3 Dys fter injetion Trns-well migrtion (reltive units) 1 1 MMP1/ COX/MMP1/ /MMP1/ COX/ Prentl LM LM No EC HUVEC HPMEC Figure 3 Geneti inhiition of, MMP1, MMP nd COX prevents metstti extrvstion., Single nd omintion knokdown ells were inoulted intrvenously into mie. Lung metstsis ws mesured y ioluminesene nd quntified. Left: effets of single knokdowns versus ontrol; right: effets of omintion knokdowns versus ontrol with representtive ioluminesent imges of mie injeted with ontrol nd -treted ells t dy 35. n 5 5; error rs indite s.e.m.; sterisk, P,.5; sed on two-sided rnk-sum test ompred to shrna ontrol LM ells., or -trgeted ells were pulsed with ell trker green nd injeted into the til vein of mie. Forty-eight hours fter tumour ell inoultion, nimls were injeted intrvenously with rhodmine-onjugted letin. Whole lungs were then extrted fter neropsy nd imged y two-photon onfol mirosopy t 33 mgnifition. Three-dimensionl reonstruted imges of tumour ells (green) reltive to lung pillries (red) re shown., Indited ell lines were seeded into trns-well inserts with or without (No EC) n endothelil monolyer. Imges of ells migrting were ptured t 31 mgnifition nd quntified with ImgeJ softwre. Trns-endothelil migrtion ws performed with either humn umilil vein endothelil ell (HUVEC) or humn pulmonry mirovsulr endothelil ell (HPMEC) monolyers with similr results. n 5 1; error rs indite s.e.m.; doule sterisk, P,.1; sed on two-sided Student s t-test. 1 7 Nture Pulishing Group outgrowth of ells esping knokdown nd re-expressing these genes (dt not shown). Sttistil tests identified multiple synergisti intertions etween, COX, MMP1 nd MMP in the erly progression of lung metstsis (Supplementry Fig. ). The speifiity of this phenotype ws onfirmed y overexpressing MMP1, MMP nd COX, whih resulted in signifint reovery of lung metstti tivity (Supplementry Fig. 3). To visulize lung metstsis events during the initil dys fter inoultion, whole lungs were extrted nd snned y onfol mirosopy. Within dys of entering the irultion, ontrol tumour ells ould e visulized outside of the lung pillries, showing tht they effiiently extrvste into the lung prenhym (Fig. 3, left pnel). Conversely, when deteted, the knokdown ells in whih ll four genes were trgeted y shrnas were trpped within vessels s single ells, seemingly inple of rehing the lung endothelium (Fig. 3, right pnel). In n in vitro ssy of trns-endothelil migrtion, the migrtory pity of LM ells through n endothelil monolyer ws inhiited y the omined knokdown of ll four genes, whih did not entil generlized defet in ell motility (Fig. 3 nd Supplementry Fig. 7). Thus, lthough severl mehnisms nd outomes hve een proposed for intertions etween EGFR/HER, MMPs nd COX 3, our results provide evidene tht the expression of, COX, MMP1 nd MMP y ner ells n olletively promote metstti extrvstion in the lungs. Comined drug inhiition of tumour growth nd dissemintion These metstsis-promoting tivities n lso e phrmologilly trgeted using previously hrterized doses of the nti-egfr ntiody etuxim, the rod-spetrum MMP inhiitor GM1 5 nd the COX inhiitor eleoxi. We used n orthotopi model to ssess the effiy of these drug omintions s interventions during the nturl formtion of lung metstsis from mmmry tumours (Fig. ). When used s single gents, these drugs minimlly inhiited tumour growth of LM ells in the mmmry glnds. However, onsistent with our geneti studies, tretment with the etuxim/eleoxi nd etuxim/eleoxi/gm1 omintions redued the rte of primry tumour growth (Fig. ). This ws ompnied y vsulr defets tht preipitted tissue hypoxi nd ensuing tumour ell poptosis (Supplementry Fig. ). This vsulr phenotype is lso onsistent with the ility of etuxim nd eleoxi to inhiit ngiogenesis y mens of oth utorine nd prrine mehnisms 7,. We exmined whether the vsulr defets eliited y these drugs lso resulted in impired tumour ell intrvstion from the primry site. To this end, the presene of irulting tumour ells ws ssessed y mesuring the reltive expression of humn-speifi GAPDH in lood from treted mie. Notly, oth drug omintions (etuxim/eleoxi nd etuxim/eleoxi/gm1) diminished the presene of irulting tumour ells, with the effet of the etuxim/ eleoxi omintion