89

Bwchtmtca et Bwphyvtca Acta, 1032 (1990) 89-118 Elsevier BBACAN87222

Tumor interactions with the vasculature: angiogenesis and tumor metastasis Christine H. Blood and Bruce R Zetter Department of Surgery, Chtldren's Hospttal and Department of Cellular and Molecular Physzology. Harvard Medtcal School, Boston, MA (U S .4 ) (Received 13 November 1989) (Rewsed manuscript recewed 19 February 1990)

Contents I

Introducuon

90

II

The primary tumor A Tumor anglogenesxs 1 Hlstoncal perspectwe 2 Cellular aspects of tumor anglogenesls 3 Anglogemc factors 4 Anglogemc factor redundancy 5 Role of accessory cells m tumor ang~ogenes~s 6 Inlubltors of anglogenesls B Properues of the tumor vasculature 1 Cellular components of tumor blood vessels 2 Extracellular matnx of tumor blood vessels 3 Tumor vessel permeability C Tumor cell &ssocmtlon 1 Modulalaon of extracellular matrix integrity 2 Modulation of cell adhesion 3 Modulatton of cell motthty D Tumor cell entry into the bloodstream

90 90 90 91 92 96 97 98 98 99 99 99 99 100 100 100 101

III

Circulating tumor ceils A Surwval of tumor ceils in the bloodstream B Interactions between tumor cells and platelets C Interacuons between tumor cells and leukocytes

102 102 102 103

IV

Tumor cell adhesion to the vessel wall A Adhesion to the endothehum B Adhesion to the subendothehal basement membrane

103 103 104

V

Tumor cell exat from the vasculature A Degradation of basement membrane components 1 Metalloprotemases 2 Plasrmnogen acttvators

105 105 105 106

AbbrevlaUons CAM, chonollantolc membrane, EGF, epidermal growth factor, HBGF, hepann-bmdmg growth factor, FGF, flbroblast growth factor, HIV, human lmmunodeflcaency varus, VEGF, vascular endothehal growth factor, PDGF, platelet-denved growth factor, ECGF, endothehal cell growth factor, TGF, transforming growth factor, TNF, tumor necrosis factor, GAG, glycosarmnoglycan, PMN, polymorphonuclear leukocytes, LFA, lymphocyte funcuon associated anUgen, TIMP, tissue mtubltors of metalloprotemases, TPA, 12-O-tetradecanoylphorbol 13-acetate, RGD, Arg-Gly-Asp Correspondence B R Zetter, Enders Bldg, Room 1017, Ctuldren's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA 0304-419X/90/$03 50 © 1990 Elsevier Science Pubhshers B V (Biomedical Dlxaslon)

90 107 107 107 107

3 Hepantlnases 4 Cathepslns 5 Elastases B Tumor cell motlhty and chemotaxls

VI Cellgrowth control at the secondary site

108

VII Conclusions

109

References

109

I Introduction The study of tumor biology has, in recent years, focused on the abnormal regulation of growth control an neoplastic cells and on the genes that control tumor cell proliferation Although cell growth as a crucial component of the malignant phenotype, several additional processes are required for successful completion of the malignant process Foremost among these are a variety of interactions that must take place between the tumor cell and the host vasculature, interactions that are essential for both tumor expansion and for metastatic tumor spread Without the ablhty to recruit new blood vessels, or to enter and exit from the bloodstream, it is likely that most tumors would never grow beyond a diameter of 1 - 2 m m and would remain locahzed to the primary site The study of t u m o r / b l o o d vessel Interactions thus becomes essential for our understanding of the mahgnant process In tins article, we will review the recent progress in our understanding of the nature of these various interactions

1I. The primary tumor It has been demonstrated that tumor spheroids grown m vitro or m vivo in the absence of blood vessels will grow until they reach a size at winch passive diffusion can no longer effectively provide nutrients for all the cells nor can waste products adequately diffuse out of the spheroid into the surrounding medium or matrix [1] In either case, an equihbraum condition is reached at which the growth of cells on the tumor periphery as equivalent to the rate of cell death In the spheroid interior [2] Commonly, the diameter of a sphere in such an equilibrium state will be between 3 and 4 m m an vitro [3] and less than 2 m m in SltU in the rabbit cornea [2] Such tumors can remain in this equilibrium state for several years an vitro [4], whereas in situ, it is possible to see small tumors remain viable and avascular for decades [5,6] Metastases are rarely associated with such avascular tumors If, however, these tumors become vasculanzed at some later tame, their morphology and growth patterns change dramatically [2] Rapid outgrowth now occurs and the tumor ceUs begin to grow in a cylindrical pattern around the new rmcrovessels [7] It

has been estimated that m a rapidly growing, vascularlzed tumor, no tumor cell will be found more than 100 /~m away from the nearest blood vessel [8] Such rapidly growing, vascularized tumors will frequently gave rise to metastatic colonies [9] Because of the importance of neovasculanzation for tumor growth and metastasis, the mechanisms by winch tumors attract new blood vessels have been the subject of extensive scientific inquiry [10-12] H-A

Tumor angwgenests

The term 'ang~ogenesis' refers to the development of new blood vessels Although the term can effectively be used to describe any form of vascular development, it has, in current usage, been applied to the development of vessels by a process of sprouting from pre-existing vessels The de novo development of blood vessels from precursor cell types that takes place during formation of the earhest embryonic blood vessels is more frequently referred to as 'vasculogenesls' A tinrd type of vascular growth that is most properly referred to as 'vascular expansion' occurs when existing small or closed vessels enlarge an diameter and are consequently able to transport greater quantmes of blood, tins type of expansion commonly occurs an the formation of collateral blood vessels, such as those that appear after cardiac tissue damage [13,14] T u m o r anglogenesls refers to the directional sprouting of new vessels toward a solid tumor The stamuh that promote tumor ang~ogenesls may be provided directly by the tumor cells themselves or indirectly by host Inflammatory cells that are attracted to the tumor site In recent years, enormous progress has been made in our understanding of the cellular and molecular basis of tins process II-A 1 Htstottcal perspectwe One of the first techniques developed for the purpose of studying blood vessel formation was the transparent ear chamber, devised [15] to study the development of new vessels at the sate of wounds Tins method was adapted by Ide and colleagues to study the vascularlzataon induced by tumor cells [16] Using the same system, Alglre and Chalkley [17] observed that tumor tissue implanted into the ear chambers elicited the appearance

91 of new blood vessels whereas normal tissue did not Algxre further suggested that the ablhty to attract new blood vessels rmght be a prereqmsIte for continued tumor growth [18] Tins proposal ehclted a long and only recently fruitful search for the tumor-derived cell molecules that promote capillary formation Nearly twenty years after Algxre's proposal, several groups were able to demonstrate that the anglogenlc agent produced by tumor cells could diffuse across a 0 45 /~m filter [19-21] suggesting that such a factor maght diffuse away from a tumor mass m VlVO and ehcit production of capillary sprouts from nearby host vessels Much of the impetus for research m tumor anglogenesls in the 1970's came from the experiments and ideas of Folkman and Ins colleagues The inltml interest in the system was stimulated by studies that confirmed that tumors grown in organ culture stopped growing at a density of 1 - 2 m m [22] Avascular tumors, kept alive by perfuslon, grew only at the periphery and developed a necrotic center [23] In contrast, tumor spheroids placed into the ocular chamber wltinn 6 m m of the m s blood vessels, reduced neovasculanzatlon, grew rapidly and metastaslzed [2,24] In a 1972 review [25], Folkman elaborated a hypothesis that stated that spherical sohd tumors were absolutely dependent on anglogenesls for growth beyond 2 mm, that for every increase m tumor diameter there must be a corresponding increase in vascularlzatlon of the growing tumor He further proposed the concept that lninbltlon of anglogenesxs might be sufficient to cause inhibition of tumor growth and metastasis [9,26], a proposition that has stimulated cons~derable research effort in the quest of finding effective anglogenesls minbitors In the course of their studies, Folkman, Glmbrone and their colleagues developed two in VlVO b~oassays that have become mainstays of anglogenesis research The first is the corneal pocket assay [24] which takes advantage of the fact that the cornea is an avascular Ussue surrounded by accessible blood vessels in the hmbus of the eye When an anglogemc factor is transplanted into the cornea w~tinn a few m m of the hmbus, capdlary sprouts grow dlrectlonally into the cornea from the hmbus Because the cornea is transparent, these new vessels are easily detected by the investigator The second assay entails placing lmmoblhzed anglogenlc factor onto the chonoallantoic membrane (CAM) of the cinck embryo [27-29] Again, the new vessels are readily vasualized on the transparent membrane, although the presence of normal vessels on the developing C A M presents a greater background than that found in the rabbit cornea Of the two assays, the chick C A M is less expensive and requires less surgical skill It is therefore useful for obtaining large amounts of data as in screening studies to test the anglogenlc activity of a variety of compounds The corneal mlcropocket assay IS, in contrast, costly and more cumbersome, but it is more accurate and more

easily interpreted due to the absence of background vessels One potential drawback of the corneal assay is that the induction of vessel growth in tins unusual avascular site m a y not be identical to the processes that occur in the more normal, vascular tissue that commonly surrounds a tumor site Both assays are subject to comphcations arising from the presence of inflammatory or lmmunogemc compounds since both processes have an associated anglogemc component The chick C A M and rabbit cornea are both suscepUble to the appearance of false positive results based on a secondary inflammatory response rather than a primary anglogenlc response to the test substance For tins reason, ~t is essential to analyze the results of all in VlVO anglogenic assays by histological examination of the relevant tissues in order to rule out or confirm the presence of inflammatory cell types such as neutropinls, monocytes or macrophages The anglogenesls assays described above have been used for a variety of purposes They have been used to detect ang~ogemc activity in malignant and in premahgnant lesions [30-33] They have also been used to screen purified test substances for angiogenlc activity In conjunction with light and electron rmcroscopy, they have been used to elucidate the cellular events that comprise tumor anglogenesls [34] and vascular regression [35] More recently, these assays have been used to great advantage m detecting the activity of ang~ogenes~s minbltors [36,37] Although the m VlVO Inoassays have proved extremely useful for detecting anglogemc actwlty m crude and purified test fractions, early attempts to purify tumor-derived angiogemc factors with these and other bioassays were only partmlly successful [38,39] Among the drawbacks of these assays for purification is the fact that the assays generally take several days, along with considerable labor and, in the case of the corneal assay, expense In add~tlon, quant~tatxon of these assays has proved to be difficult, although recent improvements m such measurements have been reported [40,41] W~thout accurate, rapid and reproducible quantitauon, such assays can only be used to detect the presence or absence of activity but not to compare fracuons from a punficauon protocol H-A 2 Cellular aspects of tumor angtogenests T u m o r anglogenesls takes place by a process of capdlary sprouting from preexisting macrovessels When these events are observed in VlVO using electron rmcroscopy, a consistent pattern of cellular events is seen [34,42-45] The first apparent response to an ang~ogentc stimulus from a tumor is the dissolution of basement membrane surrounding a preexastlng vessel, usually a post-capillary venule Next endothelial cells begin to rmgrate out of the old vessel, toward the tumor The leading endothehal cells do not appear to divide, whereas

