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[CANCER RESEARCH 46,1-7, January 1986]

Tumor Invasion and MÃ©tastasesâ€”Roleof the Extracellular Matrix:Rhoads Memorial Award Lecture1

Lance A. Liotta

Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20205

Metastasis is the major cause of morbidity and death forcancer patients. Treatment modalities such as surgery, chemotherapy, and radiotherapy can now cure approximately 50% ofthe patients who develop a malignant tumor. The majority of thepatients in the treatment failure group succumb to the directeffects of the mÃ©tastasesor to complications associated withtreatment of mÃ©tastases(1-5). The dispersed anatomic location

of mÃ©tastasesand their heterogeneous cell composition preventsurgical removal and limit the response to systemic anticanceragents. Consequently a major challenge to cancer scientists isthe development of improved methods to predict the metastaticaggressiveness of a patient's individual tumor, prevent local

invasion, and identify and treat clinically silent micrometastases.Many laboratories are studying the fundamental mechanisms ofinvasion and mÃ©tastases,with the hope of identifying specificbiochemical factors which can be the basis for such diagnosticor therapeutic strategies. Over the last several years, significantprogress has been made toward this goal.

The complexity of the metastatic process has forced investigators to focus on one step at a time in order to reduce thenumber of variables to a reasonable level. In recent years, ourlaboratory has focused on the interaction of metastatic tumorcells with the extracellular matrix. The metastasizing tumor cellmust interact with the matrix at many stages of tumor invasionand mÃ©tastases. One particular type of matrix, the basementmembrane, appears to play a crucial role during the progressionof invasive tumors and during hematogenous dissemination.

Defective Interaction of Invasive Tumor Cells with BasementMembranes

The extracellular matrix is a dense latticework of collagen andelastin, embedded in a viscoelastic ground substance composedof proteoglycans and glycoproteins. It is a supporting scaffoldwhich isolates tissue compartments, mediates cell attachment,and influences tissue architecture (6-9). The matrix acts as a

selective macromolecular filter and plays a role in mitogenesisand differentiation. Interactions between normal cells and thematrix may be altered in neoplasia, and this may influence tumorproliferation and invasion (10). The vertebrate organism is separated into tissue compartments bordered by the basementmembrane and interstitial stroma (6). The basement membraneis a meshwork of type IV collagen, specific glycoproteins suchas laminin and entactin, and heparin sulfate proteoglycans (11-

17). Type IV collagen is the structural backbone of the basementmembrane. Type V collagen and other types of collagen mayexist at the interface between the basement membrane and thestroma. For most tissues, the organ parenchymal cells secrete

Received 8/9/85; accepted 9/26/85.'Presented on May 23, 1985, at the Seventy-sixth Annual Meeting of the

American Association for Cancer Research, Houston, TX.

and assemble the basement membrane. General and widespreadchanges occur in the distribution and quantity of the epithelialbasement membrane during the transition from benign to undif-ferentiated invasive carcinomas (18-21). Benign pathological

disorders with epithelial disorganization or proliferation are usually characterized by a continuous basement membrane separating the epithelium from the stroma. In contrast, invasivecarcinomas consistently exhibit a defective extracellular basement membrane adjacent to the invading tumor cells in thestroma. The basement membrane is also defective around tumorcells in lymph node and organ mÃ©tastases(21). In certain regionsof well-differentiated carcinoma, basement membrane formation

by differentiated structures can be identified. Even in theselocations, the basement membrane is often abnormal because itis discontinuous or focally reduplicated. Electron microscopyreveals focal defects in the continuity of the basement membranelamina densa of carcinoma in situ. These defects may be theearliest stages of progression to invasive carcinoma because inzones of actual microinvasion the basement membrane is markedly fragmented or absent altogether. Defective basement organization and loss may be due to decreased synthesis or toabnormal assembly of secreted components. Alternatively theloss may be due to increased breakdown caused by tumor orhost derived proteases. Normal epithelial cells and benign proliferating parenchymal cells are thought to require a basementmembrane for anchorage and growth (6, 7, 9). Invasive tumorcells may lack such a requirement.

Once the tumor cells escape the primary tumor, they mustinteract with preexisting host basement membranes at manystages in the metastatic cascade. This occurs, for example,during tumor cell entry or exit from blood vessels, the invasionof muscle (22) or nerve, or the traversal of most epithelialboundaries.

