Cancer Res 1974 Folkman 2109 13

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1974;34:2109-2113.Cancer ResJudah Folkman

Tumor Angiogenesis Factor

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[CANCER RES EARCH 3 4, 2 10 9-2 11 3, A ug ust 1 97 4]Tumor Angiogenesis Factor1J ud ah F olk ma nT he Dep ar tm en t o f S ur ge ry , C hi ld re n's Hos pi ta l M ed ic al C en te r, a nd t he Har va rd Med ic al S ch oo l, B os to n, Mas sa ch us et ts 0 21 15

SummaryRecent evidence from our studies indicates that solidtum or grow th is n ot con tinuous but that it can be separatedinto tw o stages, avascular and vascular. In the avascularstage, tum ors rem ain dorm ant at diam eters of 1 to 2 mm.Further grow th is possible only after new capillaries havebeen elicited from the host an d hav e penetrated the tum or.This capillary proliferation is stim ulated by a diffusiblefa cto r, t umo r a ng io ge ne sis fa cto r, re le as ed by s olid tumors ,a nd by neopl as ti c c el ls i n cul tu re .T he biology and isolatio n of tum or angiogenesis factor

are discussed. Evidence for the m echanism of tum or dorm ancy in the absence of angiogenesis w ill be presented.T herapeutic im plications of the inhib ition of tum or angio ge ne sis w ill b e men tio ne d b rie fly .IntroductionT here is increasing evid ence that tum or cells communicate with normal host cells. NGF2 is an example (14).C ertain m ouse sarcom as secrete a factor that stim ulatesg rowth in n eig hb orin g sen so ry an d symp ath etic n erv e cells.How ever, the secretion of NGF is lim ited to a few tum ortypes. Furtherm ore, N GF is not necessary for continuedtumor growth; in a sense, it is a side product or a luxury

molecu le . T he majo rity o f u nu su al mole cu les an d in ap propriate horm ones thus far known to be secreted by tum orsare each restricted to a sm all group of tumors and are oflittle im portance to the successful grow th o f the tum or.By contrast, the ability of malignant solid tum ors tostim ulate proliferation of new capillaries is common to awide variety of neoplasm s and appears to be an essentialreq uiremen t fo r p ro gres siv e tumo r g rowth (1 ,4 ). N o d ou btit w ill b e fo un d th at o th er fo rm s o f commun icatio n b etw eentum or and host m ay be im portant for tum or survival (13).T herefo re, it seem s tim ely to ex am in e o ur p resen t u nd erstanding of the relationship between solid tumor andvascular endothelium and to focus on the concept thatin fo rm atio n ex ch an ge b etw ee n th ese 2 tiss ues o perates in 2d irectio ns . N ew cap illaries in du ced b y tumo r in tu rn ap pearto p lay a k ey ro le in reg ulatin g tumo r g rowth .

1 Pr es en te d a t t he Thi rd Con fe re nc e o n Embr yo nic a nd F et al Ant ig en sin Cancer, November 4 to 7, 1973, Knoxville, Tenn. This work w assupported by Grant l ROI CA 14019-01 from the National CancerIn sti tu te , G ra nt DT -2A f rom th e Amer ic an C an ce r S oc ie ty , a nd g if ts f romAlza Corporation.2 Th e ab brev iation s u se d are : NGF, n erv e g ro wth fac to r: TAF , tu mo rangiogenesis factor .

The R ela tio ns hip b etw ee n Tumor and Vas cu la r Endoth eliumNew in fo rmatio n h as b ecome a vailab le in th e p ast d ecad econcerning tum or vessels and th eir endothelial cells. Forexam ple, it has been shown that from 10 to 40% of a solidtum or m ay consist of vascular endothelial cells (1). Furthermore, we now realize that the life of a solid tumorcan be separated in to 2 stages: before vascu larization andafter v ascu la rizatio n (6 ). Most so lid tumo rs seem ca pab le o fliving by sim ple diffusion until they reach a diam eter of afew mm , after w hich n ew v esse ls p en etrate th em . In a nim alsthis conclusion is based on studies of tum or grow th in therabb it ear cham ber and the ham ster cheek pouch (18 20). Inm an, this inform ation derives from studies of biopsy andautopsy specim ens of tiny m tastasesthat have not yetvascula rized (5) .Furtherm ore, we know that solid tum ors cannot m akec ap illa rie s o n th eir own but mu st e lic it t he se v es se ls from th ehost. This generalization is supported by 3 kinds ofex perim en ts, (a) When tumo rs are implan ted in to th e rab bitear cham ber, the ham ster cheek pouch, or the rat dorsal airsac, n ew v essels alw ay s arise from th e h ost an d g row towardth e tumo r; v essels n ev er ex ten d from th e tumo r in to th e h ost(3, 18). (b) Sm all tumor im plants or inoculations of tum orcells from tissue culture inserted into the cornea of therabbit eye attract new blood vessels from the lim bus at the