92 the traalang cells undergo D N A synthesis and cell dwlslon Endothelial cell division is not, however, an absolute requirement for angiogenesis Sholley and coworkers [46] have shown that when tumors are placed m irradiated rabbit eyes where no endothelial cell division can take place, capillaries can still grow over a distance of 1 - 2 m m m the absence of endothehal cell prolaferatmn In contrast, endothelial cell rmgration appears to be required for angiogenesis Once the immature new vessels have elongated, they undergo a process of canalization (lumen formation) along with branching and formation of vascular loops to form a complete vascular network The final stage of capillary development is the formation of a mature capillary bed in wluch the capillaries are surrounded by basement membrane components [47] and, in some cases, by a layer of perivascular cells called pencytes [48] Formation of a basement membrane and investment of capillaries with perlcytes are generally associated with the end of the proliferative stage and the begmmng of the mature or quiescent stage of capillary function [49] Progress m the identlflcataon and purification of angiogenlc factors was accelerated by the development of in vitro assays using cultured endothelial cells Angiogenesls occurs as the growth of new capillaries Because endothehal cells from macrovessels differ significantly from endothehal cells from larger vessels [50,51] it became apparent that the culture of capillary endothelial cells would faclhtate development of in vitro anglogenesls assays Beginning an the mad 1970's [52-56], reports of short-term capdlary endothelial cell cultures began to appear, and by 1979 [57-62] it was possible to grow capillary endothelial cells in long-term culture Using the cultured capillary endothelaal cells, it has been possible to develop assays for each of the cellular steps In the angiogenlc process Endothelial production of collagenase and plasmmogen activator was shown to rise in response to angiogenlc factors [63-65] Endothehal cell migration [66-69], and proliferation [57] are also induced Under certain conditions, capdlary endothehal cells in vitro will form three-dimensional networks that resemble capillary networks in vivo [70] Tlus process is dependent on both the nature of the growth factors present and the nature of the extracellular matrix to winch the cells are attached [71-74] The development of tlus variety of assays has facilitated the ident~ficatlon and purification of a large number of anglogenlc factors [75,76], as well as the detection of human tumors based on their elaboration of angmgenlc factors Into surrounding tissues and fluids [77,78] and has permitted a greater understanding of the cellular and molecular mechanisms underlying the anglogenic process [79,80] One important concept to emerge from tlus work is the realization that not all of the factors that cause angiogenesls an vavo have the same set of effects on pure cultures of capillary endothelial cells in

vitro Some anglogenic factors cause a pleiotyptc response of enzyme production, nugrat~on and prohferatlon [81,82], some cause endothehal cell nugration only [83-86] Still others appear to have no direct effects on endothelial cells [87] and are therefore considered to exert their in vivo angiogenic effects by attracting secondary cells such as macrophages that, in turn, produce endothelaal-actlvatang angiogenlc factors [88,89]

H-A 3 Anglogemc factors At the present time, more than a dozen purified molecules have been shown to be angiogenac when assayed in the chick or rabbit bioassays With the exception of the discovery of angaogenln, most of the anglogemc molecules were first purified on the basis of some unrelated activity and then later were shown to be angiogemc as well Most of these molecules are polypeptldes, although the last also includes hpads, nucleotides and one vItanun Whether each of these molecules causes anglogenesis to occur naturally in VlVO is currently a matter of speculation but it is reasonable to expect that the following last contains at least some physiologically important angiogemc factors The current status of anglogenic factors has been reviewed extensively [12,75] and will be discussed only briefly in tlus review A summary of the angiogemc factors discussed an tins review is presented in Table I (a) Angzogenm Anglogenin is a basic polypeptlde, with a molecular weight of 14 400, that is produced and secreted by murlne and h u m a n carcinomas [90-92] Although anglogenln is a potent angmgenlc factor when tested in the cluck C A M or rabbit cornea, and can Induce activation of an endothehal cell phosphomosltide-specific phosphohpase-C [93], it does not appear to have any rmtogenic or chemotactic activity on cultured endothelial cells Tins suggests that the angiogemc activity of angtogenm may be mediated through an accessory cell type that produces an endothehal activating factor Angiogenln possesses a partial homology to a class of rlbonucleases and has some limited ribonuclease activity [94,95] that appears to be related to its anglogenlc activity Blocking RNAase activity with bromoacetate [96] or with placental RNase inhibitor [97] dirmmshes the anglogenlc activity of the molecule Angiogenin is not restricted to tumors The primary site of angiogenin m R N A synthesis in the rat appears to be m adult hver [98], although it IS also produced by normal fibroblasts and as found in bovine and human plasma [99,100] Because of its wide &stributlon and multiple activities. it is possible that the primary role of tins molecule is other than the regulation of anglogenes~s (b) Angmtropm Angmtropln IS an intriguing angiogenlc factor isolated from peripheral blood monocytes that has been proposed to play a role in normal wound healing [101-103] It is unusual in that the angmgenic activity appears to depend on an R N A component of a

93 polynbonucleoprotem having a total molecular weight of 4500 [104] Angmtropm is a potent angmgemc agent when tested m vivo, and is a potent stimulator of endothehal cell rmgratlon in vitro [85] In addition, angmtropln admamstratlon in vivo produces a transient vasoddatlon It has no effect, however, on endothehal cell prohferauon Because migration alone can give rise to a full blown angdogemc response, this type of factor should sttU be considered a direct-acting angiogemc factor When added to confluent endothehal cell cultures, anglotropm can Induce the formation of three-dl-

menslonal networks that resemble capdlanes [85] Another macrophage denved angtogemc factor that is chemotactic for endothehal cells [83,105] has been reported by Banda and colleagues Such molecules must be considered prime candidates for a role m stimulating the anglogenesls associated with normal wound heahng [103] and may be useful in the future in the treatment of chromc wounds or ulcers (c) Epldermal growthfactor Epidermal growth factor (EGF) is a well-charactenzed polypeptlde that stimulates eplthehal cell prohferatton tn vitro and has a

TABLE I

Angmgemc molecules Factor

Source

Ang~ogenesis assay

Stimulates endothehal prohferation

rmgratlon

Ref

Anglogemn

Munne and human carcinomas, normal plasma

Cluck CAM, rabbit cornea

no

nt

90-100

Angtotropm

Penpheral blood monocytes

Cluck CAM, rabbit cornea, rabbit ear lobe

no

yes

85,100-109

Macrophagederived factor

Macrophages

Rabbit cornea

no

yes

105

Epidermal growth factor (EGF)

Mouse parotld gland, saliva, urine, rmlk

Hamster cheek pouch, rabbit cornea (modest response)

yes

nt

106,107

Fibrin

Tumors, wound granulation tissue

Cluck CAM, rabbit cornea plexaglass chamber implants

no

yes

112-118

Basic hbroblast growth factor (bFGF)

Broad distribution

Cluck CAM, rabbit cornea

yes

yes

119,121-137 140-142 144-151

Acidic hbroblast growth factor (aFGF)

Primarily neural tissue

Cluck CAM, rabbit cornea

yes

yes

120,138,139 143

Ntcotmarmde

Walker carcinoma

Cluck CAM, rabbit cornea

nt

nt

172

Platelet-denved endothelial cell growth factor (PD-ECGF)

Platelets

Cluck CAM, rat cornea

yes

nt

173-175

Transforrmng growth factor a

Transformed hbroblasts, tumors, macrophages

Hamster cheek pouch

yes

nt

108

Transforrmng growth factor fl

Platelets, bone

Cluck CAM, newborn mouse skin

no

no

200

Tumor necrosis factor a

Activated macrophages

Cluck CAM, rat cornea

no

yes

86,206

Vascular endothehal growth factor

Tumors Pituitary cells

Cluck CAM, rat cornea

yes

nt

208-212

n t = not tested

94 variety of m wvo effects [106] E G F ~s not a potent endothehal cell rmtogen, although it can have hrmted actlvaty on endothehal cells m the presence of exogenous protemases such as t h r o m b m [107] E G F has angmgemc actxwty when injected into the rabbat cornea but the act~wty is modest and Is exceeded by that of a related molecule, transforrmng growth factor et [108] that interacts with the same cell-surface receptor Thus, whale E G F is capable of stimulating endothehal cells and inducing angaogenesls, thas does not seem to represent a primary role for this molecule (d) Fibrin Although the functmn of fibrin m blood coagulation is well known, fibnn is also found in wound granulauon tissue as well as in sohd tumors [109-111] Because both processes have an anglogemc component, Dvorak and colleagues have investigated whether fibrin or fibrmopeptldes maght have any role in the anglogenlc process [112] In early expenments, fibrin gels implanted into the subcutaneous space of gumea pigs were observed to become vascularlzed and invested with a collagenous stroma [113] These results were confirmed by others using the chick CAM assay [114] along with the rabbit cornea [115] although m the latter case there was a considerable inflammatory reaction to the fibrin stimulus In more recent work, Dvorak and colleagues have devased an improved assay to quantify the vascular response to fibnn in VlVO In these experiments, porous cyhndncal plexaglass chambers were filled with fibrin or control substances and ~mplanted into guinea p~gs for a 6 day period [116] Neovascularlzatlon was measured by counting the number of vessels that invaded into the chamber The authors found that ang~ogenes~s proceeded m the presence of fibrin alone but was potentiated by the a d d m o n of the chemotactlc pept~de fMetLeu-Phe or the platelet-derlved growth factor Anglogenes~s was not induced m chambers containing either agarose or type I collagen While the mechamsm of fibrin-reduced anglogenesls is not completely understood, fibrin does have a &rect effect m stimulating endothehal cell motdlty [117] and vascular cells cultured in fibrin gels form three-&menslonal networks resembhng capillaries [118] For these reasons, fibrin may be considered a potential &rect-actlng anglogemc factor (e) Heparm-bmdmg growth factors Purification of ang~ogemc factors was faohtated by the finding that a class of molecules capable of reducing anglogenesls m the rabbit cornea or cluck CAM displayed a tugh affinity to the glycosarmnoglycan hepann [119-123] These heparm-bmdlng growth factors were found to be plelotypic activators of capillary endothehal cells capable of stimulating protemase production and chemotaxas, as well as endothehal cell dWlSlOn [81,124,125] Two general types of hepann-bmdlng factor were d~stlngmshed on the basis of xsoelectrlc point with one being basic ( p I 9-10) and the other being acidic ( p I 5 0) [126,127]

Although the two forms of growth factor have nearly identical biological activity, they differ in their patterns of &stnbutlon [128] Basic H B G F has been found in nearly every tissue exarmned [81,129-137], whereas acidic H B G F is preferentially locahzed m neural tissues such as the brain, hypothalamus, pituitary and retina [120,138,139] A m i n o acid [140-142] and nucleotlde [143,144] sequence analysis revealed that the hepannbinding factors were similar m composition to a previously discovered group of endothehal mltogens called fibroblast growth factors ( F G F ) [145,146] Although the h e p a r m - b m d i n g growth factors (HBGFs) are extremely active angmgemc factors, their role m tumor angtogenesls m vwo is not well-understood One point whach has puzzled investigators concerns the fact that these factors have no signal peptlde and do not appear to be secreted by the cells into the extracellular fluid Thus, it IS not certam how these factors could represent the 'diffusible' angxogemc factors first described by Greenblatt and Shublk [19] Two potential means have been proposed for the release of these factors from solid tumors D ' A m o r e and Klagsbrun [12] have postulated that H B G F s maght be released from lysed cells in lschemac tissue such as extsts in a necrotic tumor center Because the pressure differential within tumors forces solutes from the center to the periphery, such factors could then be released to the tumor environment An alternative explanation derives from the finding [147-150] that H B G F s are found In basement membranes in VlVO and are deposited into extracellular matrices by endothelial cells in vitro where they remain bound to heparln untd released by hepannase treatment [151,152] Because tumors frequently release hydrolytic enzymes, including hepanndegrading enzymes [153,154], it is possible that tumors release stored H B G F s from basement membranes m vavo and these molecules then diffuse toward pre-exastmg vessels where they eliot an angaogemc response A series of h e p a n n - b m d m g molecules that are secreted from tumor cells has recently been described In these stu&es, a group of oncogenes, including the mt and hst genes has been found to represent FGF-related molecules that differ from a o & c or basic FGF, m that they possess signal peptldes and are secreted from the cells that produce them [155-161] The relatmnshap between transformatmn and the mtracellular locahzat~on of growth factors was demonstrated by the elegant experiments of Rogelj et al [162] m whach N I H 3T3 cells were transfected with a basic F G F c D N A wluch had been coupled to DNA encoding a signal pepUde In thas case, the cells were transformed and haghly tumongemc whereas cells transfected with F G F lacking a signal peptlde were not tumorigenic These data suggest that H B G F s m a y have a dual role m neoplasm, acting as growth and transforming factors for the tumor cells

