Review: Basic Mechanisms of Metastasis

Review: Basic Mechanisms of Metastasis BY JAMES SANCHEZ, MD,

VICKI BAKER, MD,

ABSTRACT: Metastatic disease is responsible for the majority of deaths caused by cancer. The process of metastasis is an orderly, stepwise process that results in the selection of cells that possess the capability to establish viable metastases. These cells must be locally invasive and be able to survive the physical traumas of dissemination and normal host defenses. Once metastatic cells have been arrested in a capillary bed, they must be able to invade the host organ parenchyma and survive in that milieu. Studies in a number of model systems have documented the phenotypic alterations in cells that have "metastatic potential." These differences may stem from normal tumor cell heterogeneity and surprisingly reflect only minor differences in gene expression. The role of activated oncogenes in metastasis is unclear, but a number of laboratories have documented that transfection with activated Ha-Ras results in increased metastatic potential. An increased understanding of the genetic basis of metastatic potential may suggest new directions for intervening in this deadly process. KEY INDEXING TERMS: Cancer; Metastasis; Tumor Heterogeneity. [Am J Med Sci 1986; 292(6):376-385.]

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alignant disease is responsible for approximately 35% of American deaths 1 and is the second leading cause of death in this country. Currently available therapy for this disease includes surgery, chemotherapy, and radiotherapy that can now cure approximately 50% of cancer patients. 1 However, the majority of the remaining patients succumb to the effects of metastatic disease. The underlying mechanisms of tumor metastasis have been the subject of intensive study over the past two decades. We have gained much new information about From the Birmingham VA Medical Center and Comprehensiue Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama. The authors gratefully acknowledge the able assistance of Mrs. Sheila Chastain in the preparation of this manuscript and the helpful discussion ofDr. Max Wicha. Reprint requests: Donald M. Miller, MD, PhD, Division of Hematology/Oncology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294.

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DONALD M. MILLER, MD, PhD

the manner in which tumor cells metastasize, grow, and exert their effects on the host.2-10 Unfortunately, this new information has not translated to more effective means of treating metastatic disease. It remains extremely difficult to detect very small metastases or even to predict the metastatic tendency of an individual tumor. We have had even less success in the prevention oflocal tumor invasion and the treatment of individual metastases. The Metastatic Process

It has become very clear that metastasis is a stepwise, selective process. The same steps must occur for the metastasis of any tumor type. Local invasion is the first step that must occur in initiation of metastases. Tumor angiogenesis with the supply of ade. quate oxygen and nutrients for tumor growth is a prerequisite for this to occur. After a tumor invades host tissue barriers, it must traverse the vascular wall or lymphatic channels to become disseminated. Once it reaches the circulation, it must be able to evade a number of host defenses, including Natural Killer (NK) cells, activated macrophages, and lymphocytes. The tumor cell must also survive the physical trauma of blood flow and be able to be arrested in the venous or capillary bed of the target organ. Tumor cell arrest is mediated by mechanical obstruction of small vessels, entrapment in platelet or fibrin clots, and interaction with exposed basement membranes. Once the tumor cell has been arrested, it must again pass through the vascular wall to enter the host organ parenchyma. It must then be able to grow in this organ to form an established metastasis. This complicated sequence of events is responsible for the fact that only a very small percentage of potentially metastatic cells are able to form metastases. However, despite the inefficiency of this process, large tumors shed enormous numbers of cells so that even a very small proportion of successfully metastatic cells may result in a large number ofmetastases. Each of the steps of the metastatic process represents a site at which selection occurs. Each step also represents a potential site for therapeutic intervention. Tumor Invasion

The first and most critical step in the development of metastases is the invasion of local host tissues to December 1986 Volume 292 Number 6