rehing sttistil signifine (Fig. ). Despite the inhiitory effets on intrvstion, we notied tht some tumour ells hd lredy olonized the lungs of nimls efore the initition of phrmologil tretment (dy ), s seen y immunofluoresent stining (dt not shown). Moreover, signifint numer of tumour ells were still detetle in the lungs of treted mie (Fig. d). Confol imging fter stining for vsulr endothelium nd ner ells showed tht vehile-treted mie hroured lrge lung metstti lesions tht hd effiiently extrvsted, wheres mie treted with the etuxim/eleoxi nd etuxim/ eleoxi/gm1 omintions exhiited strong is towrds smller mirometstses tht remined trpped within the lung vsulture (Fig. e). Digitl imge quntifition of multiple lung setions estlished sttistilly signifint inhiitory effets for oth drug omintions on the overll lung metstti urden, s well s on the size distriution of the lung metstsis lesions (Fig. f, g). GM1 ddition did not inrese the inhiitory effets of the etuxim/eleoxi omintion. 77
ARTICLES NATURE Vol 1 April 7 d Drug omintion +Drugs dys 17 dys Tumour volume (mm 3 ) Reltive humn GAPDH mrna levels (per 3 ml lood) 1, 1 GM1 Celeoxi Cetuxim Cetuxim + eleoxi Three drugs 1 1 Dys fter drug initition Three drugs Inhiiting lung oloniztion y metstti ells When used s single gents, these drugs lso minimlly inhiited pulmonry outgrowth of LM ells diretly inoulted into the til vein, wheres in omintion these drugs prevented metstsis in mnner tht mimiked the effets of geneti knokdown (Fig. 5 nd Supplementry Fig. 9). An exeption to this trend ws the ntgonisti intertion etween eleoxi nd GM1, perhps refleting the ft tht the ltter is rod-spetrum MMP inhiitor, likely to ffet oth pro- nd nti-metstti MMPs 9. Although the intertions etween these gents did not hieve sttistil thresholds for synergy, the omintion of etuxim nd eleoxi, s well s of etuxim nd GM1, resulted in supr-dditive inhiition of lung metstsis (Supplementry Fig. 9). Consistent with the geneti inhiition studies, omined phrmologil intervention resulted in the pillry entrpment of the remining tumour ells even when visulized weeks fter tumour ell inoultion (Fig. 5). Interestingly, the inhiition of extrvstion ws reversed on disontinution of drug tretment, with most of the remining lesions expnding into olonies tht invded the lung prenhym (Fig. 5, right pnel). We performed similr experiments with mlignnt ells freshly otined from the pleurl fluid of two ptients with dvned rest Cetuxim + eleoxi f 1, 9 3 Figure Phrmologil inhiition of tumour growth nd dissemintion in orthotopilly implnted primry tumours., Shemti representtion of the time ourse for tumour ell implnttion, primry tumour growth nd therpeuti tretment to ssess effets on primry tumour nd lung metstti progression. Animls were treted with vehile ontrol, etuxim, eleoxi, GM1, or the indited omintions., Tumour volume mesurements of mie treted with vehile ontrol or trgeted therpies, either individully or in omintion. n 5 ; error rs indite s.e.m.; sterisk, P,.5; doule sterisk, P,.1; triple sterisk, P,.1; sed on two-sided Student s t-test, ompred to ontrol-treted nimls., Blood from mie ws isolted nd red lood ells lysed. RNA from the remining ells ws extrted for qrt PCR. The presene of irulting tumour ells ws ssessed s funtion of humn-speifi GAPDH expression reltive to murine Bm, in 3 ml of mouse lood perfuste. n 5 7 ; rs indite medin GAPDH expression; sterisk, P,.5; sed 7 Lung metstti re (µm ) e NS g Perentge of metstti re (µm ) Three Cetuxim drugs + eleoxi 7 Nture Pulishing Group 1 75 5 5 Drug omintion Three drugs Cetuxim + eleoxi < 1, >1, Size distriution (µm ) on two-sided Student s t-test. d, Stining for tumour ells in lung ryosetions using humn-speifi vimentin ntiody. Nulei were visulized using,-dimidino--phenylindole (DAPI). Arrowheds indite tumour ell lusters in representtive 31 imges of lungs from ontrol (left) nd nimls treted with ll three drugs (right). e, Confol imging t 33 mgnifition of tumour ells (vimentin, green) nd lung vsulture (letin, red) from ontrol (left) nd treted nimls (three drugs, right). Sle rs, mm. f, Digitl quntifition of lung metstti urden. Fluoresein isothioynte (FITC)-stined ner ells in the lungs were quntified