Three-Step Theory of Invasion

The interstitial stroma of most tissues does not normallycontain preexisting passageways for cells. The basement membrane is an insoluble continuous but flexible structure which isimpermeable to large proteins (11). These types of extracellularmatrix become focally permeable to cell movement only duringtissue healing and remodeling, inflammation, and neoplasia. Cellinfiltration of the matrix undoubtedly depends on multiple factorsincluding properties of both the infiltrating cell and associatedhost cells, as well as properties of the matrix itself.

We have proposed a three-step hypothesis (Chart 1) (23)

describing the sequence of biochemical events during tumor cellinvasion of the extracellular matrix. The first step is tumor cellattachment via cell surface receptors which specifically bind tocomponents of the matrix such as laminin (for the basementmembrane) and fibronectin (for the stroma) (24, 25). The an-

CANCER RESEARCH VOL. 46 JANUARY 1986

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Research. on August 15, 2020. © 1986 American Association for Cancercancerres.aacrjournals.org Downloaded from

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TUMOR INVASION AND METASTASES

HT.ptÂ¡\ cullittnÂ«Â»

Step 1: Attachment Step 2: Dissolution

Step ÃŒ:Locomutiun

Chart 1. Three-step hypothesis of tumor cell invasion of extracellular matrix. Schematic diagram (not to scale) of tumor cell invasion of the basement membrane. Step

1 is tumor cell attachment to the matrix. This process may be mediated by specific attachment factors such as laminin, which form a bridge between the cell surfacelaminin receptor and type IV collagen. Step 2 is local degradation of the matrix by tumor cell-associated proteases. Such proteases may degrade both the attachmentproteins as well as the structural collagenous proteins of the matrix. Type IV collagenase makes a single cleavage 25% of the distance from the amino terminus of typeIV collagen. Proteolysis may be localized at the tumor cell surface where the amount of active enzyme outbalances the natural protease inhibitors present in the matrix.Step 3 is tumor cell locomotion into the region of the matrix modified by proteolysis. The direction of locomotion may be influenced by chemotatic factors. Continuedinvasion of the extracellular matrix may take place by cyclic repetition of these three steps.

chored tumor Å“il next secretes hydrolytic enzymes (or induceshost cells to secrete enzymes) which can locally degrade thematrix (including degradation of the attachment components).Matrix lysis most probably takes place in a highly localized regionclose to the tumor cell surface. The third step is tumor celllocomotion into the region of the matrix modified by proteolysis.Continued invasion of the matrix may take place by cyclic repetition of these three steps.

Laminin Receptor

Laminin, a major glycoprotein of basement membranes (26),plays an important role in the interaction of tumor cells with thebasement membrane. Laminin binding to the surface of cells canbe mediated by specific high affinity receptors (27-33), or by

membrane glycolipids (34) at a lower affinity. Laminin has beenshown to regulate a variety of biological phenomenon includingcell attachment, growth, morphology, and cell migration (25, 35-

37). The multifunctional biological properties of this moleculemay relate to its large size and multidomain structure (Table 1).Laminin is a cross-shaped molecule with three short arms andone long arm (34, 38-43). All four arms have globular endregions. The cross-shaped intersection of the short arms con

tains numerous disulfide bonds and is relatively protease resistant (38, 41). The domains of laminin are heterogeneous infunction and composition. The carbohydrate structure of theglobular end regions of the molecule are different from the rod-

shaped regions (40). The function of the carbohydrate groups isas yet undefined. The protease-resistant central region of laminin

contains the binding site for the laminin receptor presumably onone or more of the rod-shaped regions of the short arms. The

long arm of laminin contains a heparin binding site (34, 43) at theglobular end region. The long arm also stimulates neurite outgrowth (36). One or more globular end regions of the short armspromote cell spreading, bind to plasma membrane sulfatides(34), and also bind to type IV collagen (42-45). The type IVcollagen-binding site for laminin is located approximately 125 nmfrom the carboxyl-terminal globular domain (Chart 1).

Table 1

Functional domains of laminin

The reference citations for these findings are given in the text.