95 themselves, and as mltogens and chemoattractants for endothehal cells recruited in the process of angtogenesis Considerable Interest has been shown in the factors that regulate growth of cells from Kaposfs sarcoma, a common tumor in males infected with Human Immunodeflclency Virus (HIV) A variety of factors have been shown to be secreted by Kaposfs sarcoma cells [163,164] and some of these are able to stimulate the growth of the Kaposl's cells themselves Perhaps the best characterized of these is a hepann-bmdlng factor that shares sequence homology with F G F ( K F G F , Refs 156 and 157) The major difference between this factor and the F G F molecules produced by normal cells ~s that K F G F contains a signal peptlde and is secreted by the Kaposl's cells whereas acidic and basic F G F have no signal peptlde and are not actively secreted by the cells in which they are synthesized [143,144] Finally, it should be noted that not all heparln-bmdmg growth factors are related to the fibroblast growth factors Ferrara and Henzel [165] have recently reported the isolation by heparln affinity of a caUomc (pI 8 5) 45 000 molecular weight dlmer from pituitary follicular cells The sequence of this factor does not bear significant resemblance to either basic or acidic F G F This factor, discussed below as the 'vascular endothelial growth factor' (VEGF) is highly specific in that it stimulates prohferatlon of endothelial cells but not eplthehal cells, keratmocytes or fibroblasts (f) Ltpld-dertved anglogenlc factors In addition to the polypeptlde angtogemc factors, a class of hpid-based angmgemc factors has also been discovered Some of these agents are well characterized molecules such as prostaglandins E 1 and E 2 [166-168], and some are undefined denvatives of the arachidonic acid pathway which are blocked by the action of mdomethacin [169171] In general, prostaglandms have not been found to have direct effects on endothelial cell growth or migration but are well known inflammatory cell attractants For this reason, it is hkely that the angmgemc activity of these factors depends on the attraction of macrophages and other accessory cells that, in turn, release direct-acting endothelial cell stimulators (g) Ntcotmamtde An unexpected finding was obtained when ethanol extracts were prepared from rat Walker 256 carcinoma cells and anglogemc activity was found associated w~th a low molecular weight fraction Analysis by desorptlon-electron impact mass spectrometry, nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry revealed that the active factor was nlcotlnamide [172] The result was confirmed by showmg that purified preparations of mcotmamide caused an ang~ogemc response when tested m either the rabhit cornea or chick CAM assay No evidence was presented in this study to demonstrate whether nlCotlnamide rmght have any effect on endo-

thehal cell proliferation or nugratlon, and consequently the mechanism of nlCotlnarnlde's angmgemc activity is currently unknown (h) Platelet-dertved endothehal cell growth factor Initial attention on mltogenic factors from platelets was focused on the platelet-denved growth factor [173-175], a potent mltogen for fibroblasts and smooth muscle cells that had no significant activity on endothelial cells There were, however, some early reports of endothehalstimulating activity in crude P D G F preparations [176] or in platelet extracts from which P D G F had been removed [177] This suggested the presence of an additional platelet-denved growth factor with endothehal specificity One such molecule, called the platelet-derived endothehal cell growth factor (PD-ECGF) has now been isolated, sequenced and its gene cloned [178,179] P D - E C G F is a slngle-cham acidic polypept~de, with a molecular weight of 45 000, that does not brad to hepann It is an endothelial mitogen and potent anglogemc factor that ~s not active on all cultured fibroblasts These characteristics suggest that P D - E C G F may stimulate endothelial repair and anglogenlc processes associated with wound healing It is not yet known whether this factor might be produced by some tumor cells and thus have a role m promoting tumor anglogenesls (0 Transformmg growth factor a T G F - a Is a 5500 molecular weight polypepUde that was first isolated from transformed fibroblasts [180,181], but has subsequently been found m a variety of tumor cells, as well as normal cells such as macrophages [182,183] T G F - a bears a 40% amino a o d homology with epidermal growth factor and its activaty is mediated through bindmg to the E G F receptor [184,185] T G F - a directly sUmulates prohferatlon of cultured vascular endothehal cells and is a potent ang~ogenlc factor when tested m vavo [108] T G F - a consequently can be considered to be an anglogemc factor produced directly by tumor cells themselves and additionally by macrophages that may be attracted to a wound site Its mode of acuon is at least partly due to direct effects on vascular endothehal cells (I) Transforming growth factor fl Although T G F - a and -fl were both ongmaUy isolated from tumor cells on the basis of their ability to induce a transformed phenotype m normal cells, the two molecules differ dramatically in both structure and function TGF-fl is a homodlmenc polypeptlde with a molecular weight of 25 000 [186] The molecule is found m a variety of forms in different locations and the variations m form may be related to variations in function among different forms of TGF-fl [187] Originally found m tumor cells, TGF-fl ~s present in many other tissues including platelets, placenta, kidney, cartilage and bone [188,189] TGF-fl is generally produced in a latent form which can be activated by proteolysls or acidification [190-192] A1-

96 though TGF-fl is mitogemc for certain cells, it is more commonly found to be inhibitory to cell growth in vitro [193,194] Whereas T G F - a promotes endothehal cell growth, TGF-fl is inhibitory [195-199] Despite this difference, both factors are angiogemc when tested in VlVO [108,200] This result lmphes that the inhibitory action of TGF-fl on endothelial cells either is Inoperative in vivo or is blocked by other mltogenlc factors present in the tumor or wound environment These positive effectors could be denved from accessory cells such as fibroblasts and monocytes that are attracted to a tumor site by the chemotactic activity of TGF-fl [201] (k) Tumor necrosis factor a T u m o r necrosis factor a was ongmally isolated as an agent that could cause necrosis and subsequent regression of some solid tumors [202] The molecule exists as a 154-157 amino acid polypeptlde having a molecular weight of 17 000 [203] T N F - a bears a 28% homology to a lymphotoxln produced by stimulated lymphocytes which has been called TNF-fl [204] T N F - a is primarily produced by tumor cells and by activated macrophages [205] It is cytotoxlc for certain tumor cells and exogenous T N F - a will cause hemorrhagic necrosis in some sohd tumors [202] Although, like TGF-fl, T N F - a inhibits endothelial cell proliferation [206,207], it stimulates endothelial cell Imgration and tube formation in vitro [86] In VlVO, T N F - a is a potent anglogenlc stimulator [86,206], suggesting again that the inhibitory effect seen on prohferation in vitro does not occur in vavo T N F - a and anglotropin may, together, be responsible for the angiogenesis assooated with activated macrophages in wound healing, inflammation and neoplasia The anglogemc activity of T N F - a presents an apparent contradiction A molecule that stimulates endothehal cell migration in vitro should stimulate anglogenesls and thus potentiate tumor growth in VlVO Yet, T N F - a Injected into a sohd tumor will cause tumor regression Folkman and Klagsbrun [76] have suggested that the paradoxical activities of certain angaogemc factors may be regulated by the direction from which the factors impinge on the blood vessels in vivo When delivered to the inner surface of the vessel wall via the blood stream, T N F promotes coagulation, hemorrhage and necrosis When presented to the exterior of a blood vessel after production by a tumor cell or macrophage, it induces endothelial nugratlon and anglogenesis Thus, the in VlVO activity of this anglogemc factor may depend on its source and its route of delivery t~ a particular tissue (1) Vascular endothehal growth factor A recent addition to the hst of anglogemc factors has been made by the discovery of an endothehal cell growth factor in media conditioned by bovane pituitary follicullostellate cells [165,208,209] The factor is a dimer ( M r 45000) comprised of two identical subunlts, each with a molecular weight of 23 000 The molecule does bind to heparln,

but is not structurally Sllmlar to the F G F family of angiogenlc factors In addition to pituitary cells, this angiogemc factor is also produced by tumor cells such as mouse NB41 neuroblastoma cells [210] and guinea pig hne 10 tumor cells [211] The intact molecule is imtogenlc for endothelial cells but not for a variety of other cell types It does, however, share some structural similarity with the fl chain of the platelet derived growth factor [212] V E G F elicits a potent angdogemc response when tested in the cluck C A M [208] or in the rat cornea [211] It is of interest that this factor was originally identified as a tumor product that promotes increased vascular permeabihty, a property not necessarily shared by other anglogenic factors [211,212]

II-A 4 Anglogemc factor redundancy One question that arises regarding angiogenesls is why there are so m a n y different factors which can cause it when it occurs so seldom in normal tissues One possibility is that the in vitro and in vavo assays used to detect anglogenesis are overly sensitive and that some of these putative ang~ogemc molecules may not normally cause angiogenesxs in SltU A second hypothesis maintams that the occasions when angtogenesis must occur, in inflammation and wound repair, are so important that a large degree of redundancy has been provided to insure that wounded tissues are repopulated with blood vessels The assumption that all of these factors may indeed play a role in anglogenesis in vivo, rinses the question of how angjogenesls is constitutively repressed m normal tissues, even though seemingly every cell in the body is poised to produce a surfeit of anglogenic stimuli This control appears to take place at several levels Some anglogenlc factors are only produced by stimulated cells such as activated macrophages, some, such as T G F - f l are produced by normal cells m an inactive form that must be further modified to obtain activity, still other factors, such as acidic and basic F G F , are not secreted in soluble form by most normal cells but are stored in depots in basement membranes Finally, some factors, such as T N F - a , will only induce angiogenesis in vessels that they approach from the ablununal side and not when they circulate in the blood stream In contrast to the Inactivity of anglogemc factors in most normal tissues, tumor cells have a large repertory of means to induce anglogenesis They can secrete angiogenin, T G F - a , TNF-ct and direct these factors toward the abhimmal side of the existing vessels They can secrete modified FGF-like molecules such as the mt and hst oncogene products that can directly stimulate endothehal cells, and they can release hydrolytic enzymes that can liberate normal F G F from basement m e m b r a n e stores Tumors may also release proteolytic enzymes that activate TGF-fl to its pro-anglogenlc state Finally, tumor cells attract other cells, such as mast cells

97 and activated macrophages, that release additional angtogenic factors and thus amplify the angaogenic potenual of the tumor H - A 5 Role o f accessory cells m tumor angtogenests

Tumor cells frequently reside in direct contact with host cells that have been recruited to the tumor site Virtually every type of circulating cell type, including neutrophlls, monocytes, macrophages, lymphocytes, eoslnophlls, basoplils and mast cells, have been found in increased numbers m at least some tumors Two of these, macrophages and mast cells, have been particularly implicated m potentmtlng the tumor angiogenlc response Macrophages can act alone to stimulate neovasculanzaUon [213] by secreting anglogemc factors including anglotropln [103], tumor necrosis factor a [86,206] and basic fibroblast growth factor [214] Mast cells are commonly found in proximity to mature blood vessels [215-218], and are found with increased frequency in the vicinity of tumors [219-228] and in lymph nodes that have been infiltrated by tumor cells [229] Although mast cells are not always found within the tumor parenchyma [230,231], they are frequently found in high numbers m the connective tissue immediately surrounding the tumor [232,233] When found within the tumor stroma, mast cells are most frequently located proximal to the tumor capillaries [223,234] A potential relationship between mast cells and tumor angaogenesis has been proposed by several Investigators In 1964, Glani reported that mast cell density increased m the most highly vascularlzed areas of tumors [235] Subsequently, Ryan hypothesized that mast cells potentinted the growth of vascular endothehum in psoriasis [236] Smith and Basu observed that a high density of mast cells accumulated at the site of a corneal allograft several days before new blood vessels arrived at the graft site [237] Finally, Glowackl and Mulhken [238,239] have investigated the appearance of mast cells m hemanglomas, endothehal cell tumors that grow rapidly in young children and then regress spontaneously after 2 - 3 years They found that the density of mast cells in rapidly growing hemanglomas was at least 5-times the density found in normal skin and that the mast cell numbers fell to normal at the t~me of tumor regression The kinetics of mast cell accumulation was investigated by Kessler et al [240], who implanted tumors on the c l i c k C A M and compared the rate of appearance of mast cells to that of the newly induced blood vessels Their results showed that mast cells appeared at the tumor site witlin 24 h after tumor implantation, whereas new vessels did not converge on the implant until 2 days after the appearance of mast cells The accumulation of mast cells at the tumor site is probably due to mast cell chemotaxls since these cells are motile and respond chemotactlcaUy to undefined factors found in tumor cell conditioned medium [241] Mast cells also