s metstti lesions, nd normlized to the re of nuler DAPI stining. Shown on the grph is the verge lung metstti re per 5 3 1 5 mm DAPI re. n 5 1; error rs indite s.e.m. g, Perentge of the lung metstti urden tht ws oupied y lesions of the indited sizes. f, g, Asterisk, P,.5; doule sterisk, P,.1; NS, not signifint, sed on two-tiled Wiloxon rnk-sum test. ner nd dignosis of lung metstsis who were undergoing routine therpeuti proedures t our institution. Crinom ells were isolted from these smples sed on the epithelil ell mrker EpCAM, under institutionlly pproved protools 3. One smple (CN3) ws sujeted to in vivo seletion for metstsis-forming ells 1. As with the MDA-MB-31 ell line, this proess yielded supopultion () tht expresses high levels of, COX nd MMP1 ompred with the unsorted CN3 popultion (Fig. 5). This ws ssoited with n elevted lung olonizing tivity of ells in mie, whih ould e inhiited y the dministrtion of etuxim nd eleoxi (Fig. 5d, e). A smple from different ptient (CN1) ws used without prior experimentl seletion for metstti ells. CN1 ells expressed higher levels of nd COX thn did MDA-MB-31 ells (Fig. 5), nd hd sl lung olonizing tivity tht ould lso e inhiited y the eleoxi/etuxim omintion (Fig. 5d). In ll ses the metstsis inhiitory effets of this omintion were oserved from the erliest stges of metstti oloniztion (dy ) nd were sustined over 5 weeks (dt not shown). These results support the generl relevne of these genes in lung oloniztion, nd the ility to interfere with this proess y omining their phrmologil inhiitors.
NATURE Vol 1 April 7 ARTICLES mrna (reltive level) d Normlized photon flux 1 Normlized photon flux 1, 1 1 MDA-31 LM-175 CN3 CN1 1 1 CN3 GM1 Cetuxim Celeoxi 1, Celeoxi/GM1 Cetuxim/GM1 Cetuxim/eleoxi Three drugs 1 1 1 3 1 3 Dys fter injetion.7 +drug mrna (reltive level) CN1 + Drug omintion MDA-31 LM-175 CN3 CN1 CN1 +drug COX Disussion Our urrent findings identify, COX, MMP1 nd MMP s suset of LMS genes tht re o-opted y rest ner ells nd reonstitute multi-funtionl vsulr remodelling progrmme e Dy Dy 5 1 CN3 9 o mrna (reltive level) 1 MDA-31 LM-175 CN3 CN1 MMP1 Off-drug +drug 3.9 3.. 1..... 1 Figure 5 Trgeted inhiition of metstti extrvstion nd lung oloniztion y LM nd primry mlignnt ells., Mie were pre-treted with the indited gents, dys efore tumour ell inoultion. After intrvenous injetions of LM ells, drug tretment ws mintined nd lung metstsis quntified s in Fig.. Left: effets of single gents versus ontrol; right: omintion drug tretments versus ontrol. n 5 5; error rs indite s.e.m.; sterisk, P,.5; doule sterisk, P,.1; triple sterisk, P,.1; lulted using two-sided rnk-sum test ompred to ontrol treted nimls., Lungs from ontrol or etuxim/eleoxi/gm1 (drug omintion)-treted mie were olleted t dy. Tretment ws terminted in suset of nimls nd mie were monitored for n dditionl weeks. Lungs from these off-drug mie were then olleted fter neropsy. Lung setions from vehile-treted, drug-omintion-treted nd off-drug mie were proessed for immunofluoresent detetion of tumour-speifi vimentin (green) nd CD31 (red). Sle rs, mm., Primry rest rinom ell popultions CN3 nd CN1 were isolted from the pleurl effusion of ptients. The CN3 derivtive,, ws otined y in vivo seletion of highly metstti ells in mie. Expression of, COX nd MMP1 ws ssessed y qrt PCR nd ompred to prentl MDA-MB-31 nd LM ell lines. n 5 3; error rs represent 95% onfidene intervl. d, CN3, nd CN1 were injeted intrvenously, mie were treted with etuxim nd eleoxi, nd lung oloniztion ws mesured vi ioluminesene. n 5 5; error rs indite s.e.m.; sterisk, P,.5; sed on two-sided rnk-sum test. e, Representtive luminesene imges. 