edge of the cornea. These are host vessels; m icroscopicstud y of the cornea has never rev ealed v essels arising fromth e tumo r impla nt (1 1). T hese are h ost v essels; m icro sco picstu dy o f th e co rn ea h as n ev er rev ealed v es sels arisin g fromthe tum or im plant (11). (c) W hen a tum or graft is placedw ith in an in cisio n in th e c hick ch orio allan to ic membran e,no new vessels penetrate the tum or until approxim ately 3days after im plantation. The tum or rem ains pale and un-vascularized (D . K nighton, D . A usprunk, D . Tapper, andJ. Folkm an, unpu blished data). R epeated m icroscopic observations of such a graft show that vessels previouslyresid en t w ith in th e tumo r g raft b eg in to d isin te grate w ith in24 hr after transplant to the chorio allantoic m embrane (D .A usp ru nk , u np ub lish ed d ata; R ef. 1 5). T his d eg en eratio noccurs despite the fact that the tum or graft is taken froma w ell-vascularized portion of a rapidly grow ing anim altum or. In contrast, w hen grafts of norm al em bryonic tissues such as m uscle or endocrine glands are carried to thechorioallantoic m embran e, their ow n vessels not on ly surv iv e b ut also b eg in to co nn ect w ith th e v essels o f th e ch orioallantoic m em brane w ithin 24 hr. Therefore, in a generalsense, norm al tissues, whether grafted to a syngeneic orto a n a llo ge ne ic h os t, u su all y c on ta in v as cu la r e ndoth eliumbelonging to the donor tissue; while a tum or graft ulti-

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J . F olkma nmate ly contains vascular endothe lium belonging to the host.It is also becoming clear that vascular endothelium inadult animals is a re lative ly quiescent ti ssue , renewing itsel fv ery s low ly w ith a labe ling inde x o f about 0 .5% or le ss (1 6).The mo st rapidly g rowing v es sels in the cho rio allanto icmembrane of the 7- to 10-day chick embryo may reach apeak labeling index o f 2 7% (2 ), but in the neig hbo rho od o fa tumor implant, w hether placed among developing vessels or mature vessels, the labeling index may be 37% ormo re. A ltho ugh the precise m echanism by which so lid tumors c ontinual ly e lic it new capillarie s from the ho st i s no tc lear, there is evidence that th is information is transmittedfrom the tumo r to the vascular endothelial cells by a diffusible factor.Tumors s eparate d from the vas cular bed by a Milli po refilter w ill induce new capillary g rowth on the o ther side o fthe f il te r ( 12 ). In separate experiments us ing the dorsal s .c .air pouch in the rat (3) and the anterior chamber of therabbit ey e (1 1), w e w ere able to show that the distance o verwhich tumo r can s timulate endo thelial mito sis is g reaterthan the 25-hic kne ss o f the M illipo re filte r and c an be asgreat as 3 to 5 mm.

More re cently , we have be en able to s eparate a c ell-fre ef rac tion f rom tumor cel ls that w il l induce the growth of newcapillary sprouts . This frac tion, c alle d TAF, has no t be endetected in normal tissues or f ibroblasts in primary culture ,w ith the possible exception of trophoblast and mousesal ivary g land (7) , al though the poss ibi li ty i s no t precludedthat the TAF may eventual ly be found in o ther nonneoplas -tic tissues at v ery low lev els. TAF is mito genic to endo thelium o f capillaries and small v enules. It is a pro te in w ith amo lecular w eig ht o f appro ximately 1 00 ,0 00 ; it co ntainsabout 3 parts RNA and is inactivated by RNase. TAFactivity has been found in both the cytoplasm and thenuc le us o f s ev eral animal and human tumors , although themajority of our work has utilized the Walker 256 car-cino sarcoma. The material can now be iso lated bo th fromtumor in the solid or ascites form and from tumor cells inc ulture . Whe re tumor nuc le i are us ed as the s ourc e o f TAF,angiogenesis activity is associated w ith the nonhisto neproteins of chromatin; w hile the histone proteins lackac tiv ity (1 7). Ang iogene sis ac ti vity in the nonhis tone proteins has been resolved to approximately 20 proteinmoieties by acry lam ide g el electro pho resis. We are presently engaged in purifying TA F obtained both from thenonhisto ne fractio n and from fractio ns iso late d from thecytoplasm of intac t c el ls in cul ture .B ioassays for TAF