respond to the chemotactlc activity of the tnpeptlde Gly-Hls-Lys [241], as well as to lntefleulon-3 [242], although the role of these particular factors in the attraction of mast cells to tumors has not yet been demonstrated If ~t is clear that mast cells precede blood vessels to tumors, it is less clear how mast cell accumulaUon could influence an~ogenesls Much work in this regard has focused on the role of heparln m anglogenesls The first indication that h e p a n n might have a role in the anglogenic process came from experiments which showed that h e p a n n released from mast cells could stimulate the mxgratlon of capillary endothelial ceils [243] T l i s led Taylor and Folkman [244] to test the effects of heparln on angtogenesis in VlVO on the click C A M They found that although heparln was not angaogenlc by itself, it could dramatically potentiate the anglogenlc response to hmlting amounts of a tumor-derived anglogenlc factor The mode of action of hepann in potentinting ang~ogenesis is not known In wtro, heparln has been shown to stabihze acidic fibroblast growth factor from degradation and inactivation [245,246] T l i s mechanism may play some role in VlVO but is probably not sufficient to explain the marked potentiation of anglogenesis caused by heparln A possible model for the role of mast cells in anglogenesis emerges from this data In tlis model, tumor cells release at least two d~stlnct factors The first is a chemoattractant for mast cells, the second is a factor that stimulates endothelial cell migration and prohferat~on In response to the chemoattractant, mast cells would nugrate toward the tumor Morphological evidence suggests that h e p a n n is released from the mast cells d u n n g tins rmgrauon and deposited m the tissue stroma [247] Endothehal cells may then begin to move toward the endothelial cell chemoattractant released by the tumor ceils Their movement does not require mast cells, but if mast cells have gone before, the endothelial cell migration wall be enhanced due to the prewous digestion of tissue stromal elements by the mast cells [248,249] and due to the deposition of h e p a n n This would result in a situation in wlich more vessels will reach the tumor more quickly than they would in the absence of mast cells It is important to note that this model portrays the mast cell as an 'aid' to anglogenesis but not a requirement for it Mast ceils are not universally associated with angaogenesis They are absent in the normal retina and are not found in areas of retinal anglogenesis associated with diabetic retlnopathy [250] Prehnunary in vavo experiments on the role of mast cells In anglogenesIs are consistent w~th the hypothesis that they potentiate ang~ogenesls and tumor growth m VlVO Induction of mast cell degranulatlon in the rat mesentery is sufficient to induce neovasculanzation [251], and anglogenesls is retarded in situations when mast cells are absent For example, when m u n n e B16-

98 BL6 melanoma cells were injected into mast cell deftclent W / W v mace, ang~ogenesls was found to be slower and lmtmlly less intense than m + / + nuce [252] The repmr of the deficiency by mjecuon of mast cell precursors into the bone marrow of the W / W v mace resulted m increased numbers of Ussue mast cells along with increased ang~ogenesls and increased tumor metastases

H-A 6 Inhlbttors of angtogenesls If anglogenesls ~s a critical determinant of tumor growth, then mhlblUon of anglogenesxs should lead to an m i n b m o n of tumor growth [25,253] For tins reason, a great deal of investigative effort has gone into the identification and ~solatlon of anglogenesls lninbltors The earhest efforts were designed to determine whether avascular t~ssues possessed vascular mhlb~tors The first can&date was cartdage, a normal ussue that as poorly vascularlzed Elsenstem and colleagues reported m 1973 that when cartilage was placed on the developing chick CAM, avascular zones formed around the cartdage pwces [254], suggesting that a cartilage-derived factor was bloclong the formatxon of normal vessels in the C A M Tins result was extended by Brem and Folkman [255] who showed that cartilage could mhlblt the anglogenlc response to tumors placed on the C A M and by Langer et al [256] who showed that tumors implanted in the mouse cornea faded to become vascularlzed when the mice were refused w~th preparations of a cartdagederived inhibitor Purification of tins factor has proceeded slowly but is now complete [257-260] The factor is slrmlar to a class of collagenase inhibitor present m cartilage [261] Tins may imply that one mtght block anglogenesls by m i n b m n g the process of matrix degradauon brought about by mtgratmg endothehal cells [65] Other poorly vasculanzed tissues also contain angaogemc lninbltors Among these are the eye lens [262], wtreous humor [263,264] and ovarian follicular fluid [265] A second approach to the discovery of anglogemc minb~tors as to attempt to antagomze angaogemc stimulators For example, because certain prostaglandms can act as anglogemc factors, it as not surprising that anglogenes~s can sometimes be blocked by the addition of prostaglandm synthetase minb~tors [266] As discussed above, heparln Is a potentlator of anglogenesas and ~t is of interest that hepann antagomsts, such as protamlne, platelet factor IV, and the eosmopinl major basic protern, can also block the anglogemc response [244] It is expected that anubodws or antagonasts to partacular angtogemc growth factors maght also inhibit anglogenesis m wvo, but tins has not yet been demonstrated A variety of steroids have been shown to minblt ang~ogenesls Some minb~t~on can be observed m the presence of steroid alone [267,268] but an general the ant~-ang~ogemc activity of the steroid ~s potentiated by

the a d d m o n of h e p a n n or related molecules [36,269,270] The anta-anglogenlc activity of the steroids is dlssocmble from their glucocortlcold actxvaty, and the term 'anglostatlc steroid' was corned to describe steroidal substances such as tetrahydrocortlsone that have the ablhty to block anglogenesls [271] A potential mechanism for the antl-anglogemc activity of these steroids has been described by Ingber et al [272] These authors demonstrate that the first observable response to the admlmstratlon of heparm plus steroxd to the chick C A M is the fragmentation and dissolution of basement membrane components from the perlvascular area of growing capillaries Because capillary funcUon and maintenance ~s dependent on an appropriate basement membrane, ttus may lead to a blockage of new vessel formation and to a regression of pre-formed vessels Further evidence for tins hypothesis is prowded by the finding that anglogenesls can also be lninblted by the administration of agents that block the formation and deposition of collagen into new vessels growing m the cluck C A M [273] The actwlty of heparln as an antl-angxogemc agent is not dependent on the antacoagulant actwlty of heparm and can be substituted by hepann fragments or by synthetic, heparln-hke molecules [266,269,274] The activity of heparln with regard to anglogenesls is therefore b~modal In combination wxth hnutlng amounts of ang~ogemc factor, h e p a n n potentmtes and enhances the ang~ogemc response However, when heparm ~s present along wxth an anglostauc steroid, the anglogemc response is blocked Two other anglogemc minbltors have recently been described In one case hyaluronate was shown to cause the f o r m a u o n of avascular zones m developing cluck hmbs [37] Whale the mechanism for tins effect as unclear, it is hkely that tins Is another example of a treatment winch causes ~mproper mteracUons between endothelial cells and their extracellular matrix In another intriguing report, Rastlnejad et al [275] have purified a 140 000 molecular weight glycoprotem found m hamster cells or human-hamster hybrids The aninb~tory actlwty was tightly hnked to the presence of an active cancer suppressor gene m transformants and revertants These results imply that one way to achieve cancer suppressaon ~s to cause cells to express an anglogemc lninbitor

H-B Properttes of the tumor vasculature Although most sohd tumors are Inghly vascular, these tumor vessels are not identical to normal vessels In mature tissues The distraction between tumor and normal blood vessels includes differences m the cellular composatlon of tumor vessels, differences m the basement m e m b r a n e composition, differences m permeabdlty and &fferences m vessel stablhty These differences

99 have been reviewed prewously [276] and wall be discussed bnefly here II-B 1 Cellular components of tumor blood vessels The vessels that grow in response to a tumor stimulus are p n m a n l y comprised of endothehal cells They differ, m this regard, from normal capdlanes wtuch often contain a second cell type, the vascular pencyte Pencytes grow adjacent to the endothehal cells on the exterior side of the vessel Although the function of pencytes has long been unclear, recent ewdence indicates that pencytes act as a maturation s~gnal that prevents endothelial cell prohferatlon [277] More recent evidence suggests that the mechamsm for th~s effect revolves the elaboration of activated transforming growth factor-fl, a known inhibitor of endothehal cell prohferatlon [278] Activation of the TGF-fl apparently takes place by the acUon of a proteolytlc activity produced by mixtures of endothehal cells and perlcytes [279] II-B 2 Extracellular matrtx of tumor blood vessels Endothehal cells are known to produce a variety of extracellular matrtx molecules including fibronectln, heparan sulfate proteoglycan, and several types of collagen [280,281] In wvo, capdlary endothehal cells generally sit on a basement membrane comprised of lanunln, heparan sulfate proteoglycan and types IV and V collagen, although the total amount of basement membrane and the relative amount of each component may vary m capillaries found in different Ussues [282] The cellular source of the various components of the capdlary basement membrane m wvo xs not completely understood but ~s thought to be contributed by both the perlcytes and the vascular endothehal cells The basement membrane surrounding new tumor blood vessels is markedly reduced relatwe to mature normal blood vessels Although most of the same extracellular components are present m reduced quanUty, there do appear to be some changes m the synthetic pattern of glycosamlnoglycans (GAGs) [47,283] In general, the newly formed tumor vessels have relatively high concentrations of hyaluromc acid and low concentrat~ons of sulfated proteoglycans Mature capillaries, m contrast, produce low amQunts of hyaluromc acid and relatwely high amounts of sulfated GAGs The mature pattern of G A G synthes~s IS most frequently found m capdlanes that have become Invested with pencytes, suggesting that perlcyte contact enhances capillary dffferentmtlon and maturation H - B 3 Tumor vessel permeablhty Among the most notable attributes of the tumor vasculature ~s ~ts increased permeablhty relative to normal mature blood vessels [284-289] Several theories have been proposed to account for this phenomenon

Blood vessels may be mtnns~cally leaky due to their altered basement membrane arctutecture d~scussed above [290,291] It xs also known that tumors, whale highly vascular due to tumor anglogenesxs, are poorly invaded by lymphatic channels and normal dralmng lymphatics m surrounding Ussues are sometimes obstructed by tumor cells [292,293] This reduces drainage of fluids from the tumor space and can cause accumulation of fluid m the tumor space and edema in the surrounding tissue This, m turn, contributes to further exudation of flmd from the tumor blood vessels m response to the increased mtersutlal pressures [294,295] Finally, ~t has been suggested that the tumor cells themselves may contribute to tlus effect by elaborating vascular permeabdlty factors that promote fluid transfer out of the tumor capillaries [211,296] One such vascular permeabdlty factor has been isolated from many, but not all, human and gmnea pig tumor cell hnes [297] This protein has a molecular wexght of 38000 and ts characterized by an abdlty to bind to lmmoblhzed heparln A slrmlar M r 45 000 tumor-derived permeabdlty factor was ~solate by Lobb et al [298] It is hkely that both of these factors are related to the V E G F permeabdlty factor described in section II A 3 [211,212] These molecules cause leakage of htgh molecular weight solutes from normal dermal rmcrovessels after rejection into the slon of normal guinea pigs Most recently, it has been shown that the permeabdxty of vessels wlttun a given tumor ~s not uniform but vanes extenswely In general, the vessels at the tumor periphery have the highest permeabdlty actlwty, whereas vessels that pass through the tumor core are the least permeable [299] An ~mportant mediator of the tumor vascular environment ~s the increased mterstltlal pressure found m sohd tumors As described recently by Jmn [294], the possible causes of tlus increased pressure include, (1) the absence of functional lymphatic vessels m the tumor stroma, (2) the increase in cell mass due to prohferatlon of tumor cells in a confined space, and (3) the increase in tumor vessel permeablhty relative to normal vessels One consequence of the increased tumor mterstltml pressure xs occlusion of the tumor interior blood vessels with consequent lschemla and eventually necrosis tn the tumor core In addition, the increased interstitial flmd pressure may facdltate exat of tumor cells from the primary tumor into the blood stream or surrounding tissue The increased pressure may also prevent the delivery of exogenous therapeutic agents, such as drugs or antibodies, and even bar the entry of normal host immune cells that could possibly htmt the tumor burden H-C