7 Nture Pulishing Group tht promotes metstti progression. In orthotopi primry tumour ssys, these ftors olletively medite pthologil ngiogeni progression, with n ensuing inrese in vsulr permeility nd tumour ell intrvstion. Remrkly, this set of genes is lso required to reh the lung vsulture nd enle extrvstion of ner ells on dissemintion of these ells from mmmry tumours to the lungs. Although the individul trgeting of these meditors ws insuffiient to prevent suh iologil tivities, their omined inhiition resulted in profound redutions in sequentil steps of metstti progression. The enggement of ommon set of ftors in distint steps of metstsis qulifies them s metstsis progression genes, whih we define s genes tht fulfil ertin rte-limiting funtions in primry tumour growth nd other speifi funtions in metstti oloniztion. Metstsis progression genes re thus distinguished from onogenes tht fulfil ell-utonomous trnsforming funtions through the ourse of mlignnt disese, nd from metstsis virulene genes, defined s those genes tht prtiipte in metstti oloniztion ut not in primry tumour development. Despite the mjor linil dvnes provided y ytotoxi, hormonl nd trgeted therpies, the medin survivl fter dignosis of metstti rest ner with viserl orgn involvement remins less thn yers 31 33. For the most prt, the trget of ommonly used hemotherpy drugs ontinues to e the prolifertion of ner ells. As the moleulr understnding of the iologil funtions neessry for metstsis inreses, it my eome possile to develop ntimetstti strtegies tht trget not only the intrinsi growth of disseminted tumour ells, ut lso their neessry intertions with newly dopted miroenvironments. Our urrent oservtions demonstrte tht inhiition of EGFR nd COX n te lung metstti progression in linilly relevnt model of rest ner. Colletively, these results identify extrvstion s n essentil step in metstti progression tht n e inhiited y omintoril therpies formulted on the sis of iologil insights. METHODS SUMMARY MDA-MB-31 nd its lung metstti derivtive LM-175 hve een desried previously 1,17. CN3 nd CN1 rinom ells were isolted from the pleurl effusion of ptients with metstti rest ner treted t our institution on otined written onsent in ordne with IRB regultions, s previously desried 3. Metstti ell supopultions were otined y in vivo seletion in mie 1.KnokdownofMMP1, MMP nd COX ws hieved using pretrosuper tehnology 1.For trgeting, n lterntive vetor ws used (psm derivtive), whih expresses the short hirpin emedded in lrger mirorna sequene 3. All niml work ws done in ordne with the MSKCC Institutionl Animl Cre nd Use Committee. BALB/ nude nd NOD/SCID femle mie (NCI) ge-mthed etween 5 7 weeks were used for xenogrfting studies. For inhiitor studies, 1 mg etuxim ws injeted intrperitonelly iweekly, whih yields plsm drug onentrtions within the orresponding rnge in etuxim-treted ner ptients 35. GM1 (Ryss L) ws injeted intrperitonelly t dose of mg kg 1 dily, whih is effiious in prelinil mouse models 5. Celeoxi (LKT lortories) ws mixed with powdered rodent how diet (Reserh Diets) t onentrtion of 1, prts per million, nd provided ontinuously during the ourse of the experiment. Celeoxi serum onentrtions in mie treted within this rnge re linilly ttinle nd suffiient to inhiit inflmmtion nd prostglndin synthesis in humns,,3. The Methods setion provides dditionl informtion inluding ell ulture, mlignnt ell isoltion from pleurl fluids, genertion of retrovirl gene-knokdown vetors, infetions nd trnsfetions, nlysis of RNA nd protein expression, trnsendothelil migrtion ssys, niml inoultion nd ioluminesene imging, soures nd use of phrmologil inhiitors, in vivo intrvstion ssys, tumour histologil nd immunohistohemil nlyses, vsulr permeility ssys, extrvstion visuliztion, nd imge quntifition. Full Methods nd ny ssoited referenes re ville in the online version of the pper t www.nture.om/nture. Reeived 19 Deemer ; epted 1 Mrh 7. 1. Fidler, I. J. The pthogenesis of ner metstsis: the seed nd soil hypothesis revisited. Nture Rev. Cner 3, 53 5 (3). 79
ARTICLES NATURE Vol 1 April 7. Hynes, R. O. Metstti potentil: generi predisposition of the primry tumor or rre, metstti vrints or oth? Cell 113, 1 3 (3). 3. Gupt, G. P. & Mssgué, J. Cner metstsis: uilding frmework. Cell 17, 79 95 ().. Perou, C. M. et l. Moleulr portrits of humn rest tumours. Nture, 77 75 (). 5. vn de Vijver, M. J. et l. A gene-expression signture s preditor of survivl in rest ner. N. Engl. J. Med. 37, 1999 9 ().. Chng, H. Y. et l. Gene expression signture of firolst serum response predits humn ner progression: similrities etween tumors nd wounds. PLoS Biol., E7 (). 7. Pik, S. et l. A multigene ssy to predit reurrene of tmoxifen-treted, nodenegtive rest ner. N. Engl. J. Med. 351, 17 ().. Wng, Y. et l. Gene-expression profiles to predit distnt metstsis of lymphnode-negtive primry rest ner. Lnet 35, 71 79 (5). 