The bio assay s av ailable fo r TAF are carried o ut in v iv oand are only qualitative. We do not as yet have aquantitative or an in vitro assay . Tw o assay systems areroutine ly used as fol lows.Chor ioallantoic M embrane of the Chick Embryo. Awindow is made in egg shell at Day 8 of incubation andthe chorioallantoic membrane allow ed to drop. The testfrac tion is then added on Day 10.The frac tion to be te ste d,usually an eluate from a S ephadex column, is first steri

lized by Millipo re filtration, dialyzed against distilledw ater, and lyophilized. A ppro ximately 0.01 ml of 0.9%NaCl solution is added to the few crystals that remain(usually in the range of a 5 to 25 ig f total protein). Thissolution is soaked up into 3 tiny pads made of glass fiberfilte r pape r pre vious ly impre gnate d w ith 5% po lyac ry la-mide ge l and then washed in Ringer's lc lateso lution. Eachpape r is plac ed on the chorio al lanto ic membrane o f a s eparate e gg toge the r w ith a c ontro l filte r pape r c ontaining nopro te in o r inac tiv ated pro tein. The filter paper is placedover 3 or 4 tiny holes made in the chorioallantoic membrane w ith a N o. 30 hy podermic needle just befo re positio ning . This g uarantees that the ecto dermal lay er o f thechorioal lanto ic membrane wil l no t be a barri er to di ffus ionof the test fraction. When fractions are positive, this isdetectable in 2 to 4 days by new vessels, which grow tow ard the filter paper, forming a "red spoke w heel" appearance. The density of the vessels in this spoke w heelallows a qualitative grading of 1+ or 5 +. No new vesselreactio n is ex cited by the co ntro l filter. This assay w ill respo nd to appro ximately 5 /ig o f to tal pro tein (co ntainingangiogenesis activity).Rabbit Cornea. F ractions of protein to be tested areinserted into pockets made in the cornea as previouslydescribed (1 1). H ig hly co ncentrated fractio ns can be inserted directly into this pocket, w hich holds a vo lume ofapproximate ly 20 1 .l te rnative ly , the se f rac tions can bedispersed in 5% po ly acry lam ide g el and injected into thepocket, allowing a slower release of the protein. Thepo lyac ry lam ide g els are o cc as ionally inflammato ry in thec orne a unle ss the y are buffe re d to 7 .4 w ith /V -2 -hydroxy -e thylpiperaz ine -jV'-2-ethanesulfonic ac id and made isoos-

moti c. Approximate ly l / ig o f to tal pro te in can be di spersedin 20 //I of acrylamide gel in such a pocket. The eye isex amined daily w ith a stereo sco pic slit lamp. N ew v esselsgrow out f rom the l imbus , traverse the cornea, and ente r thepocket. Inactiv e fractio ns do no t attract vessels. In thistechnique, the rate of vessel grow th can be measured.Capillary growth reaches a maximum of 0.3 mm/day.This assay is used as a supplement for the chick embryoassay.Inhibition of Angiogenesis

If neov ascularization w ere only a side effect of tumorgrowth, then the e ff ort to i so late , charac te ri ze , and inhibi tTAF might s eem unimportant. However, we are be ginningto appreciate that the induc tion o f new capi ll ari es by tumoris c ritic al to progre ss iv e tumor growth. In te rms o f tumorprogression, TAF may be one of the most importantmole cule s s ec re te d by a tumor, be caus e w ithout TAF mos ttumors would remain avascular and dormant at a diamete ro f a few mm. This ide a turns upon 4 pie ce s o f e xpe rimentale videnc e, as summariz ed in Chart 1 .The 1st information comes f rom tumor implants grown inisolated perfused organs (7 ). W e have show n that smallorgans such as rabbit thyroids , when perfused through the irarterial tree in isolated chambers, w ill support tumorgrowth. However, vascular endothe lial cel ls in these organs

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