Tumor cell dtssoctatton

Single tumor cells or small groups of cells must separate from the p n m a r y tumor mass m order to form metastauc colomes at anatormcally distant s~tes As

100 tumors progress toward mahgnancy, the surrounding extracellular matrix is slowly degraded and the strength of the adhesive interactions between tumor cells is diminished As enzymatic destruction of extracellular matrix continues [300,301], nugratory tumor cells may effectively traverse tissue barriers, and increased interst~tml pressures within the tumor m a y also faclhtate tumor expansion into adjacent tissues and into blood vessels within the tumor Reductmns m cell-cell adhes~xqty have been proposed to be closely hnked to acqmsltlOn of the invaslve phenotype associated with metastatic tumors I1-C 1 Modulatton of the extracellular matrix mtegrtty Metastatic tumor cells produce a variety of degradatlve enzymes that effectively weaken or destroy extracellular matrix molecules [302] Among the matrix molecules degraded by tumor-derived enzymes are types I and IV collagens [303,304], heparan sulfate proteoglycans [153,154,305], other proteoglycans [306] and elastin [307-310] In addltmn to enzymes produced by the tumor cells, an advancing front of mvaslve tumor cells can also induce secretion of hydrolytic enzymes from adjacent non-tumor tissue As exastlng extracellular matrix molecules are degraded, new matrix proteins are synthesized by the tumor cells and these may in turn be less effective at maintaining tumor cell adhesion M a n y tumors, for example, produce a form of heparan sulfate that carries fewer sulfate groups than the corresponding molecule found m normal tissues [311-315] Extracellular matrix destruction at the tumor periphery may be faclhtated by degradatlve enzymes produced by host cells, such as neutrophlls and monocytes, that are attracted to the tumor site [316] T u m o r angmgenesis may further promote matrix degradation because the migrating endothehal cells [65] and co-migrating mast cells [317] will modify the extracellular matrix via the productmn of hydrolytic enzymes or by the deposition of new matrix molecules [47,318] I I - C 2 Modulation of cell adhesion The extracellular matrix in most tissues contains several distract molecules that m a y influence tumor cell adhesion [319,320] As noted above, degradaUon of these molecules will reduce tumor cell adhesion and potentially enhance exat of metastatic cells from the primary tumor site In addmon, alterations m the number or affimty of tumor cell surface receptors for adheswe molecules will also affect cellular adheslwty Among the best characterized of the adhesive matrix molecules are fibronectln and larmnln, two large molecules with mulUple domains for bmchng to cells as well as other adhesion molecules, such as collagen and hepann [321,322] Considerable progress has been made on defining the peptlde domains which me&ate cellular adhesion to adhesive molecules and also on the ~solatlon

and characterization of the cellular receptors that bind these matrix molecules These topics have been reviewed extensively [323,324] and won't be treated in detail here Some of the peptlde sequences to which tumor cells bind have been identified, such as an R G D site in fibronectin, vitronectin and von Willebrand's factor [325], and a Y I G S R site in lamlnln [326] but other evidence exasts for distinct binding sites in other adhesion molecules as well as alternate sites in fibronectln and lamlnln [327,328] A class of receptor that mediates binding to a vartety of extracellular matrix molecules has been defined and termed mtegrlns to denote their properties as integral m e m b r a n e proteins that hnk the cytoskeleton to the extracellular environment [323] All integnns contain two chains The fl chain ~s currently thought to exist in three forms (ill. f12, flJ), whereas the a chain can exist in m a n y different forms Because the recogniUon of a particular adhesive peptlde is governed by the interaction of the two lntegrln chains, different molecules are recognized by different lntegrlns depending on the nature of the two chains Certam mtegrms recogmze adhesion sequences other than R G D in diverse adhesion molecules including collagen and lamlnln [328] Evidence now exists that binding of tumor cells to fibronectln is dlrmnlshed by alterations in the mtegrln composition of the cell surface [329] In this case, the lnl'ubltlOn of m t e g n n expression is accomphshed at the transcriptional level In addition, cell-substratum adhesion can be effected by a transformation-induced decrease in the a f h m t y of these receptors to their matrix hgand [330] Not all cell adhesion receptors are integrms, however A 67 000 molecular weight molecule has been identified [331,332] which is reported to mediate binding to the Y I G S R moiety in lamlmn [326] A s~rmlar molecule that binds to a hexapeptlde sequence m elastm, as well as to galactoside sugars, has also been purified [333-335] A potentially important but not extenswely stu&ed posslblhty for the reduced homotyplc adhesion of primary tumor cells may be found m the reducuon of intercellular junctions reported in cells after neoplastic transformation Nlcolson and colleagues [336] have noted a correlation between loss of intercellular junctmns and metastatic potentml of m a m m a r y adenocarcinoma In these studies the number of effective intercellular junctions was determined by quantifying the amount of dye that could be transferred between adjacent tumor cells Slrmlar studies reveal that cultured normal rat kidney ( N R K ) cells exbablt reduced levels of intercellular dye transfer after transformation mediated by the oncogenes v-ras, v-mos and v-src [337] H - C 3 Modulatton of cell motthty The dissociation of primary tumor cells and their eventual invasion into the blood stream may be en-

101 hanced by events that lead to increased tumor cell motihty [338-340] Molecules that influence tumor cell moUhty can be davided into two classes, those produced by the tumor cells themselves, and those generated by the degradation or modification of the extracellular environment Molecules that are produced by tumor cells and subsequently stimulate their own motlhty were ftrst isolated by Hayasha and colleagues m the early 1970's [341,342] This topic has been investigated more recently by Llotta et al [343] who have designated such agents as 'autocrlne motility factors' and have purified one such factor from human melanoma cells Autocrlne motihty factors have also been reported to be produced by rat mammary adenocarcmoma cells in culture [344] Also of mterest with regard to tumor cell dissociation are agents called 'scatter factors' wtuch are produced by fibroblasts and promote the dispersal of epithehal cells from tightly packed clusters to separated single cells [345] Such factors, if produced by carcinoma cells m vavo or by nelghbonng stromal cells, could readdy enhance the dispersal of cells from the primary tumor and their eventual entry into the circulation Many tumor cell chemotactlc factors appear to be degradation products of extracellular matrix molecules including fibronectln [321,346], lamlnIn [326,347], collagen [348], elastin [349], and as yet undefined products of resorbmg bone [350] With fibronectm, the adhesion promoting peptlde RGD also appears to be responsible for the rmgratlon-stimulatlng actlwty of the molecule [351,352] These fragments may be generated by the acUon of tumor-derived hydrolytic enzymes [353] or by enzymes produced by inflammatory cells attracted to the tumor s~te by tumor-derived chemotactic factors [354-356] H-D

Tumor cell entry mto the bloodstream

Tumor cell dlssemmaUon proceeds by either of two routes, spread in the lymphatic circulation (lymphatic dissemination) or spread through the bloodstream (hematogenous dissemination) [357] Because the tumor mterstmum generally lacks functional lymphatics [292,293,358], exit of tumor cells into the circulation generally takes place vaa intravasatIon of cells into the tumor blood vessels Invasion into the lymphaUcs can occur through several routes by invasion of the tumor front Into lymph nodes or lymphatic channels, by invasion of lndwldual cells extruded from the tumor mterStltium into the surrounding tissue, or by passage of tumor cells from the bloodstream into the lymphatics vm the numerous lymphatlcovenous junctions [359,360] For this reason, both lymphauc and hematogenous spread of tumor cells are facditated by the presence of new capillary blood vessels that result from the process of tumor ang~ogenesls

Pr)mary S~te

..........

. . . . . .

)c

........

Fig 1 Tumor cell mteracuons with the vasculature The drawing depicts the multiple interactions of tumor cells with the vasculature m wbach tumor cells at the primary s~te intravasate into newly formed vessels and circulate as single cells or as cell aggregates (tumor emboh) The tumor emboh are often non-selectively trapped in precapdlary artenoles whereas single cells continue on to the postcapdlary venules The processes that are necessary for successful metastas~s include adhesion to the venular endothehum and subendothehal basement membrane (A), degradation of extracellular matnx molecules and cellular magratlon into the parenchymal space (B), cell growth at the secondary site (C) and new capdlary growth (anglogenes~s) accompamed by increased tumor growth (D)

Estimates of the number of tumor cells wluch can gain access to the bloodstream reflect the relative ease with lntravasatlon is accomphshed Butler and Gulhno [361] have shown that 3-4 106 cells are shed into the bloodstream in 24 h for each g of tumor load by rats bearing mammary adenocarcinomas In a smular study, using a transplanted MTW9 mammary carcinoma, Llotta et al [362] showed that the number of tumor cells shed into the bloodstream increased from 1 4 103 cells per 24 h on day 5 after implantation to 1 5 105 cells per 24 h on day 15 High numbers of circulating tumor cells were also observed in nuce carrying the B16 melanoma or Lewts lung carcinoma [363] Tumor cell entry into the bloodstream generally takes place through the tumor-induced capdlanes or through existing small veins and venules (Fig 1) Although large veins may be invaded by baghly aggressive tumors, arterial invasion rarely occurs Direct invasion of tumor cells Into the lumen of exastlng blood vessels requires that the cell penetrate any subendothehal basement membrane surrounding the vessel This poses less of a problem m tumor capillaries wtuch, as noted in secUon II-B 2, have little orgamzed extracellular matrtx, or for entry into lymphatic vessels wbach also lack an orgamzed basal lamina Invasion through intact basement membrane of extstmg mature vessels would, in contrast, require degradation of type IV collagen, lamlmn and heparan sulfate [302,362-366] A correlation between tumor metastasis and the production of type IV collagenase by the metastatic tumor cells has been found in several studies [304,367,368] but the particular rele-

102 vance of tins process to mtravasatlon as opposed to later extravasatlon in secondary sites is unclear It is hkely that the majority of tumor ceils enter the bloodstream through immature new blood vessels at the tumor periphery and that degradation of basement membrane molecules wdl play a greater role in the later process of extravasatlon III. Circulating tumor cells III-A

bloodstream mclude cytotoxac T lymphocytes [381-383], mononuclear phagocytes [384-388] and natural kdler cells [389-397] It is of considerable interest that recent evidence suggests that tumor cells are more susceptible to the actions of kaller ceils when they are circulating m suspension than when they are adherent to endothelial cells or extracellular matrix components [398] Tins suggests that the survival of circulating tumor cells wdl be enhanced if they can adhere to vascular or subvascular surfaces

Survwal of tumor cells in the bloodstream I I I - B lnteracttons between tumor cells and platelets

The bloodstream is a hostile enwronment for tumor cells Using radiolabeled melanoma cells, Fldler [369] reported that 24 h after entry into the circulation, less than 0 1% of the cells were viable For tins tumor, less than 1% of the cells released Into the bloodstream ever form a metastatic colony and m a n y other tumors are even less efficient at forn~ng metastases [370-372] The estabhshment of successful lung metastases appears to depend on the absolute number of cells shed into the circulation, and is enhanced if the tumor cells circulate m small clusters [373-375] The reasons for the short half-hfe of tumor cells in the bloodstream are not completely understood but several mechanisms are thought to have a role The simplest explanation is simply that most ceils do not survive in an environment of 100% plasma or serum Sato has reasoned that most cells in tissues exast in an environment contmmng a rmcrofiltrate of plasma components that diffuse or are transported across the capillary bed in each tissue The nature of the filtrate will &ffer from Ussue to tissue depending on the properties of the capillary endothehum in that tissue and on its proxamaty to other organs that produce hormones or growth factor Tins hypothesis has led to the development of defined growth media for different cell types that are comprised of a &stmct set of nutrients and hormones for each cell type [376] In addition to nutrient requirements, passage through the vasculature requires that cells be able to change shape m order to squeeze through small capillaries Deformability ~s consequently a common property of blood cells that must constantly traverse the vasculature However, many tumor cells are less deformable and consequently more sensitive to mechanical injury in the circulation [377-379] For the purposes of tins rewew, it is important to realLze that unhke blood or endothehal cells, most tumor cells are derived from sohd ussues and have no particular ablhty to survive for long periods of time in a plasma environment They must rapidly escape from the bloodstream m order to form a successful metastatic colony A second contributor to the denuse of tumor cells in the clrculaUon is the action of protective cell types that have anti-tumor acuvlty [380] The cells most commonly responsible for removing tumor cells from the