9. Bild, A. H. et l. Onogeni pthwy signtures in humn ners s guide to trgeted therpies. Nture 39, 353 357 (). 1. Mssgué, J. Sorting out rest-ner gene signtures. N. Engl. J. Med. 35, 9 97 (7). 11. Bernrds, R. & Weinerg, R. A. A progression puzzle. Nture 1, 3 (). 1. Minn, A. J. et l. Genes tht medite rest ner metstsis to lung. Nture 3, 51 5 (5). 13. Minn, A. J. et l. Lung metstsis genes ouple rest tumor size nd metstti spred. Pro. Ntl Ad. Si. USA 1, 7 75 (7). 1. Cilleu, R., Young, R., Olive, M. & Reeves, W. J. Jr. Brest tumor ell lines from pleurl effusions. J. Ntl Cner Inst. 53, 1 7 (197). 15. Gupt, G. P. et l. Identifying site-speifi metstsis genes nd funtions. Cold Spring Hr. Symp. Qunt. Biol. 7, 1 1 (5). 1. Kng, Y. et l. A multigeni progrm mediting rest ner metstsis to one. Cner Cell 3, 537 59 (3). 17. Minn, A. J. et l. Distint orgn-speifi metstti potentil of individul rest ner ells nd primry tumors. J. Clin. Invest. 115, 55 (5). 1. Wry, K. K., Thkker, G. D., Humtsoe, J. O. & Yng, J. Anlysis of VEGF-responsive genes involved in the tivtion of endothelil ells. Mol. Cner, 5 (3). 19. Crmeliet, P. & Jin, R. K. Angiogenesis in ner nd other diseses. Nture 7, 9 57 ().. Krysn, K. et l. Prostglndin E tivtes mitogen-tivted protein kinse/erk pthwy signling nd ell prolifertion in non-smll ell lung ner ells in n epiderml growth ftor reeptor-independent mnner. Cner Res. 5, 75 1 (5). 1. Dnnenerg, A. J., Lippmn, S. M., Mnn, J. R., Surmih, K. & DuBois, R. N. Cylooxygense- nd epiderml growth ftor reeptor: phrmologi trgets for hemoprevention. J. Clin. Onol. 3, 5 (5).. Howe, L. R. et l. HER/neu-indued mmmry tumorigenesis nd ngiogenesis re redued in ylooxygense- knokout mie. Cner Res. 5, 1113 1119 (5). 3. Pi, R. et l. Prostglndin E trnstivtes EGF reeptor: novel mehnism for promoting olon ner growth nd gstrointestinl hypertrophy. Nture Med., 9 93 ().. Goldstein, N. I., Prewett, M., Zuklys, K., Rokwell, P. & Mendelsohn, J. Biologil effiy of himeri ntiody to the epiderml growth ftor reeptor in humn tumor xenogrft model. Clin. Cner Res. 1, 1311 131 (1995). 5. Gijels, K., Glrdy, R. E. & Steinmn, L. Reversl of experimentl utoimmune enephlomyelitis with hydroxmte inhiitor of mtrix metlloproteses. J. Clin. Invest. 9, 177 1 (199).. Joy, R. F., Seiert, K., Cole, C. E., Kelloff, G. & Luet, R. A. The ylooxygense- inhiitor eleoxi is potent preventive nd therpeuti gent in the min mouse model of denomtous polyposis. Cner Res., 5 5 (). 7. Gtely, S. & Li, W. W. Multiple roles of COX- in tumor ngiogenesis: trget for ntingiogeni therpy. Semin. Onol. 31, 11 ().. Krshim, T. et l. Inhiition of ngiogenesis y the ntiepiderml growth ftor reeptor ntiody ImClone C5 in ndrogen-independent prostte ner growing orthotopilly in nude mie. Clin. Cner Res., 153 1 (). 9. Overll, C. M. & Kleifeld, O. Tumour miroenvironment opinion: vlidting mtrix metlloproteinses s drug trgets nd nti-trgets for ner therpy. Nture Rev. Cner, 7 39 (). 3. Gomis, R. R., Alron, C., Ndl, C., Vn Poznk, C. & Mssgué, J. C/EBP t the ore of the TGF ytostti response nd its evsion in metstti rest ner ells. Cner Cell 1, 3 1 (). 31. Giordno, S. H. et l. Is rest ner survivl improving? Cner 1, 5 (). 3. Solomyer, E. F., Diel, I. J., Meyerg, G. C., Golln, C. & Bstert, G. Metstti rest ner: linil ourse, prognosis nd therpy relted to the first site of metstsis. Brest Cner Res. Tret. 59, 71 7 (). 33. Bselg, J. & Norton, L. Fous on rest ner. Cner Cell 1, 319 3 (). 3. Silv, J. M. et l. Seond-genertion shrna lirries overing the mouse nd humn genomes. Nture Genet. 37, 11 1 (5). 35. Luo, F. R. et l. Correltion of phrmokinetis with the ntitumor tivity of Cetuxim in nude mie ering the GEO humn olon rinom xenogrft. Cner Chemother. Phrmol. 5, 55 (5). 