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gain access to the circulation. During tumor cell invasion, malignant cells must actively cross two types of extracellular matrix; basement membranes and interstitial stroma. After penetration of the epithelial basement membrane (to form an invasive tumor) the cells traverse the stroma to reach the lymphatics or blood vessels (intravasation) in which they disseminate to distant sites. The tumor cells must also cross basement membranes when they migrate out of blood vessels (extravasation). They must migrate out of the circulation into the target organ, and then pass into the host organ parenchyma to establish a viable metastasis. In each of these steps, the metastasizing tumor must alter the extracellular matrix to allow it to attain metastatic capability. These alterations are apparent in the widespread changes that occur in the epithelial basement membrane during the transition from benign to invasive tumors. Benign disorders may result in disorganization of underlying stroma but are always characterized by a continuous basement membrane separating the epithelium from stroma. However, it has been consistently noted that there is no basement membrane formed around invading tumor cells in stroma. This observation also applies to lymph node and organ metastases. Malignant tumors have been shown to modify extracellular matrix in at least three different ways. 1. Digestion of matrix components. This occurs by the secretion of a number of collagenolytic and proteolytic enzymes by metastasizing cells. 13-15 2. Augmented accumulation of matrix components by host cells in response to tumor cell stimulus. IS-IS 3. Synthesis of matrix components by tumor cells. Synthesis of matrix components such as collagen allow the tumor cells to create their own basement membrane. This usually occurs in amounts much lower than amounts secreted by normal cells and is dependent on the differentiated state of the tumor cell. 19-21 Each of these mechanisms of extracellular matrix modification appear to interact to allow local invasion by selected tumor cells. Because the normal host extracellular matrix does not normally contain cellular passageways, it provides a natural barrier for tumor cell invasion. In fact, basement membrane is impermeable to even large proteins. The basement membrane becomes permeable to cell movement only during tissue remodeling, wound healing, inflammation, and tumor invasion. The ability of cells to traverse the extracellular matrix depends on a number of factors including properties of both the invading cell and the specific extracellular matrix. Liotta and co-workers have devised a three-step hypothesis that describes the biochemical events that characterize tumor cell invasion of the extracellular matrix.s These steps are tumor cell attachTHE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

ment, secretion of hydrolytic enzymes, and tumor cell migration. Tumor cell attachment may be mediated through specific surface glycoproteins such as laminin and fibronectin and/or through tumor cell receptors for these molecules. 22-30 Fibronectin is a major surface glycoprotein that promotes attachment of cells to collagen. The concentration of fibronectin has been inversely related to tumorgenicity, but not metastatic capability. Laminin is another attachment factor that has stimulated considerable interest. This protein (MW 1,000,000) is found exclusively in the basement membrane and appears to be selectively used by metastatic cells for attachment. A specific receptor for laminin has been documented in a number of tumor cell types. The concentration of laminin receptors is increased as much as 50-fold in highly metastatic cells. The second step is the secretion of hydrolytic enzymes by the tumor cells or induction of enzyme secretion by host cells, which results in the local degradation of extracellular matrix. 13-16 Lysis of the matrix is thought to take place in a localized region close to the tumor cell's surface. Local dissolution of the basement membrane can be observed electron microscopically in areas adjacent to invading tumor cells. 31 The ability to digest type IV collagen (by secretion of collagenolytic activity) has been shown to correlate with metastatic activity in a number of cell lines.32 The secretion of collagenocytic activity by these cells33-36 appears to facilitate transit of metastasizing cells. The third step is tumor cell migration into the region of modified matrix. This is thought to be at least partially in response to chemotactic (or hapotactic) factors secreted either by the tumor cell itself or by the host parenchymal cells. 37-40 The ability to migrate may provide another selection criterion for metastatic cells. Tumor Cell Dissemination

In contrast to the detailed information available about invasion, much less is known about transport of tumor cells once they have entered the circulation or lymphatic vasculature. It is presumed that the cells are passively transported to the site of their potential arrest. It has been shown that less than 1% of the millions of tumor cells that may escape from a primary tumor survive to become viable metastases, while the majority of cells will die. 41 .42 However, this small proportion is adequate for the initiation of many metastases in patients with relatively large tumors. Most clinical metastases do not occur until the primary tumor has reached a volume of at least ·1 cms (approximately 109 cell).41 The high rate of tumor cell death may be attributed to a number of factors including mechanical shear forces, destruc377