Molecule orfragment Structural features Biological functions

Whole laminin

tShort arms, 35 nm Promotes cell attach-Long arms, 76 nm ment, spreading,Rich in a-D-mannopyr- migration, growth

anosyl residues morphogenesis,and mÃ©tastases

Short armdomainRod-shaped

intersectionofshortarmsLong

arm4-4-Globular

ends rich in Â«-o-galactosylendgroupsArms

may be composed of morethanone

type ofchainDisulfide-bonded

â€¢knot"Relatively

protease resistantContains

mannose-ter-minatedoligosaccha-ride

unitsMay

containÂ«-helixstructureProtease

labileGlobular

ends bind totype IVcollagenand

sulfatidesPromotescell attach

ment andspreadingContains

laminin receptor-binding do

mainInhibitscell attach

ment and mÃ©tastasesBinds

heparinsulfateproteoglycanPromotes

neurite outgrowth

Many types of normal and neoplastic cells contain high affinitycell surface binding sites (laminin receptors) for laminin (27-31 ).

Our laboratory found that laminin in solution would bind via suchspecific receptors to suspended or attached cells or to isolatedcell membranes (Chart 2A). In time course experiments, theplateau of laminin binding is reached after 30 min at roomtemperature. With increasing concentrations of labeled lamininadded to the cells or the membranes, the binding is saturableand occurs with a high ratio of specific binding to nonspecific


2




246125I LAMININ nM

Chart 2. Binding of laminin to human breast carcinoma plasma membranes andhuman melanoma cells. A, saturation curve of specific binding to breast carcinomaplasma membranes as a function of increasing concentrations of 125l-laminin.Incubations are performed at 25Â°C for 40 min. Specific binding represents the

difference between binding in the presence (O) and the absence (A) of 200 nMlaminin. Inset, displacement of specifically bound 125l-lamininby increasing concentrations of laminin. Following incubation with 125l-laminin as described above,

unlabeled ligand was added and the incubation was continued for 1 h. The ligandunits are laminin nM. Specific binding is the percentage maximum labeled lamininwhich can be displaced with 500-fold excess unlabeled laminin. The proportion ofspecific binding to nonspecific binding judged by competition or displacement wassimilar. The free unlabeled ligand concentration at which 50% of the bound labeledligand is displaced is approximately 2 nM (32). B, Scatchard plot of the specificbinding data for suspended living human A2058 melanoma cells is linear (r = 0.98)yielding Kd 0.90 nM.

binding. Scatchard analysis is linear (Chart 2B). The bindingaffinity constant is in the nanomolar range with 10,000 to 100,000receptors per cell. We isolated the laminin receptor by lamininaffinity chromatography (27, 28, 32) (Fig. 1). It has a molecularweight of slightly less than 70,000, contains interchain disulfidebonds, and has an isoelective pH value of 5.2. The isolatedreceptor retains the ability to bind laminin but not fibronectin orany type of collagen. A laminin receptor with a similar molecularweight and binding coefficient has been reported by Malinoff andWicha (29) and Lesot ef al. (30).

In order to study mechanisms regulating the expression andfunction of the laminin receptor, we developed a library of mAbs2in collaboration with Dr. Jeffrey Schlom's group. The mAbs were

prepared against the purified laminin receptor extracted fromhuman breast carcinoma plasma membranes (32,33). Two mAbs(LR1 and LR2) were found to differ in their effects on lamininbinding to the receptor. By solid phase radioimmunoassay, LR1and LR2 bound with equal titer to the purified receptor. Usingimmunoblotting (Fig. 1), both LR1 and LR2 recognize a single M,67,000 component among all the proteins extracted from themembranes of breast carcinoma tissue. The antibodies alsobound with equal titer to isolated microsomal membranes or

2The abbreviation used is: mAb, monoclonal antibody.