3. Niedererger, E. et l. Celeoxi loses its nti-inflmmtory effiy t high doses through tivtion of NF-kB. FASEB J. 15, 1 1 (1). Supplementry Informtion is linked to the online version of the pper t www.nture.om/nture. Aknowledgements We thnk A. Minn, D. Pdu, C. Vn Poznk, L. Norton, C. Hudis, Y. Pylyev, P. Gupt, T. Westrook nd Z. Lzr for insightful disussions nd tehnil suggestions. We lso thnk S. Tulley nd memers of the Moleulr Cytology Core Fility for expert tehnil ssistne. J.M. ws funded y Ntionl Institutes of Helth grnt, nd y grnts of the W. M. Kek Foundtion nd the Kleerg Foundtion. G.P.G. is supported y n NIH Medil Sientist Trining Progrm grnt nd Deprtment of Defense Brest Cner Reserh Progrm pre-dotorl wrd. D.X.N. is Berlex postdotorl fellow of the Dmon Runyon Cner Reserh Foundtion. A.C.C. ws supported y n ASCO Young Investigtor Awrd nd y the Chrles A. Dn Foundtion. J.M. is n Investigtor of the Howrd Hughes Medil Institute. Author Contriutions J.M. designed nd supervised experiments. G.P.G., D.X.N. nd A.C.C. designed experiments; G.P.G., D.X.N., A.C.C., P.D.B. nd J.Y.K. performed experiments; C.N. nd R.R.G. isolted metstti ells from linil smples; K.T.-M. supervised histologil nd onfol mirosopy imging; J.M., G.P.G., D.X.N. nd A.C.C. nlysed dt nd wrote the mnusript. All uthors disussed the results nd ommented on the mnusript. Author Informtion Reprints nd permissions informtion is ville t www.nture.om/reprints. The uthors delre no ompeting finnil interests. Correspondene nd requests for mterils should e ddressed to J.M. (j-mssgue@ski.msk.org). 77 7 Nture Pulishing Group
doi:1.13/nture57 METHODS Cell ulture. MDA-MB-31 nd its lung metstti derivtive LM-175 hve een desried previously 1,17. All tumour ell lines were ultured in DMEM supplemented with 1% FBS, glutmine, peniillin, streptomyin nd fungizone. CN3 nd CN1 rinom ells were isolted from the pleurl effusion of ptients with metstti rest ner treted t our institution upon written onsent otined following IRB regultions s previously desried 3. Briefly, pleurl effusion smples were entrifuged t 1, r.p.m. for 1 min, ell pellets were re-suspended in PBS nd treted with ACK lysis uffer to lyse lood ells. A frtion of these ells underwent negtive seletion to remove leukoytes (CD5 1 nd CD15 1 ells), nd EpCm-positive ells were sorted from the popultion upon reovery in tissue ulture for h. HUVEC (SienCell) endothelil ells were ultured in omplete ECM medi (SienCell), wheres primry humn pulmonry mirovsulr endothelil ells (HPMECs, Cmrex) were ultured in EGM- (Cmrex). HUVECs nd HPMECs were used etween pssges 3. The retrovirl pkging ell line GPG9 ws mintined in DMEM ontining 1% FBS supplemented with puromyin, G1, doxyyline, peniillin, streptomyin nd fungizone. Trnsfetions were done using stndrd protools with Lipofetmine (Invitrogen). After trnsfetion, GPG9 ells were ultured in DMEM ontining 1% FBS nd sodium pyruvte. Genertion of retrovirus nd knokdown ells. Knokdown of MMP1, MMP nd COX ws hieved using pretrosuper tehnology trgeting the following 19-nuleotide sequenes: 59-AGCGGAGAAATAGTGGCCC-39 (MMP1), 59- GGACGGACTCCTGGCTCAT-39 (MMP) nd 59-GGGCTGTCCCTTTACTT- CA-39 (COX). Knokdown of IL13RA, SPARC nd VCAM1 ws hieved s previously desried 1. For trgeting, n lterntive vetor ws used (psm derivtive), whih expresses the short hirpin emedded in lrger mirorna sequene 3. The two trget sequenes used in the gene were 59-CCCAATATATTCTGACCGTTAA-39 nd 59-ACCACAAATGCATAAAT- GCATA-39. To produe retrovirus for omintion knokdown, multiple hirpin vetors were trnsfeted s pools into the GPG9 mphotropi pkging ell line. Viruses were olleted nd 7 h fter trnsfetion, filtered, nd onentrted y ultrentrifugtion. Conentrted retrovirus ws used to infet ells in the presene of mgml 1 polyrene, typilly resulting in trnsdution rte of over %, nd infeted ells were seleted with puromyin. Beuse the totl mount of plsmid DNA used for o-trnsfetion of multiple hirpins ws the sme s tht used for single hirpin trnsfetion, the omintion knokdown retrovirl titres were similr to titres ttined when generting single knokdown virus. Moreover, we hve demonstrted tht up to four different vetors n e delivered nd expressed effiiently in MDA-MB-31 ells using this protool. Thus, knokdown ells otined fter drug seletion were injeted s pooled popultion without suloning. To generte knokdown-resue ell lines, we used similr method to produe virus enoding omplementry DNAs for overexpression of the RNAi-trgeted genes, long with hygromyin seletle mrker. Comintion overexpressing retrovirus ws used to