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tion by host derived circulating immune cells, oxygen toxicity, and the potential loss of growth factors present in the local environment. .Resistance to one or more of these factors may play a role in selecting cells that are able to form viable metastases. Several groups have shown that in at least several instances (including RAW lymphosarcoma and B16 melanoma) tumor cells with high metastatic potential have an increased resistance to cytolysis by activated macrophages or Natural Killer (NK) cells. 43 .44 There are a number of experimental systems for studying the behavior of circulating tumor cells and their survival. It is clear from work in these systems that the presence of tumor cells in the circulation is not adequate, per se, for metastasis. It is also clear that the metastatic cell must have a number of properties that allow it to metastasize successfully. Fidler has proposed that survival and eventual metastasis ofB16 melanoma cells is dependent on unique properties of tumor cells and that the metastatic process is not a random phenomenon. 45 .46 Genetic instability of the parent tumor may allow the development of tumor cells that are able to survive in the circulation. There are a number oflines of evidence suggesting that NK cells play an important role in host resistance against metastases. 47-52 The fact that NK cells are primarily responsible for the elimination of intravenously inoculated tumor cells was initially suggested by the finding that cells from the lung metastases of a transplantable tumor in mice were more resistant to NK activity than were locally growing primary tumor cells. 48 It has also been well documented that suppression or augmentation of NK activity of the mouse is associated with parallel alterations in resistance to artificial metastases after intravenous inoculation. 49 .5o It is likely that activated macrophages, NK cells, and possibly other immune mechanisms coordinate to create host resistance to hematogenous metastases. Tumor Cell Arrest

The vast majority of tumor cells are arrested in the first capillary bed they encounter. 53 .54 This is reflected by the fact that tumor cells injected into the tail vein of rats result in a 60-100% concentration in the lungs. 53 If cells are injected into the portal vein, the first encountered organ is the liver, and fibrosarcoma cells are almost completely retained in this organ.55 Fidler and Nicolson, however, injected melanoma cells into one of a parabiotic pair of mice and observed metastases in the noninjected mouse. 56 This observation indicates that at least a few cells survive passage through the first capillary bed and are able to reach distant organs. It is apparent that tumor cell aggregates are more efficient than single tumor cells in establishing metastases. 57 .58 This is probably due to cellular pro378

tection of the aggregate as opposed to single cell exposure to traumatic influences. A number of investigators have studied the fate of tumor cells that have lodged in the lung.53.59.6o After 24 hours 90 to 95% of these cells had disappeared and after 72 hours less than 1% remained. Disappearance appears to be due to cellular degeneration and death as opposed to redistribution. Cellular adhesiveness may play a role in the organ-specific arrest of metastatic cells. A number of studies with the B16 melanoma have documented that lung-specific B1G cells are retained more avidly in the lungs following a tail vein injection than the parent B16 cells.53.61.62 When the lung-specific B16 cells were mixed with dispersed lung cells, they tended to form aggregates more readily than with the parent cell line. Moreover, these cells adhered more strongly to 3T3 cell mono layers suggesting that they may bind more avidly to endothelial cells. Interestingly, the B16-F10 (lung metastasisspecific) adhered more readily to monolayers of B16 cells suggesting that they may tend to form aggregates more readily, which might explain why they are more easily arrested than single cells. Attempts to correlate cell membram~ glycoprotein content with metastatic potential or to modify cell surface antigens with neuraminidase 01' trypsin have given mixed results, and there is no evidence of a consistent effect.63-55 Blood coagulation also plays a major role in the development of viable metastases. There are clear associations between metastases and blood coagulation, including the occurrence of thrombi around embolic tumor cells66-68 and the intravascular coagulation of many patients with disseminated cancer. 69 .70 Early observations documented that intravenous injection of tumor cells into thrombocytopenic animals resulted in lower numbers of pulmonary metastases.71.72 The investigators speculated that this was due to the absence of platelets and not other effects of the induced thrombocytopenia. Platelet aggregating activity has also been shown to increase the efficacy for a number of tumor cell types to cause metastases. Experiments with fibrin formation and fibrinolysis have given contradictory results. It is clear that coumarin derivatives and other vitamin K antagonists are able to cause decreased numbers of both artificial and spontaneous metastases. 7ll-75 However, coumarin derivatives, in addition to their anticoagulative action, have a number of other activities that may be responsible for this observation. Indeed, it appears that the antimetastatic effect of vitamin K depletion may be independent of blood coagulation. 76. 77 Heparin and other sulfated polysaccharides probably act to inhibit metastases by preventing thrombogeneration. The treatment of host animals with December 1986 Volume 292 Number. 6

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Heparin or Heparin-like substances usually results in decreased numbers of metastases. 78-80 Not only inhibition of fibrin formation but also stimulation of fibrinolysis have been shown to have antimetastatic effect. 81 . 82 It is not clear that this relates to tumor cell coating, but it may provide a measure of protection to the arrested tumor cells. Implantation