The development of circulating aggregates of tumor cells and platelets has been recognized for over 50 years [399] The role of platelets m the metastatic process is complex Some stu&es have shown that platelets potentiate the metastatic response, whereas others show no effect [400-402] A potential Involvement of platelets m metastasts was suggested by the work of Gasic and Gaslc [403,404] who reported that the number of experimental metastases formed after intravenous rejection of tumor cells was reduced in thrombocytopemc mace relative to normal ammals S~rmlarly, fewer spontaneous metastases were formed from subcutaneous tumors implanted into thrombocytopemc mace [405] Treatments that prevent p l a t e l e t / t u m o r cell mteracuons have also been shown to decrease metastatic frequency [406-408] M a n y tumors possess a procoagulant activity that leads to the generation of t h r o m b m and subsequent platelet activation [409-414] Tumor-induced platelet aggregation is frequently blocked by t h r o m b m minb~tors [415,416] One well documented coagulant produced by tumor cells is tissue factor, a phosp h o h p o p r o t e m that acts at an early stage in the coagulation cascade [413,414,417] Other tumors produce prothrombmase winch directly catalyzes the conversion of p r o t h r o m b m to t h r o m b m [418] Still other tumors appear to activate platelets by a thrombm-mdependent m e c h a m s m [402] Bastida et al [419] have, for example, shown that activation of a hpoxygenase pathway m certain tumor cells leads to exposure of a glycoproteln that facilitates t u m o r / p l a t e l e t aggregation Several mechamsms have been proposed to explain the occasional enhancement of metastasis by p l a t e l e t / tumor e m b o h z a u o n [400] The embolus may, for example, form a protective 'cocoon' around the tumor cells that offers protecUon from cytotoxac lymphocytes and natural I d l e r cells [420-422] Tins mechanism would apply if the embolus formed while the cells were circulating or, as is sometimes the case, after the tumor cells have arrested in the vasculature [423,424] Alternatively, the embolus may potentmte the arrest of tumor cells m the vasculature at a secondary s~te It ~s known, for example, that platelets can facdltate tumor cell adhesion to extracellular matrix vm the platelet glyco-

103 proteins Ib and I I b / I I I a [425-427] Entrapment of tumor emboh with subsequent enhanced adhesion would, however, result primarily m arrest of the embolus at the first organ downstream from the primary tumor site Since many tumors cannot colomze every organ [428,429], embohzatlon would only potentiate metastasis in cases where the first site encountered was, in fact, a preferred site for growth of that particular tumor In other cases, the arrest of a tumor embolus in a hostile organ may actually reduce the number of eventual metastases by preventing the tumor cells from reaching a more favored location Finally, tumor emboh are most hkely to become arrested in the precaplllary arterioles, where exit from the vasculature is somewhat more difficult than It is from the post-capillary venules The total interactions of platelets with tumor cells may be considered something of a mixed blessing, protecting the tumor cells and promoting their adhesion, but sometimes leading to arrest in an inappropriate environment I I I - C lnteracttons between tumor cells and leukocytes P o l y m o r p h o n u c l e a r leukocytes (neutrophlls or PMNs) are the most abundant form of circulating white blood cell and play a potentially important role in regulating the vmbillty and mvaslveness of circulating tumor cells Exposure of P M N s to tumor ceils can cause P M N actwatlon, resulting in the release of proteolytlc enzymes, oxygen radicals and various metabohtes that are normally stored in the P M N granules [430] The reported effects of PMNs on tumor cells are diverse, some are cytotoxac and would be expected to reduce metastatic efficiency whereas other effects are potentially protective for the tumor cells In one report, for example, PMNs were reported to accelerate pulmonary clearance of circulating B16 melanoma cells, leading to a reduction in metastatic potential [431] Tumor-associated PMNs are frequently, however, neither cytotoxlc nor cytostatlc for the tumor cells themselves, and have been found to promote rather than hinder experimental metastasis [432] C n s s m a n et al [423] have shown that P M N s rapidly bound to circulating B16 or Lewis lung carcinoma cells and remained in contact throughout the process of arrest and extravasatlon P M N s from rat peritoneum increased the attachment of rat hepatocarcinoma cells to endothehal monolayers suggesting that PMNs maght facihtate the early steps of tumor cell arrest and extravasatIon [433,434] Recently, a leukocyte adhesion molecule that mediates cell adhesion has also been found on certain tumor cells [435] Such molecules may therefore mediate the binding of neutroptuls to tumor cells and perhaps of tumor cells to endothelial cells in the vascular wall The majority of recent studies have shown that interactions of tumor cells with P M N s serves to enhance metastatic efficiency rather than to tunder it Orr and

colleagues [434] have shown an increase m pulmonary metastasis after oxygen radical-mediated damage to endothehal cells in the lung vasculature, suggesting that PMN-Induced endothehal injury may facilitate the passage of tumor cells into the extravascular space Recent studies by Welch et al [432] have also shown a potentiation of metastasis by neutroptuls In these studies, the investigators showed that co-inoculauon of PMNs and tumor cells Into mouse tall veins s~gniflcantly Increased the number of pulmonary metastases formed P M N s that are isolated from sohd tumors in VlVO show an increased ability to promote the Invaslve and metastatic potential of other tumor cells, suggesting that the association of P M N s with tumor cells reduces P M N activauon or differentiation Tumor-ehcited neutrophlls make a variety of degradatlve enzymes that rnlght accelerate the destruction of the subendothehal basement membrane and allow more rapid tumor cell passage into the tissue space These cells produce type IV collagenase, heparltinase and neutrophll elastase, a broad spectrum protelnase produced by P M N s that can degrade m a n y components of the subendothehal extracellular matrix [436] Because metastasis is an inefficient process, any event that makes it easier for the tumor cells to exit from the primary site or to gam entry to the secondary site should significantly enhance the metastatic potential In the case of neutrophils, the net degradatwe effects on endothelial cells and on the subendothehal basement membrane act to promote metastases appear to outweigh any direct cytotorac effect that the P M N s might exert directly on the tumor cells IV. Tumor cell adhesion to the vessel wall

Circulating tumor cells, mult~cellular tumor thrombl and aggregates of tumor cells w~th host cells will form metastases only if they escape kflhng by the host immune system and destruction by mechanical shear forces associated with passage in the blood stream Those that survive must then arrest m the microvasculature at a secondary site and pass out of the vessel into the organ interstmum The tumor cells can adhere to the luminal surface of the vascular endothelium and, after endothehal retraction, to the subendothelial basement membrane (Fig 1A) The short half-hfe of tumor cells m the circulation suggests that tumor cells capable of mlnlnuzlng their time in the bloodstream by rapid adherence and passage through the vessel wall should have an Increased probablhty of successful metastas~s 1V-A Adhesion to the endothehum One of the earliest and most ~mportant adhesive events in the process of metastasis ~s the adhesion of tumor cells to the vascular endothehum in the secondary site Although m a n y tumor cells, especially those

104 m aggregates, become trapped in the first vascular bed encountered, considerable evidence demonstrates that passive cell trapping is not sufficient to promote tumor metastasis [359,437-440] In many cases the majority of tumor cells wall lodge m the first organ encountered, but metastatic colonies will be found only in organs downstream from the s~te of first arrest This has led some investigators to speculate that a specific cellular adheswe interaction may initiate the formation of metastatic colonies at a particular secondary site [441,442] Several early studies utilizing endothelial cells from large blood vessels showed that metastatic tumor cells could adhere and invade through cultured endothelial cell monolayers [443,444], and that these properties were enhanced m highly metastatic tumor cells [445,446] Because recent comparatwe studies have shown that rmcrovascular endothelial cells differ from large vessel cells, and that endothehal cells from one organ may differ from those found m another [51], it was felt important to test the adhesion of tumor cells to nncrovascular endothelml cells asolated from a varaety of organ sites This work has primarily been carried out by Auerbach and colleagues [447,448] who have convincingly demonstrated that, in many cases, metastatac tumor cells adhere selectively to capillary endothehal cells derived from their preferred secondary s~te Other work that compared the adhesion of lung-homang melanoma cells and hver-homlng lymphoma cells to lung or liverderived endothehal cells has confirmed this specificity [449,450] The molecules that medmte the adherence of tumor cells to mlcrovascular endothehal cells are only starting to be elucidated Much of the information we have is derived from studaes on the homing of lymphocytes to specific endothelaal cells an lymph nodes or Peyers patches [451] In a number of studies from a variety of groups [452,453], a picture has emerged of a set of cell adhesion molecules on endothehal cells that interact specifically with a set of adhesive receptors on the lymphocyte cell surface Because of the potential role of these molecules in deterrmnmg the site of lymphocyte extravasatlon, they have been gwen the name 'vascular addressms' One such molecule found on mouse lymphocytes is a 90000 molecular weight glycoprotem that contains a carbohydrate binding domain winch mediates binding to endothehal cells in peripheral lymph nodes [454-457] A human homolog for this homing receptor [458] shows a sagnlficant degree of homology to an endothehal-derlved molecule that acts as a binding molecule for polymorphonuclear leukocytes [459] A role has also been suggested for the lymphocyte hormng molecules in the targeting of metastatic lymphoma cells that colonize the hver [460] as anubodles to these receptors can block experimental metastasas of certain lymphoid tumors However, not every llver-honung lymphoma cell contains the same type of receptor for

binding to endothelial cells Another molecule that may medaate tumor cell adhesion to organ-speofic determinants is the lymphocyte function associated antagen (LFA) [461] Roos and colleagues [435] have shown that the adhesion of l y m p h o m a cells to hepatocytes can be blocked by the ancubataon of those cells wath antlbodaes to L F A Similarly, l y m p h o m a cells that lack LFA are deficient m laver metastasis [462] The authors suggest that the adhesion of ttus type of lymphoma is dependent on the binding of L F A to an as yet undefined hgand on the endothelial cell surface IV-B Adhesion to the subendothehal basement membrane

After tumor cells adhere to the endothelial cell surface, endothelial cell retraction is often observed [463,464] This results in exposure of the subendothehal basement membrane, a structure wtuch contains a variety of adheswe molecules, Including lamlnm, types IV and V collagen, vltronectln and heparan sulfate proteoglycan Considerable data has accumulated regarding the importance of tumor cell interactions with lamxnln m the process of pulmonary metastasis [465] Some tumor cells use cell surface lamlnm to attach other matrix molecules in VlVO In ttus case, tumor cells pre-incubated with antibodies to lanunln will have reduced adhesive efficiency and consequently show reduced metastatac potential [466-470] Adhesion of tumor cells to basement membrane lammln is also an important component of the metastatic pathway Two forms of lamlnln receptor have been reported on tumor cells The first is an M r 67000 glycoprotem [331,471] that appears to bind to at least two distinct deterrmnants in the lamlnln molecule, the pentapeptade Y I G S R [326] and the hexapeptlde L G T I P G [335] Involvement of the Y I G S R sequence in the metastatic process is suggested by experiments in which pretreatment of tumor cells with the peptlde causes reduced experimental metastas~s when the cells are rejected intravenously into syngenelc mace [472] The larmnm receptor has been cloned and the gene sequenced [473,474] It IS synthesized as a low molecular weight form and later processed to its commonly found M r 67 000 form This same receptor apparently also mediates cellular binding to elastan [335] and perhaps to other matrix molecules as well [334] A second lamlnin receptor that has the properties of an integrln has recently been identified on human ghoblastoma cells [328] Not all tumor cells adhere to lamlnin, however, [475,476] and the role of this molecule in mediating tumor metastasas to organs other than the lung has not yet been elucidated T u m o r cells bind to a variety of other extracellular matrix molecules including flbronectln [477,478], type IV collagen [479,480] heparan sulfate proteoglycan [481] and elastin [482], but the importance of these processes m facilitating metastasis is not yet clear Preincubatlon