super-infet previously generted knokdown ells tht were susequently seleted with hygromyin. Anlysis of mrna nd protein expression. Totl RNA from suonfluent MDA-MB-31 ells ws olleted nd purified using the RNesy kit (Qigen). Five-hundred nnogrms of totl purified RNA ws sujeted to reverse trnsriptse retion ording to the SuperSript III first-strnd synthesis system (Invitrogen). DNA orresponding to pproximtely 5 ng of strting RNA ws used for eh of four replites for quntittive PCR. Humn, MMP1, MMP, COX nd -mirogloulin (s n endogenous ontrol) were mplified with ommerilly designed Tqmn gene expression ssys (Applied Biosystems) nd the Tqmn universl PCR mster mix (Applied Biosystems). Quntittive expression dt were quired nd nlysed using n ABI Prism 79HT Sequene Detetion System (Applied Biosystems). For immunolotting, ells were wshed with PBS nd lysed in RIPA uffer (5 mm Tris-HCl, ph 7., 1% NP-,.5% N-deoxyholte, 15 mm NCl, 1 mm EDTA) supplemented with 5 mm NF, mm -glyerophosphte, nd omplete protese inhiitor oktil (Rohe). Proteins were seprted y SDS PAGE nd trnsferred to nitroellulose memrnes (Bio-Rd) tht were immunolotted with mouse monolonl ntiodies tht reognize COX (Cymn Chemils) nd -tuulin (Sigm). To filitte detetion of endogenous COX protein, ells were lso pre-treted with ng ml 1 TNF- for 5 h efore lysing (R&D Systems). For nlysis of sereted protein expression, ells were plted in triplite t 9% onflueny in 1-well pltes, inuted in DMEM.% FBS, nd onditioned medi ws olleted 7 h lter. Medi ws lered of ells y entrifuging t, r.p.m. for 5 min. Pro-MMP1, pro-mmp nd VEGF-15 onentrtions were nlysed in onditioned medi using ELISA kits (R&D Systems). Animl studies. All niml work ws done in ordne with protool pproved y the MSKCC Institutionl Animl Cre nd Use Committee. BALB/ nude nd NOD/SCID femle mie (NCI) ge-mthed etween 5 7 weeks were used for xenogrfting studies. For primry tumour nlysis, 1 3 1 vile single ells were re-suspended in 1:1 mixture of PBS nd growth-ftor-redued Mtrigel (BD Biosienes) nd injeted orthotopilly into mmmry glnd four in totl volume of 5 ml s previously desried 1. Primry tumour growth rtes were nlysed y mesuring tumour length (L) nd width (W), nd lulting tumour volume sed on the formul plw /. For experimentl metstsis ssys, 3 1 5 ells were re-suspended in.1 ml PBS nd injeted into the lterl til vein. Lung metstti progression ws monitored nd quntified using non-invsive ioluminesene s previously desried 1. Phrmologil inhiitors. Cetuxim (ImClone) ws otined from the MSKCC phrmy. For inhiitor studies, 1 mg etuxim ws injeted intrperitonelly i-weekly. Injetion with etuxim t doses etween.5 mg nd 1 mg per injetion hieve plsm drug onentrtions within the orresponding rnge in etuxim-treted ner ptients 35. GM1 (Ryss L) ws injeted intrperitonelly t dose of mg kg 1 dily, whih hs previously een shown to e effiious in prelinil mouse models 5. Celeoxi (LKT lortories) ws mixed with powdered rodent how diet (Reserh diets) t onentrtion of 1, prts per million (1 g eleoxi per 1 kg how), nd provided ontinuously during the ourse of the experiment. Previous phrmokineti studies demonstrte tht eleoxi serum onentrtion in mie treted within this rnge re linilly ttinle nd suffiient to inhiit inflmmtion nd prostglndin synthesis in humns,,3. Trns-endothelil migrtion. HUVECs or primry HPMECs were seeded into ollgen-oted trns-well inserts (1 mm pore size, BD Flon) t 1, ells per well, nd llowed to grow to onfluene for dys. Tumour ells were pulsed with 5 mm ell trker green (Invitrogen) for 3 min efore eing onditioned overnight in.