It is obviously necessary for arrested tumor cells to pass through the vascular endothelium into the target organ parenchyma to form viable metastases. To do so, the tumor cell needs to interact once more with the basement membrane and matrix proteins such as laminin and fibronectin. There is evidence that tumor cells are able to cause retraction of endothelial cells to provide an avenue for diapedesis.83 They are then able to migrate through the vascular endothelium into the organ parenchyma, possibly responding to chemotactic factors present in these organs. 37~O There has been a gI:eat deal of speculation about the role of cell membrane glycoproteins in the organ specificity of experimental metastases. Fidler, Nicolson, and other groups 'h ave extensively characterized B16 melanoma and have been able to isolate organ-specific B16 celllines. 53 •8 4-87 Although there are cell surface glycoprotein differences between these sublines, it is not obvious how these differences influence organ specificity. It has been suggested that cells that have traversed the endothelium into the organ parenchyma have a markedly increased survival time relative to those cells that have been arrested in the blood vessel. It seems likely that there is some protection from host defenses afforded to cells that are able to complete this migration. Inhibition of Metastases

The obvious goal of much of the metastasis research currently being performed is to develop an effective means of inhibiting the development of metastases. There are a number of experimental manipulations that are able to inhibit various steps in the metastatic cascade, but none of them have proved useful in a clinical setting. The general absence of metastases in cartilage has prompted suggestions that, in addition to the avascular nature of this tissue, there may be characteristics of this tissue that retard growth of invading cells. In support of this concept, Folkman and coworkers88•89 have shown that cartilage contains an anti-angiogenesis factor that completely inhibits growth of new vessels in this tissue. Other workers90 have also been able to extract a low molecular weight compound from cartilage that inhibits proliferation of malignant, but not normal cells. These observations suggest that there may be organspecific means of protection.from metastases. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

Agents that induce hypothyroidism have also been shown to lower the incidence of metastases and increase survival in rats carrying the 44 Morris Hepatoma. 91 Although the exact mechanism of this effect is unknown, it may occur as a result of slower growth of the primary tumor. Isselbacher and co-workers have characterized the antimetastatic activity of a cancer-associated galactosyltransferase acceptor (CAGA) purified from human malignant effusion. 92 They found that this low molecular weight glycopeptide reduced death from metastasis by 55-90% in mice bearing sublines of the KA31 and KB521 Kirsten sarcoma-transformed mouse fibroblast cell lines. Other investigators have found that macrophages activated by heat-killed propionibacterium acnes, or Bacillus Calmette-Guerin (BCG), are able to reduce substantially the number of metastases in X5563 plasmacytoma-bearing mice. 93• 94 It has also been shown that pretreatment of Lewis lung carcinoma cells with three different proteinase,inhibitors prior to engraftment results in fewer and smaller metastases. 95 These lines of evidence suggest that inhibition of enzymes necessary .for inv~sion may reduce the capability of cells to metastasize. The role of platelets and fibrinolysis in metastasis has been discussed previpusly. 'Obviously, anticoagulation and anti platelet agents constitute a major potential means of antimetastasis treatment. Results from experiments to test this hypothesis have been conflicting. A large number of studies have tested the role of anticoagulants and platelet inhibitors in the adjuvant treatment of cancer. Studies using platelet inhibitors have given variable results, suggesting that a specific tumor response may be dependent on the tumor and the drug. Aspirin, for example, has been shown to be both active and inactive as an antimetastasis drug. 96•97 A number of newer antiplatelet drugs as well as prostacyclines have also shown variable activity, depending on the tumor model system.9S• 99 Many studies have investigated the effect of anticoagulation on metastasis. In a clinical setting, several authors have presented evidence suggesting that anticoagulation substantially reduces the incidence of metastases, with an increase in patient survival. 99-102 It has been noted that the antimetastatic effect of the coumarin derivatives may be distinct from the anticoagulant activity. This has been suggested by evidence that vitamin K depletion or treatment with inactive coumarin derivatives can provide the same antimetastatic effect. The clinical role of this treatment is currently evolving. Tumor Cell Heterogeneity

The cellular heterogeneity of neoplasms provides a major obstacle in treatment of malignancy. The phenotypic heterogeneity of tumor cells includes differences in: histologic appearance; karotype; anti379