200K

70K

43K â€”i

25K â€”

r -

B DFig. 1. Immunoblot of human breast carcinoma plasma membrane extracts with

anti-laminin receptor mAbs. Plasma membranes from human breast carcinomawere extracted with Nonidet P-40 and run (reduced) onto a 7% sodium dodecylsulfate-polyacrylamide electrophoresis gel. The gel was blotted onto nitrocellulose.The nitrocellulose was incubated with the mAbs followed by rabbit anti-mousesecond antibody. The bound antibodies were detected by '25l-protein A using

fluorography. Lane A, total extracted proteins transferred to nitrocellulose. Lane B,iodinated purified human breast carcinoma laminin receptor antigen. Lane C, mAbLR2 blotted to total protein extract (replicate extract shown in Lane A). A singlecomponent is recognized among all the protein bands. Lane D, mAb LR1 blottedto total protein extract (replicate extract shown in Lane A). A single component isrecognized. Lane E, control immunoblot using human serum. No immunoreactivityis present with mAb LR1 (shown) or LR2 (not shown). Control immunoblots of thetotal extract using IgM control antibodies, second antibody alone, or 125l-protein A

alone were completely devoid of immunoreactivity. K, molecular weight in thousands.

NT* 10-'

Monoclonal Antibody DilutionCharts. mAb inhibition of binding of 125l-laminin to human breast carcinoma

plasma membranes or MCF-7 breast carcinoma cells. The antibodies were addedwith the 125l-laminin (6 nM) at the beginning of the incubation. Specific laminin

binding was determined as shown in Chart 2. Binding of laminin to MCF-7 breastcarcinoma cells (mean of four experiments; oars, SD). mAb LR1 but not LR2inhibited specific binding. Control mAb B6.2 (C) recognizing a different antigen onMCF-7 cells failed to inhibit binding. Control NS1 cell supernatant alone was devoid

of inhibitory activity.

living breast carcinoma cells. No binding to serum componentswas evident. When added together with the labeled ligand, mAbLR1 produced a dose-dependent inhibition of specific laminin

binding to human breast carcinoma cells (Chart 3). In contrast,mAb LR2 had no effect on laminin binding. The two classes of


3




30 60Time (Minutes)

Chart 4. Monoclonal antibodies (mAb LR-1 ) to the binding domain of the humanlaminin receptor inhibit attachment of MCF-7 breast carcinoma cells to denudedlaminin-rich surfaces of human amnion basement membranes (BM). Attachmentwas conducted at 25Â°Cfor the indicated times. Means of 6 separate assays; bars,SD. mAb control, IgM control mAb, 1/100; anti-Fn, anti-fibronectin, 1/100; mAb LR-

7,lgMmAbLR1, 1/100.

antibodies may therefore recognize different structural domainson the receptor molecule. We next studied the effect of theseantibodies on the attachment of human melanoma and carcinomacells to native human amnion basement membrane surfaces(Chart 4). Human amnion membranes consist of a single layer ofepithelium, a continuous basement membrane, and an underlyingnonvascular interstitial stroma. The epithelium of the amnion canbe removed leaving a continuous laminin-rich basement membrane surface. The receptor-binding fragment of laminin compet

itively inhibits attachment of cells to the amnion basement membrane surface (46). mAb LR1 markedly inhibits attachment ofmelanoma and carcinoma cells (Chart 4) to the basement membrane surface but not to the stromal surface (which lacks laminin).Control antibodies fail to inhibit attachment. Thus the orientationof laminin in the native basement membrane is such that thecross-shaped receptor-binding domain of the short arms is ex

posed on the attachment surface.Laminin receptors may be altered in number or degree of

occupancy in human carcinomas. This may be the indirect resultof defective basement membrane organization in the carcinomas.Breast carcinoma and colon carcinoma tissues contain a highernumber of exposed (unoccupied) receptors compared to benignlesions (31, 33). The laminin receptors of normal epithelium maybe polarized at the basal surface and occupied with laminin inthe basement membrane. In contrast, the laminin receptors oninvading carcinoma cells may be distributed over the entiresurface of the cell. They may be unoccupied because of the lossof formed basement membrane associated with the invadingcells (10). Using mAb LR1, we have isolated a putative humancomplementary DNA clone for the laminin receptor.3 This should

be useful for future studies of the genetic regulation of lamininreceptor expression.