105 of tumor cells with the tetrapepUde R G D s , the adhesive deterrmnant m fibronectm, vatronectln, von Wdlebrand's factor and thrombospondln [324], causes a sagmfacant reduction in metastatic efficiency [483] Binding to fabronectln, however, as not Indispensable for metastatic colonazatmn [484] so the R G D s experiments may well affect the banding of tumor cells to one of the other RGD-contaamng adhesion molecules Although the components of the extracellular matrices in most organs are very similar, recent evadence suggests that the adhesive elements of the extracellular matrix may vary from organ to organ Reid and co-workers [485], for example, have shown that tumor cells that vary in their preferred sites of metastasis, selectively adhere and grow on extracellular matrix components extracted from their preferred organs The authors provide evidence that a proteoglycan as one of the extracellular m a t n x molecules responsible for this adhesion m the laver The extracellular matrix may also influence tumor cell adhesion to endothelml cells indirectly The expresstun of certain adhesion molecules on the endothelial cell surface can be induced by cytokmes, such as interleukln-1, tumor necrosis factor a and "/-interferon These reduced adhesion molecules increase the binding of tumor cells [486] and leukocytes [487,488] It appears that organ-specific components of the extraceUular matrix can also modulate the expression of endothehal cell-surface adhesion molecules Pauh and Lee [489] have reported that aortic endothelial cells grown on extraceUular matrix extracted from either lung or laver preferentially band to tumor cells that selectively colomze that organ Tins suggests that the expression of organ-specific adhesion molecules by vascular endothelml cells as not an mtnnsac feature of the endothelial cells in each distract organ, but is instead regulated by the c o m p o s m o n of the extracellular matrix on which the endothehal cells reside V. Tumor cell exit from the vasculature V-A

Degradanon of basement membrane components

Both the subendothellal basement membrane and the interstitial connective tissue matrtx can present a barrier to the mvasmn of tumor ceils from the vasculature into the tissue parenchyma For tins reason, the abahty to degrade matrix molecules as a hallmark of the metastaUc process Tins topic was introduced m subsection II-C 1 in our discussion of the process of tumor invasion into the vasculature In tins case, the small amount of Immature basement membrane surrounding the tumor capillaries provtdes a modest barrier to tumor mtravasatlon In the case of extravasataon into the normal tissue at a secondary site, the tumor cell must degrade intact mature basement membrane as well as the matrix com-

TABLE II Matrtx-degradmg actwmes increased m metastattc tumors

Protelnase

Substrates

Production by tumor cells

Type IV collagenase

Type IV collagen

304,367,368, 498-506

Type V collagenase

Type V collagen

497

Transin/stromelysin

Type IV collagen, elastln 363,368, fibronectm, lanunin 490-494 proteoglycans

Urokmase-type plasnunogen activator (plasrmn)

Plasrmnogen, latent collagenase

527-544

Hepartltmase

Heparan sulfate proteoglycans

153,154,546

CathepsxnB

Collagens, flbronectln larmnln, proteoglycans

547-556

Elastase

Elastln, collagens fibronectln, larmmn, proteoglycans

307-310,560, 561

ponents of the interstitial tissue In addlUon, the metastatic tumor cell must accomphsh tins without the aid of accessory cells that would be recrmted to the primary tumor site As different tumor cells are capable of selectively colonmxng different tissues, the abdxty to degrade specific m a t n x molecules could specify the nature of the tissues through winch a specific tumor cell can invade A wade variety of hydrolytic enzymes are produced by tumor cells [302] Many are carried on the tumor cell surface where they occupy a suitable position to degrade the small amounts of matrix necessary to allow movement of the individual cells [303] For the purposes of tins review, we wall concentrate on five classes of degradatave enzyme produced by tumors metalloprotemases, plasrrunogen actwators, hepantlnases, cathepsins and elastases The properties of these enzymes are summarized an Table II V-A 1 Metalloprotemases

Several investigators have reported that increased production of matrix-degrading metalloproteanases as associated with the metastatic phenotype The two enzymes that are most often found increased in metastauc cells are transm and type IV collagenase [368] Transin [490] and its homolog stromelysln [491], degrade type IV collagen, elastm, fibronectan, cartilage proteoglycans, larmmn and gelatin but not anterstmal collagens [492,493] It is increased in mouse skm that has undergone repeated treatment with carcinogens [494] Type

106 IV collagenase, as its name implies, cleaves type IV collagen fmrly specifically [495,496] For some tumor cells, a poslnve correlation can be made between metastatic potentml and levels of type IV collagenase produced by the cells [304] A separate metalloprotemase that degrades type V collagen has also been reported to be produced by certain tumor cells [497] and additional metalloprotemases are involved in ghoblastoma infiltration into the central nervous system [498] A fibronectan degrading metalloprotelnase has also been isolated from transformed chick embryo fibroblasts [499] Both type IV collagenase and transm are secreted as latent zymogens and can be actwated with treatment by other protemases, such as cathepsm B or plastron [500,501] An interesting recent study has shown that binding of the R G D sequence to the fibronectm receptor on cultured synovml fibroblasts snmulates metalloproteinase gene expression [502] If this is also true for metastanc tumor cells, ~t would suggest that lnteracnon of tumor cells w~th extracellular matrix molecules in the secondary txssue might stimulate production of enzymes necessary for invasion of that nssue An interesting correlation has recently been made between metalloprotemase production and the actwlty of certain oncogenes When the Harvey ras oncogene, for example, ~s transfected into N I H 3T3 mouse fibroblasts [503] or into human bronchial epithelial cells, increased secretion of type IV collagenase is observed [504-506] These effects can be blocked if the cells are co-transfected with the adenovlrus E l i oncogene [504] The acnvity of these metalloprotelnases is blocked by the actwlty of a class of agents called 't~ssue lnhibltors of metalloproteinases' (TIMPs) This inhibitor has been found to inhibit the lnvasmn of B16 melanoma cells through a m m o n membrane m an m vitro mvas~on assay [507] Because the protemases and the Inhibitor can be produced by the same cell, the relative amount of each agent determines the net proteolytlc activity Production of excess metalloprotemase should be observed m frankly metastatic cells, whereas cells that overexpress T I M P may lose the metastatic phenotype The amnlon invasion assay has been used to show that natural T I M P is capable of inhibiting infiltration of tumor cells across a native basement membrane [508,509] Schultz et al [507] have shown that premcubatlon of B16 melanoma cells with recombinant human T I M P prior to taft-veto mject~on into syngenelc mace significantly reduced tumor colonlzatmn of the lung In a related mvest~ganon of this phenomenon, Khoka et al [510] found that transfectmn of T I M P ant~sense R N A into non-tumongenlc Swiss 3T3 cells resulted in decreased T I M P activity, increased protemase activity, tumorlgenXclty and metastatic ability It should be noted that the cartilage derived inhibitor of angaogenesls described m secnon II A 6 is also a member of the T I M P family [260]

V-A 2 Plasmmogen activators

The correlation between the neoplastic state and the production of agents that cause the activation of plasrmnogen to plasrmn has been thoroughly reviewed [511] We will therefore consider only the evidence that plasmmogen activators may have a role in the metastatic process Of the two types of commonly studied plasmlnogen activators, tissue plasrmnogen activator (t-PA) and urokmase-type plasmanogen activator (u-PA), it is u-PA that is most often ~mphcated m the metastatic process Many cells brad this actwator to a high affimty cell-surface receptor [512-515] and it can then be presented m active form at sites of matrix degradation [516-518] P u r l f e d plasmlnogen has also been shown to brad directly to extracellular matrix components [519522] where it may later be acnvated and cause matrix degradation In VlVO, u-PA can be localized to the tumor-matrix interface in areas of Lewis lung carcinoma invasion [523] The plasmln formed through the acnon of plasmlnogen acnvator may act d~rectly to degrade matrix molecules and may further activate latent protelnases such as procollagenase [501,524] and enhance tumor cell invasion through amnlon membranes m vitro [525] In fact, the combined action of plasmm and collagenase have been found to be necessary for tumor cell invasion through intact basement membranes m vitro [526] Several groups have found a correlation between the production of plasmlnogen activators and invaswe or metastatic potential of particular tumors m vitro [527530] as well as m VlVO [531-536], although such correlations do not hold true for all tumor types [537,538] Some of the earlier studies did not, however, distmgmsh between the different forms of plasmlnogen activator and consequently m a y need to be reevaluated in hght of recent evidence that u-PA is more effcient at promoting the metastatxc phenotype Inhibltors that block plasmln or PA actxvity have been reported to have antl-metastanc activity The plasmln inhibitor e-amino caprolc acid (EACA) and the u-PA inhibitor tranexarmc acid have been shown to inhibit growth and metastas~s of murlne breast carcinomas [539] In a later study, u-PA was found to enhance spontaneous pulmonary metastasis, and this enhancement was blocked by the activity of tranexamac acid [540] although the complete range of action of these inhlbltors in VlVO remains unknown The evidence for u-PA involvement in tumor invasion and metastas~s stems largely from the work of Ossowski who has studied the colonization of human HEp-3 hepatoma cells injected into chick embryos The number of HEp-3 lung colomes was reduced by adrmnlstration of antibodies to u-PA but not t-PA [541] The ~rflubltlOn appeared to take place at the level of tumor cell Invasmn of C A M mesenchyme, a necessary early step in this metastatic assay [542] Slnular experiments

107 by Heanng et al [543] showed that antibodies to u-PA lninbited B16 melanoma colonization in syngenelc mace The importance of u-PA in metastasis has been confirmed in transfection experiments in winch pulmonary metastasis of Ha-ras transfected N I H 3T3 cells was potentiated by further transfectlon with u-PA The enhanced invasion and metastasis were both reduced by u-PA antibody [544]

V-A 3 Heparttmases Heparan sulfate proteoglycans are major components of eplthehal and endothelial basement membranes and of some interstitial connective tissues Consisting of a protein core with attached glycosamlnoglycan chains, extracellular proteoglycans are first degraded by proteinases that attack the core and hnk proteins Proteoglycans are degraded by broad spectrum protelnases such as stromelysm, elastase and trypsin, as well as cathepsln B, a lysosomal peptIdase secreted in high amounts by many tumors cells [545] Specific glycosidases are then required to cleave the glycosalmnoglycan side chains Elevated levels of glycosldases have been noted in several transformed cell lines These enzymes are predominantly heparitlnases which preferentlally degrade heparan sulfate, these are distinguished from heparlnases winch preferentially degrade the related GAG, hepann Metastatic cells, placed on an extracellular matrix produced by endothehal cells, sohibxhze glycosanunoglycans by means of a cell surface heparltlnase [153,154,546] In addition to promoting degradation of the basement membrane barrier to tumor invasion, production of heparltinases may have an addmonal positive effect on tumor growth As noted previously, FGF, a known anglogemc factor, binds tightly to heparin and heparan sulfate, and can be found sequestered in basement membranes in VlVO [149] Recently, Bashkm et al [152] have reported that heparItinase treatment of subendothehal extracellular matrix releases basic F G F that had been bound to the matrix Because basic F G F is a potent angtogenlc agent, the authors suggest that tumor heparitmase may release angaogemc activity from basement membranes in vivo and thereby enhance the process of tumor anglogenesls V-A 4 Cathepsms A correlation has been drawn between the metastatic potential of a tumor and the appearance of active cathepsm B, a lysosomal cystelne protemase [547,548] In B16 melanoma cells, for example, there was a 2-7fold elevation of lysosomal cathepsln B activity in the Ingh metastatic F10 hne when compared with the B16F1 line that displays lower metastatic potential in vivo [549] Slmdar results were found with two variants of a spontaneous rat anaplastlc sarcoma, the non-metastatic variant showed low cathepsin B activity whereas the highly metastatic variant showed lugh activity [550]