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genicity; immunogenicity; biochemical properties; growth behavior; metastatic capability; susceptibility to destruction by chemotherapeutic drugs; radiation; hyperthermia; and ability to evade humoral andlor cell-mediated immune reactions mounted by the host.l03-107 Tumor cell heterogeneity is not confined to the primary tumor but is also found in metastatic lesions proliferating in different organs or even in the same organ. 103. 108. 109 Although neoplastic cellular heteroget:leity has been well documented, the actual etiology has not been determined. It is thought that most human cancers are probably derived from the proliferation of a single, transformed cell and the generation of biologic diversity is due to a rapid clonal diversification during tumor progression. 110 Foulds has postulated that the malignant tumor cell populations are subjected to various host selection pressures during progressive tumor growth resulting in the spontaneous appearance of new tumor cell variants. This generates tumors that contain phenotypically diversified cell populations. 119 Nowell has suggested that acquired genetic variability within developing tumors, along with host selection pressure, results in the emergence of new tumor cell variants that have acquired features of growth autonomy and increased malignant potential favoring continued tumor progression. ll2 The development of tumor cell heterogeneity is a dynamic process. Cellular variation at any time during tumor progression is dependent on both the number of distinct subpopulations of tumor cells in the primary tumor and the selection pressures being exerted at that time. Selection pressures on a tumor population can either be host-mediated (immune response) or artificially induced (treatment with therapeutic agents). The dynamic interplay of tumor cell subpopulations with varying degrees and types of selection pressure may generate tumors with different histologic types as well as differences in tumors of common histologic type in different patients.l03.106.113 There is solid evidence to suggest that the rate of formation of tumor cell variants is higher than the rate of variability of nonmalignant cells. In fact several investigators have determined the rates of variant formation to be from 10-2 to 10-5.104.113 These values far exceed the classical rate of mutations that generally occur at 10-6 to 10-8 mutations per generation. ll5 The isolation of subpopulations or clones has been used to compare particular subpopulations of tumor cells according to specific characteristics (eg, metastatic versus nonmetastatic, drug resistant versus nondrug resistant). Cloning magnifies particular tumor cell properties for closer scrutiny. Tumor cell cloning has allowed the observation that subpopulations of tumor cell are not completely autonomous and require the influence of other cell subpopula-

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tions. It has been demonstrated that mammary tumor cell subpopulations behave differently when grown separately as opposed to growing together with other subpopulations. ll6 These behavioral differences include chemosensitivity,117 immunogenicity, ll8 metastatic ability, 119 and growth rate.ll6.120 In addition to their behavioral differences, cloned tumor cell subpopulations have been shown to be phenotypically unstable, rapidly generating progeny with varying metastatic abilities, growth rates, and drug sensitivity.l04.106.ll3.121.122 Cloned subpopulations of B16 melanoma cells grown in isolation from other subpopulations demonstrated varying levels of metastatic potential from generation to generation. By contrast, when individually isolated clones were co-cultivated together the metastatic behavior of each clone was maintained through successive generations. 123 These experiments suggest that the phenotypic stability of tumor cells is dependent on cell to cell interaction. The mechanisms of this phenomenon of intercellular dependency is not known l23 and is not limited to the B16 melanoma, having also been demonstrated in clones of the mouse UV-2237 fibrosarcoma, mouse RAW1171ymphosarcoma, 13762 mammary adenocarcinoma, and the rat 1 ARC hepatocarcinoma. 1l3• 143 Situations that impose new selection pressures and eliminate a majority of tumor cell clones will lead to rapid phenotypic diversification. l23 This has been demonstrated by in vivo clonal analysis of a series of metastatic B16 melanoma lesions. 126-128 These studies revealed that more than 80% of metastatic foci originated from a single tumor cell but that within 45 days the metastatic lesions demonstrated a wide range of phenotypic diversity. These results have been interpreted as evidence for rapid generation of tumor cell heterogeneity within metastases stemming from the limited cellular diversity of the early metastatic lesion and the absence of restraints imposed by constituent sUbpopulations of tumor cells present in the primary neoplasm. The development of tumor cell heterogeneity via the generation and expansion of phenotypically different subpopulations during tumor progression enables tumors to survive multiple forms of adversity. Therapeutic measures, such as chemotherapy, exert a powerful selection pressure encouraging the emergence of new clonal subpopulations that possess resistant phenotypes. This increases the chances that a resistant clone will survive and reduces the possibility of cure in the host. Because of the heterogeneous nature of the responses of malignant tumor cell subpopulations to chemotherapy and other treatment modalities, a single treatment regimen is unlikely to kill all the cells in a tumor, even if multipIe agents are used simultaneously. New treatment strategies will require the identification of traits shared by all tumor cells that could be used to side December 1986 Volume 292 Number 6