The laminin receptor can be shown to play a role in hematog-

enous mÃ©tastases(46). In animal models, tumor cells selectedfor the ability to attach via laminin by Terranova ef al. (47)produced 10-fold more mÃ©tastasesfollowing i.v. injection. Wholelaminin on the tumor cell surface will stimulate hematogenousmÃ©tastases(46, 48). This stimulatory effect requires the globularend regions of the molecule (Table 1). Treating the cells with thereceptor-binding fragment of laminin markedly inhibits or abolishes lung mÃ©tastasesfrom hematogenously introduced tumorcells (46). Thus the laminin receptor can play a role in hematogenous mÃ©tastases through at least two mechanisms. If the

3 Sobel ef a/., manuscript in preparation.

receptor is unoccupied, it can be used by the cell to bind directlyto host laminin. If the receptor is occupied with laminin, the cellcan utilize the surface laminin as an attachment bridge throughthe globular end regions. The fragment of laminin which binds tothe receptor but which lacks the laminin globular end regionsinhibits both of these mechanisms.

Type IV Collagenase

In vitro studies of tumor cell invasion of the extracellular matrixhave shown that cell proliferation is not absolutely required (49).However, protein synthesis and proteolysis do seem to play animportant role. Inhibitors of protein synthesis or natural inhibitorsof metalloproteinases block invasion of the matrix (49, 50). Thusinvasion of the matrix is not merely due to passive growthpressure but requires active biochemical mechanisms.

Many research groups have proposed that invasive tumorcells secrete matrix-degrading proteinases (51, 52). Collagen is

an important substrate because it constitutes the structuralscaffolding upon which the other components of the matrix areassembled (6). Tumor-derived collagenases which degrade in

terstitial collagen types I, II, and III have been characterized by anumber of investigators (52-54). Tumor collagenases have prop

erties similar to those of classic vertebrate collagenase firstdescribed by Gross and Magai (55). They are metal ion (calciumand zinc)-dependent enzymes which function at neutral pH.

Classic collagenase produces a single cleavage in the collagenmolecule (interstitial collagen types I, II, and III) at 25Â°Cproducing

three-fourths and one-fourth size fragments (75% of the distance

from the amino terminus). The molecular weight of classic collagenase ranges from 33,000 to 80,000 depending on the source.In some studies, the amount of tumor collagenase can be correlated with the aggressive behavior of the tumor (53). Ourlaboratory extended the hypothesis of interstitial collagen proteolysis by tumor cells to proteolysis of basement membranes.Detailed studies of tumor cell extravasation (56) or invasion ofmuscle (22) demonstrated local fragmentation of the host basement membrane adjacent to the tumor cell. Consequently localproteolytic modification of the basement membrane may benecessary for (or at least augment) the migration of cells throughthis structure.

We found that tumor cells could degrade both collagenousand noncollagenous components of the basement membrane(23). Tumor cells collected from the venous effluent of a transplanted sarcoma exhibited higher basement membrane-degrad

ing activity compared to the general population of cells in thetumor. Thus tumor cells entering the circulation may be a sub-

population selected for the ability to degrade vascular basementmembranes. The selection of aggressive tumor subpopulationsis in keeping with the concept of tumor cell heterogeneity andselection of metastatic variants proposed by Fidler er al. (2). Aseries of subsequent studies have extended the finding thatmetastatic tumor cells have the capacity to degrade basementmembranes (57-63). Investigation into the proteases involved in

tumor cell destruction of basement membranes revealed thatbasement membrane collagens types IV and V differed markedlyfrom interstitial collagens I, II, and III with regard to proteolyticsusceptibility (64-66). Collagen types IV and V are not suscep

tible to classic collagenase which degrades collagen types I, II,and III. A separate family of collagenolytic enzymes were pro-


4




Tabte2Collagenasetype IV activity and in vivo behavior of tumor cellhybridsCollagenase

typeIVar*ti\/it\/Ã¤ivactivity"

(ng/10*cells)Cell

lineB16-F10""UV-2237mK-1

735 done16C3H-FPECAPECÂ«Cell

hybrid2237""xF10F10â„¢xclone16--1F10"Rxclone16-3F10RRxC3HF10RRxPECF10RRxPECw2237â„¢

xPEC,2237â„¢x PEC,Lung

metastasisformation''Nude

mice128

Â±4172Â±2219

Â±103Â±118Â±1015Â±6201

Â±31203Â±64223

Â±5622Â±1332Â±1338Â±92Â±16Â±5C57BL/6

x C3H F,mice238(2-316)(T+)c60

(1-1 58)(T+)0(T+)0(T-)0(T-)ND149(0->400)46

(9-300)300(41 >300)0(0-1)0000200(1

00 > 300)(T-t-)0(T+)0(T+)0(T-)0(T-)ND>400109(9-294)259

(22 >300)0000

(0-1)5(0-18)