Like other protelnases involved in matrix degradation, the active enzyme can be found associated with the plasma membranes of metastatic tumor cells [551,552] and the membrane-bound form is resistant to mactwatlon by minbltors of cathepsin activity [553] Cathepsln B may faclhtate collagen degradation by two mechamsms, direct hydrolysis of collagen fibrils [554] as well actwatlon of procollagenase to an active form [500] In vivo, Inghest amounts of cathepsm B have been found at the advancing edges of invading human breast carcinomas and fibroadenomas [555,556] where they may facilitate invasion by aiding in the degradation of connective tissue extracelhilar matrix

V-A 5 Elastases Elastm is an unusual hydrophoblc molecule that has a umque distribution It is present in highest concentrations in distensible tissues such as the ligaments, arterial walls, lung and skin [557] Well characterized elastolytlc enzymes have been isolated from inflammatory cells such as neutropinls, monocytes and macrophages [558,559] Although elastln is a common component of the arterial wall, it Is not commonly found in the smallest mlcrovessels through which metastatic tumor cells must pass An exception is the lung in winch large, dense deposits of elastm underlie the alveolar capdlarles and consequently may present a barrier to metastatic colonization of the hver We have therefore proposed that tumors winch form pulmonary metastases should be able to degrade pulmonary elastm in order to optimally invade into the lung parenchyma [310] Thus elastase production is not a property of all metastatic tumor cells but may be preferentially found in tumors that arise in the lung or in tumors that arise in other locations and later metastaslze to the lung Among the tumor cells that have been found to express elastln-degrading activity are the munne Lewis lung carcinoma [312], bladder carcinomas [310] and a variety of mammary carcinoma cell lines [309,311,560] as well as human mammary adenocarcinomas xn vwo [561] As discussed below, the degradation of matrix molecules, such as collagen, fibronectm, lanunm and elastln, not only creates openings in tissue barriers that may facihtate tumor cell invasion but the degradation products of these hydrolytic reactions may directly stimulate tumor cell rmgratory processes that further facdltate the metastatic process V-B Tumor cell motthty and chemotams Although some tumor cells can invade into tissues passively, though cell growth and expansion [562], active cell motility enhances metastatic potential and is a property of most metastatic tumor cells [340-342,563] Increased motlhty is frequently observed in highly

108 metastatic tumor cells relative to poorly metastatic or non-metastatic cells [564-569] After tumor cells have adhered to the endothehum or subendothehal matrix in a secondary site, the tumor cells must migrate across the vessel wall and into the tissue lnterstItlUm (Fig 1B) As noted above, ttus passage is facihtated by the activity of proteinases and other matrix-degrading enzymes produced by the tumor cells themselves In ttus regard, the extravasatlon of tumor cells from the vasculature to the extravascular tissue space has much in common with the inflammatory process Here, host cells respond to an inflammatory response by a coordinated process which includes travel in the bloodstream, adhesion to activated endothehal cells, degradation of vascular basement membrane and migration across the vessel wall and into the tissue stroma Consequently, many of the enzymes secreted by tumor cells may be analogous to enzymes secreted by inflammatory cells, and chemotactic factors that stimulate tumor cell motility may be related to factors that are chemotactic for leukocytes In addition, tumor cells attract inflammatory cells [240.570] and these tumor-associated inflammatory cells may enhance the invaslve capabilities of the tumor cells [432] Many of the factors that have been shown to be chemotactic for metastatic tumor cells are fragments of extracellular matrtx molecules that are produced by the action of the tumor-derived degradatlve enzymes discussed in the previous section Examples of such factors include degradation products of flbronectin [346,351, 571,572], laminln [326,348,573-575], collagen [349], vitronectin (serum spreading factor) [576], thrombospondin [577] elastln [310,349] and products of resorbing bone [350] Certain chemotactlc factors appear to be specific for specific tissues Hujanen and Terranova [578], for example, have shown that tumor cells which colonize specific for specific tissues will migrate in response to extracts prepared from their preferred metastatic sites but riot to extracts prepared from other sites The mefhonlsms underlying the tumor cell chemotactic response are less well understood than those controlling leuko¢yte chernotavas Chemotaxas of both cell types appears to be mediated by specific cell surface receptors that recognize chemotactic molecules The well studied chemotactlc peptide fMet-Leu-Phe, for example, binds to specific receptors on the leukocyte surface and the signal is transduced across the membrane by a G-protein that activates a phosphohpase C mediated cascade of phosphoinosotlde metabolism [579] In some instances, tumor cells respond In a similar manner In the A2058 human melanoma cell response to an autocrine motility factor, for example, the chemotactic signal is mediated by a specific cell-surface receptor and the signal transduction mechanism involves a pertussls sensmve G-protein [580] Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a repeated

sequence in elastin that has been shown to stimulate the migration of monocytes and hgament fibroblasts [581] We have identified an M r 59000 VGVAPG-blndlng protein on the surface of m u n n e Lewis lung c a r o n o m a cells [349] Responsiveness of Lewis lung carcinoma variants [582] to V G V A P G depends not upon the presence or absence of the receptor, but rather to the affinity state of the receptor [583] We have further shown that the affinity state of the receptor correlates with the level of protein klnase C activity associated with the cellular membrane Receptor affinity and corresponding chemotactlc response can be induced in apparently non-responsive cells by treatment with the klnase C activator TPA [583] Such increased motility in TPA-treated cells may explain, in part, why TPA treatment of certain tumor cells increases their metastatic potential [584,585] Although solublhzed extracellular matrix domains represent the largest known family of tumor cell chemoattractant, matrix molecules in their native, insoluble form m a y also direct tumor cell migration by the process of haptotaxls, in which cells migrate on a substratum that consists of an increasing gradient of an adhesive molecule This phenomenon has been extensively characterized for tumor cells responding to the haptotactic activity of flbronectln, laminln or thrombospondin [322,344,351,377] In this case. tumor cells will move up the gradient of immobilized chemoattractant even in the absence of an adhesive gradient

VI. Cell growth control at the secondary site The ablhty of a metastatic colony to form at a secondary site depends on several factors First, the cell must have the appropriate genetic composition to grow in a foreign environment Ttus will normally include the activation or overexpression of cellular oncogenes [586,587] or the deletion or inactivation of a cellular suppressor gene [588,589] The tumor cell then requires that the secondary site be a conduove site for cell growth containing growth factors that stimulate division of that particular tumor cell and lacking any growth lnhlbltors that m a y suppress tumor growth (Fig 1C) Of the m a n y oncogenes that have been tested for their effects on metastasis, the most abundant data concerns the role of the r a s oncogene In several studies, non-metastatic cells that possess some properties of transformed cells have been converted to metastatic tumors by transfectlon with an activated ras oncogene [590-595] It is not clear, however, whether the effects of the oncogene in these studies is on cell growth or on some later step in the metastatic cycle When cells wtuch can form primary tumors but are not shed into the bloodstream are transfected with an activated r a s gene, the cells obtain the ability to enter the bloodstream and form metastases [596] T u m o r cell growth rate IS

109 not affected by the gene transfer In tins case, it Is hkely that the primary effect of the oncogene is on tumor cell mtravasatlon into the circulation and not on cell growth Other oncogenes that enhance metastatic potential fall into the general class of genes coding for tyrosme klnases [597] Again, the primary mechanism of tins pro-metastatic effect is not certain since such genes can also effect changes m events downstream from the growth cycle, including protemase production and cell motility A suppressor gene that prevents tumor metastas~s has also been reported lacking in some metastatic tumor cells [598-600] Several growth factors have been reported to influence the growth of metastatic tumor cells Many of the factors, such as F G F , E G F and P D G F are produced to varying degrees m m a n y tissues [601] and may be expected to have a role in the establishment of metastatic colonies m a variety of tissues Other factors have a more organ-specific distribution In several stud~es, metastatic tumor cells that d~splay patterns of sitespecific tumor metastas~s have been shown to respond selectively to factors present in their preferred organ site [450,602-610] Such specificity was first suggested by Paget over 100 years ago [611] Slte-specihc growth lninbltors such as TGF-fl [187], interferon [612] and tumor necrosis factor [205] may all modulate the growth of metastatic tumor cells at the secondary site Here again, recent ewdence suggests the presence of organspecific growth minbltors such as m a m m a s t a t m , a m a m m a r y gland-specific ant~-mltogen that inhibits the growth of m a m m a r y epithelial cells as well as m a m m a r y adenocarclnoma cells [613] Finally, it should be noted that anglogenests ~s no less important to the growth of the secondary tumor colony than It was to the growth of the primary sohd tumor Again, the secondary tumor will generally grow only to a size of 1 - 2 m m if governed only by ItS endogenous ability to grow in a specific site Beyond that small diameter, further growth depends on the abdlty of the metastatic cell to attract new blood vessels Because secondary tumors often give rise to tertiary tumors m distant organs by a process termed the metastatic cascade [614], the entry of tumor cells into the neovasculature at the secondary s~te is a crucml component of the further spread of these neoplasms Thus, m a c~rcular process, primary tumor cells reqmre blood vessels to grow and spread to secondary organs, tumor cells in the secondary s~te s~mllarly require vessels to grow and to return to the bloodstream to colomze addmonal organ sites (Fig 1D)

establish new blood vessels to provade essential nutrients and oxygen for sustained tumor growth Anglogenes~s and tumor dlssermnatlon are temporally correlated, tumor cell shedding into the bloodstream rapidly follows estabhshment of blood flow m the new tumor vessels Tumor-induced blood vessels are fragile and highly permeable, with a discontinuous basement membrane Whereas direct access to the lumen of exastmg intact blood vessels m adjacent tissue would reqmre Inghly mvaswe tumor cells w~th elevated production of degradatwe enzymes, access to newly formed tumor capillaries may be more easily accomphshed Once tumor cells gain access to the vasculature, successful metastasis reqmres that circulating tumor cells (1) survwe attack by tumoncldal immune cells, (2) survive the mechanical stress of flow through the nucroclrculaUon, (3) arrest m the rmcrovasculature or adhere to specific cell attachment molecules expressed on the endothehal cell surface or in the subendothehal basement membrane, (4) exit the vasculature through the action of degradatlve enzymes, and (5) grow in the new organ site H~ghly lnvaswe tumor cells winch can independently degrade the subendothehal extracellular matrix may have an increased probabdlty of forming a successful metastatic colony, less aggresswe tumor cells may occasionally accomphsh extravasatlon by following the path of inflammatory cells that pass easdy through vessel walls and into parenchymal tissue Active nugrauon of tumor cells through the basement membrane and into adjacent stroma and organ parenchyma should also speed the exit of tumor cells from the bloodstream and consequently promote tumor cell surwval and eventual metastas~s T u m o r cells that can respond to local chemoattractants, m a manner analogous to that of inflammatory leukocytes, may be more likely to reach the organ s~te and form a metastauc colony Only a speclahzed subpopulation of tumor cells from the primary tumor will accomplish all stages of metastas~s and overcome the numerous vascular barrters, so that the secondary tumor may be composed of tumor cells that represent only a small percentage of those shed into the bloodstream T u m o r cells in metastatic colomes can attract new blood vessels, rec~rculate in the vasculature and eventually form tertiary colomes downstream from the secondary metastas~s m a process that has been termed the metastatic cascade [614] Thus, winle growth is one hallmark of neoplasm, successful interaction with the vasculature and with the cells that circulate w~tinn it are essentml at virtually every step of the metastatic process

VII Conclusions References Mahgnancy is a systemic disease, in winch tumor mteractmns w~th the vasculature are crucial to the pathologlc process An in SltU neoplasm must recrmt and

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