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step the heterogeneous responses incurred with current antineoplastic therapy. A better understanding of the molecular biology of human cancer cells and the mechanisms that regulate phenotypic diversification in specific tumor cell lineages is needed. It has been suggested that a set of genetic sequences exist that could be activated by surrounding environmental stimuli leading to widespread changes in genetic and phenotypic properties of tumor cells. l29 In addition a phenotypically complex process such as metastasis will more than likely not be determined uniquely by a single gene but the result of many genes. 104 Despite the fact that metastases represent the major cause of morbidity and mortality in cancer patients, the amount of research devoted to elucidating the underlying mechanisms of metastasis and identifying tumor cell properties required for metastatic dissemination comprises only a small fraction of the current effort in the area of basic cancer research. Molecular Basis of Cancer Metastasis

Classically tumors have been classified as either benign or malignant. Distinguishing features of malignant tumors .include the propensity for invasive growth and metastatic spread to distant organs. Benign tumors generally pose little threatto the host and are usually cured by local treatment such as surgical resection. Conversely, malignant tumors are often incurable because of their invasive character and ability to metastasize and involve mUltiple organs. A more detailed understanding of the malignant process is essential for substantial advances in cancer treatment. The multistep process of metastasis has been described with respect to the expression of a number of tumor cell and host properties.2-7 This metastatic cascade describes the malignant phenotype, but the genetic events that must be necessary in the metastatic process are unknown. There is growing evidence that the underlying basis Qf cancer is genetic; that the malignant state is maintained by genetic changes and not by other "epigenetic" regulatory systems affecting the cell. lso DNA transfection experiments have identified a set of transforming genes present in the genome of a proportion of human and experimental tumors. These genes have been identified initially as viral oncogenes (v-oncs) and later as their normal cellular homologue (c-oncs) by their ability to induce tumors.1Sl.1S2 Neoplastic transformation is believed to be a multi-step process requiring the concerted action of two or more oncogenes to transform normal cells such as embryonic fibroblasts. 133 It is not clear whether the genetic elements responsible for tumorigenesis play ·any role in producing the metastatic phenotype. There have been a number of reports of transfection experiments that have attempted to identify the THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

genetic basis of the metastatic phenotype. Transfection of tumor DNA into recipient NIH 3T3 cells has been shown by several groups to induce the met~ astatic phenotype assayed in nude mice.134.135 Transfection of the Ha-Ras oncogene alone could induce the metastatic phenotype in immunoincompetent mice assayed following both iv and sc injection. Ha-Ras transfected recipient cells were unable to produce the metastatic phenotype in immunocompetent hosts except when the presence of discrete human tumor DNA fragments were added via repeated transfection. 134 Liotta and colleagues have suggested that the metastatic phenotype may require the expression of at least two or more genes, a situation similar to that of tumorigenesis.135.13G An activated rasH oncogene may playa role in metastasis, although it is clear that this gene alone is not sufficient to induce the metastatic phenotype and another gene or genes are required in a concerted effort. 134-137 Other metastasis-associated gene products that have been implicated in the induction of metastatic potential include those involved in the immune defense of tumor metastasis. Metastatic murine tumors that are deficient in the H-2K major histocompatibility complex (MHC) class 1 antigens have been transfected with H-2Kb genes resulting in a profound decrease in metastatic capacity in immunocompetent recipient mice, but unchanged metastatic capacity in immunodeficient mice. 13B These findings have suggested that an inverse relationship exists between malignancy and expression of the MHC class 1 molecules and corroborates the observation that metastatic spread of rasH transformed cells transfected with metastatic human tumor DNA are capable of metastasis both in immunodeficient and immunocompetent mice. The technique of subtractive liquid hybridization has been used in several instances to quantitate differences in gene expression between closely related cells. 139-141 This technique has been used to quantitate the differences in gene expression between high- and low-metastatic variants of a murine fibrosarcoma. These studies have demonstrated that gene expression differs by only 2% between the highly metastatic and benign tumor sub clones despite strikingly different phenotypic behavior. 142 This difference of 2% demonstrates that not only are these two subclones closely related, but that the number of genes responsible for the metastatic phenotype must be limited to a relatively small number despite the marked differences observed in metastatic characteristics. Although the number of experiments demonstrating genetic involvement in the metastatic process is small, it is the general consensus that induction of the metastatic phenotype has a definite genetic basis that may involve activation of a coordinated set of genes. The continued application of molecular bio381