* Mean Â±SE from 3 to 10 experiments." Median number of lung nodules from 10 animals per group. The ranges are given in parentheses.0 T+ or T-, tumorigenicity. All cell hybrids were highly tumorigenic. PEC, activated (PECA)or nonactivated

(PECn/0C57BL/6 x C3H F, peritoneal macrophages;C3H-F, mouse embryo fibroblasts; ND, not determined.

ee

ffÂ«

>.

90

80

70

60

50

40

30

20

10 ,,

O

P ST TI T2 T3 T4 T5Charts. Collagenase type IV (type IV collagen-degrading metalloproteinase)

activity of NIH 3T3 parent cells (P), spontaneously transformed tumorigenic (non-metastatic) NIH 3T3 cells (S7), and five separately isolated human tumor DNAtransfectants (70) (clones 77-75) which produce mÃ©tastasesin NIH nude mice.Bars, SO.

posed to be required for catabolism of basement membranes. Insupport of this concept, a type IV collagenolytic metalloproteinase was identified in highly metastatic tumor cells and in en-

dothelial cells (67). Separate metalloproteinases were found todegrade type V collagen (66, 68, 69). Type IV collagenase has amolecular weight of approximately 62,000 to 65,000 after activation (60). It is secreted in a latent form but may also exist onthe cell surface. The level of type IV collagenase is augmentedin many highly metastatic tumor cells. Antibodies preparedagainst type IV collagenase react with invading breast carcinomacells and breast carcinoma lymph node mÃ©tastasesby Â¡mmuno-

histology (20).Type IV collagenase produces two sets of cleavage products

by gel electrophoresis (58). The size of the fragments is consistent with a single major cleavage through both chains of type IVcollagen. The substrate cleavage region for type IV collagenasehas recently been identified by Fessier et al. (70). The largercleavage fragment contains the amino terminus. By rotary shadowing electron microscopy, the type IV collagen cleavage site

was localized to a position 25% of the distance from the aminoterminus. Thus this metalloproteinase cleaves the type IV collagen molecule on the opposite end compared to classic collagenase cleaving type I collagen. Timpl ef a/. (15) and Charanios efal. (45) have hypothesized that type IV collagen molecules maybe linked at their end regions, and possibly side to side, to forma uniform hexagonal network. Type IV collagenase can effectively break down this network by cleaving each triple helicalmolecule and breaking each side of the hexagon unit (Chart 1).

Possible Genetic Linkage of Type IV Collagenase Expressionwith the Metastatic Phenotype

A metastatic colony is the end result of a complex series oftumor host interactions (1-5). It is apparent that these interac

tions involve multiple gene products. A cascade or coordinatedgroup of gene products expressed above a certain thresholdlevel may be required for a tumor cell to successfully traversethe successive steps in the metastatic process. The crucial geneproducts may regulate host immune recognition of the tumorcell, cell growth, attachment, proteolysis, locomotion, and differentiation. The specific family of gene products necessary formÃ©tastasesmay be different for each histological type of tumor.

We used DNA transfection (71) and somatic cell hybridization(72) to investigate whether type IV collagenase may be a possiblemember of this hypothetical group of metastasis-associated

gene products (Table 2; Chart 5). Transfection of tumor DNAinto recipient NIH 3T3 cells has been shown by our laboratoryand others to induce the metastatic phenotype assayed in nudemice (71-75). The metastatic phenotype was elicited in secondary and tertiary transfectants. Transfection of the ras" oncogene

alone could induce the metastatic phenotype only in certain typesof recipient cells (73) assayed following both i.v. and s.c. injectioninto nude mice. After studying this phenomenon in a variety ofdifferent recipient cells including second generation diploid ratembryo fibroblasts, we have developed a working hypothesis(73). Induction of the metastatic phenotype requires at least two(and possibly more) gene complementation groups. In the correct


5




recipient cells, one of these genes may be the activated but notthe cellular (protooncogene) form of the ras" oncogene. The

exact DMA sequence and function of the other members of thegene complementation group are under intense study by ourgroup and others (73, 74, 76). It is expected that importantmembers of the metastasis-associated gene products will be

those relating to host antitumor immune defenses (74, 76).Induction of mÃ©tastases by transfection of foreign DNA mayinvolve indirect phenomena such as induction of genetic instability and progression in the recipient cells (73, 75). Geneticcontrol of mÃ©tastasesis separate from tumorigenicity because aplasmid construct of the protooncogene which induces largeamounts of the cellular form of the M, 21,000 protein will induceNIH 3T3 cells to be fully tumorigenic but not metastatic (73).