% FBS ECM medi without growth ftors. The next dy, 5, tumour ells were seeded into trns-well inserts with or without onfluent endothelil monolyer, nd the wells were fixed in % prformldehyde fter 1 h. Cells on the pil side of eh insert were srped off nd the trns-well memrne mounted onto slides. Migrtion to the solterl side of the memrne ws visulized with Zeiss Axiopln immunofluoresent mirosope t 31 mgnifition. Pitures of 1 rndom fields ross three replite wells were ptured for quntifition using ImgeJ softwre (NIH). In generl, 15 ounts per field of LM ells were seen to migrte in the sene of monolyer, wheres 5 ounts per field were seen to migrte through n endothelil rrier. Migrtion of the indited lines ws plotted s perentge of migrting LM ontrol ells. Intrvstion. Drug-treted mie were perfused with 5 ml PBS through the left ventrile. Three millilitres of lood perfuste ws olleted from the trium nd lysed two times using ACK lysis uffer (Cmrex). Totl RNA ws extrted from the remining ells nd used for qrt PCR s desried ove. The presene of humn irulting tumour ells ws determined y the reltive expression of humn GAPDH normlized to murine -mirogloulin. Tumour nd lung immunostining. Mie were killed nd perfused with PBS nd % prformldehyde through the left ventrile, efore tumours were extrted, fixed nd prffin-emedded. Immunohistohemil stining for CD31 (Snt Cruz), phospho-histone H3 (Upstte) nd leved spse-3 (Cell Signling) ws performed on prffin-emedded tumour setions y the MSKCC Moleulr Cytology Core Fility. Brightfield mirosopi imges were olleted using n Axiopln mirosopy system (Zeiss). Tumour ell prolifertion (ph3) nd poptosis (leved spse-3) were quntified using omintion of Adoe Photoshop (Adoe) nd ImgeJ softwre (NIH). In rief, the olour-piker funtion ws used to identify mnully the most drkly stined region of interest, with onstnt fuzziness ftor. The seleted regions were fethered nd expnded in uniform mnner, nd thresholded into inry imges, whih were susequently nlysed in ImgeJ. Morphometri nlysis of CD31-stined vessels ws hieved with Photoshop nd Imge Proessing Tool Kit (Reindeer Grphis In.) sed on previously desried protool 37. Angiogeni properties were then sored s funtion of vessel density, verge vessel length, verge numer of rnh points per vessel, nd lumen formtion. On verge, immunohistohemistry quntifition ws performed y tking pitures from five rndom fields per tumour, imging t lest three tumours per smple set. For immunofluoresene, tumours were fixed nd frozen in OCT. Periyte overge of vessels ws identified y doule stining for the periyte mrker NG (Chemion) nd CD31 endothelil ell mrker (BD Biosienes Phrmingen), followed y detetion with fluoresently onjugted seondry ntiodies (Jkson Immunoreserh). Permeility of tumour lood vessels ws ssessed y intrvenous injetion of rhodmine-onjugted dextrn (7 kd, Invitrogen) t mg per g ody weight. After 1 h, mie were killed, tumours extrted, nd 3-mm setions exmined y fluoresene mirosopy for vsulr lekge. Immunofluoresent stining for pimonidzole dduts in 7 Nture Pulishing Group
doi:1.13/nture57 primry tumours ws performed ording to the Hypoxyproe-1 stining kit (Chemion). To oserve metstti extrvstion within the first h of irultory entry, tumour ells were lelled with 5 mm of ell trker green (Invitrogen) for 1 h nd inoulted into mie. Before srifie, mie were injeted intrvenously with rhodmine-onjugted letin (Vetor Lortories) to stin the lung vsulture. Lungs were perfused with PBS, inflted through intr-trhel injetion, nd extrted en lo. Whole lungs were then snned y two-photon onfol mirosopy t 33 using Lei TCS SP mirosope (DM IRE inverted stnd). Representtive three-dimensionl imges of stined pillries nd tumour ells were proessed using Voloity v.3. (Improvision). To exmine extrvsting ells in the drug-treted mie, n lterntive protool ws used. In this se, tumour ells did not retin ell trker lel during the extended time frme of the experiment nd were lterntively o-stined with monolonl ntiody tht seletively detets humn vimentin (Novostr); nti-cd31 ntiody (BD Biosienies Phrmingen) ws used to visulize lung pillries. After fluoresent seondry ntiody inution, imges were ptured with Lei TCS SP mirosope (DMRXA upright stnd) nd proessed using Voloity (Improvision). Quntifition of lung metstsis ws performed y reting montge imges of whole-lung setions t 31 mgnifition using n Axiovert M imging system equipped with motorized inverted stnd (Zeiss). Imge quntifition ws performed s desried ove using omintion of Adoe Photoshop (Adoe) nd ImgeJ softwre (NIH). 37. Wild, R., Rmkrishnn, S., Sedgewik, J. & Griffioen, A. W. Quntittive ssessment of ngiogenesis nd tumor vessel rhiteture y omputer-ssisted digitl imge nlysis: effets of VEGF-toxin onjugte on tumor mirovessel density. Mirovs. Res. 59, 3 37 (). 7 Nture Pulishing Group