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logic techniques to tumor cell biology will almost certainly provide further insights into the understanding of tumor cell metastasis. References 1. Silverberg E, Lubera A: Cancer Statistics, 1986. Ca-A Cancer Journal For Clinicians 36:9-25, 1986. 2. Poste G, Fidler IJ: The pathogenesis of cancer metastasis. Nature 283:139-146, 1980. 3. Liotta LA, Rao CN, Barsky SH: Tumor invasion and the extracellular matrix. Lab Invest 49:636-64C, 1983. 4. Sugarbaker EV, Ketchan AS: Mechanisms and prevention of cancer dissemination: an overview. Semin Oncol 4:19-32, 1977. 5. Liotta LA: Tumor invasion and metastases: Role of the basement membrane. Am J Pathol117:339-348, 1984. 6. Schirrmacher V: Cancer metastasis: Experimental approaches, theoretical concepts, and impacts for treatment strategies. Adv Cancer Res 43:1-73, 1985. 7. Nicolson GL: Cell surfaces and cancer metastasis. Hosp Pract 17:75-86, 1982. 8. Folkman J: Tumor angiogenesis. Adv Cancer Res 43:175-203, 1985. 9. Liotta LA: Tumor extracellular matrix. Lab Invest 47:112-113, 1982. 10. Roos E, Dingemans KP: Mechanisms of Metastasis. Biochim Biophys Acta 560:135-166, 1979. 11. Barsky SH, Siegal G, Jannotta F, Liotta LA: Loss of basement membrane components by invasive tumors but not by their benign counterparts. Lab Invest 49:140-148, 1983. 12. Burtin P, Chananel G, Foidart JM, Martin E: Antigens of basement membrane in the peritumorai stroma in human colon adenocarcinomas: An immunofluoresence study.lnt J Cancer 30:13-18,1982. 13. Fiszer-Szafarz B, Gullino PM: Hyaluronidase activity of normal and neoplastic interstitial fluid. Proc Soc Exp Biol Med 133:805-807, 1970. 14. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz Shafie S: Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 284:67-69, 1980. 15. Edmonds-Alt X, Quisquater E, Vaes G: Proteoglycan- and fibrin-degrading neutral proteinase activities of Lewis lung carcinoma cells. EurJ Cancer 16:1257-1261,1980. 16. Barsky SH, Rao CN, Grotendorst GR, Liotta LA: Increased content of type V collagen in desmoplasia of human breast carcinoma. Am J Pathol108:276-283, 1982. 17. Lozzo RV, Bolender RP, Wight TN: Proteoglycan changes in the intercellular matrix of human colon carcinoma: an integrated biochemical and stereologic analysis. Lab Invest 47:124-138,1982. 18. Jackson JG, Orr JW: The ducts of carcinomatous breasts with particular reference to connective tissue changes. J Pathol Bacteriol74:265-274, 1967. 19. Adamson ED, Gaunt SJ, Grahan CF: The differentiation of treatocarcinoma cells is marked by the types of collagen which are synthesized. Cell 17:469-476, 1979. 20. Alitalo K, Keski-Oja J, Vaheri A: Extracellular matrix proteins characterize human tumor cell lines. Int J Cancer 27:765-761, 1981. 21. Smith BD, Martin GR, Miller EJ, Dorfman A, Swarm R: Nature of the collagen synthesized by a transplanted chondrosarcoma. Arch Biochem Biophys 166:181-186, 1975. 22. Terranova VP, Liotta LA, Russo RG, "Martin GR: Role of Laminin in the attachment and metastasis of murine tumor cells. Cancer Res 42:2265-2269, 1982. 23. Terranova VP, Rohrbach DH, Martin GR: Role oflaminin in the attachment of PAM 212 (Epithelial) cells to basement membrane collagen. Cell 22:719-726, 1980. 24. Vlodavsky I, Gospodaronicz D: Respective roles of laminin

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