For a series of NIH 3T3 cell transfectants which exhibit metastatic propensity, all secreted high levels of type IV collagenasecompared to NIH 3T3 parent cells or spontaneously transformed(tumorigenic but nonmetastatic) NIH 3T3 cells (Chart 5).

A similar association of type IV collagenase with the metastaticphenotype was also observed following somatic cell hybridization(Table 2). The results of somatic cell hybridization must becarefully analyzed because hybrid cells are unstable and theexact karyotypic feature of each hybrid clone will be different(77, 78). However, it is possible to interpret the data derivedfrom an individual hybridization system as regards the correlationof a specific gene product with the metastatic phenotype. Themetastatic phenotype of tumor-tumor cell and tumor cell-normal

cell hybrids were compared with their type IV collagenase activity(72, 77). Fusion of metastatic tumor cells with nonmetastatictumor cells resulted in maintenance or augmentation of themetastatic phenotype. However, when metastatic cells werefused with normal cells, in this series, the metastatic phenotypewas suppressed. Furthermore the hybrids retained the ability toproduce tumors. Thus tumorigenicity was shown to be distinctfrom metastatic propensity. The levels of type IV collagenase inthe hybrid cells were altered in parallel with metastatic behavior.Suppression of mÃ©tastases resulted in suppression of type IVcollagenase. In no case was a metastatic hybrid identified whichhad lost the ability to elaborate type IV collagenase. Thus forthis particular series of hybrids, type IV collagenase may be oneof many gene products expressed concomitantly with other geneproducts necessary for formation of mÃ©tastases.

Type IV collagenase is only one member of a family of protein-

ases which participate in the physiological turnover of basementmembranes. A cascade of proteases including thiol proteases,heparinases, and serine proteases such as plasminogen activator all contribute to facilitating tumor invasion (79-82). Proteoly-sis regulation can take place at many levels including tumor cell-

host cell interactions (61) and protease inhibitors (54) producedby the host or by the tumor cells themselves. Protease secretionor activation by invading cells may also be coupled to cell shapeor locomotion (67,83). Expression of matrix-degrading enzymes

is not tumor cell specific. The actively invading tumor cells maymerely respond to different regulatory signals compared to theirnoninvasive counterparts.

ACKNOWLEDGMENTS

The work presented here is the result of enthusiastic efforts by all members ofmy research group in the Section of Tumor Invasion and MÃ©tastases and ofcollaborations with Dr. George Martin and Dr. Victor Terranova in the National

Institute of Dental Research. I thank Alan Rabson for his essential scientific supportand encouragement.

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6. Hay, E. D. Cell Biology of Extracellular Matrix. New York: Plenum PublishingCorp., 1982.

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13. Kefalides, N. A., Alper, R., and Clark, C. C. Biochemistry and metabolism ofbasement membranes. Int. Rev. Cytol., 67:167-180,1979.

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15. Timpl, R., Wiedmann, H., VanDelden, V., Furthmayer, H., and KÃ¼hn,K. Anetwork for the organization of type IV collagen molecules in basementmembrane. Eur. J. Biochern., 120: 203-212,1981.

16. Hogan, B. L. M. High molecular weight extracellular proteins synthesized byendoderm cells derived from mouse teratocarcinoma cells and normal extraem-bryonic membranes. Dev. Bid., 76:275-281,1980.

17. Carlin, B., Jaffe, R., Bender, B., and Chung, A. E. Entactin, a novel basallamina-associated sulfated glycoprotein. J. Biol. Chern., 256: 5209-5218,

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1986;46:1-7. Cancer Res Lance A. Liotta Matrix: Rhoads Memorial Award Lecture

Role of the Extracellular−−Tumor Invasion and Metastases

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