Clinical and experimental studies on the biology of metastasis

Biochimica et Biophysica Acta, 780 (1985) 227-235

227

Elsevier BBA87138

CLINICAL AND E X P E R I M E N T A L S T U D I E S O N T H E BIOLOGY O F M E T A S T A S I S DAVID T A R I N

Nuffield Department of Pathology (University of Oxford), John Radcliffe Hospital, Oxford OX3 9DU (U.K.) (Received March 1st, 1985)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

227

II.

Review of data from human autopsy studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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IIL Conclusions from human autopsy data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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IV, Experimental analysis of mechanisms influencing distribution of tumour metastases . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Distribution of tumour cells released intravascularly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Observations on patients infused intravenously with malignant cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Studies in animals and in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

230 230 230 231

V.

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Selectivity and stability of the metastatic process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References .*. . . . . . . . .

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~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Introduction When the poet Byron died at Missolonghi fighting for Greek independence and T.E. Lawrence successfully harnessed the Arab revolt to bring about the downfall of the Ottoman Empire they were illustrating opposite aspects of a general principle; that if one wishes to establish a foreign culture or community in a particular location one must have the cooperation or at least the acquiescence of the local residents. And so it is, as will be shown below, in the formation of secondary tumour colonies in distant organs by disseminating tumours, a process first termed metastasis by Recamier [1] in 1829. Metastatic behaviour confers upon the community of tumour cells able to un-

leash it from their genetic complement the potential to survive excision of the primary tumour. It constitutes a major continuing problem in the clinical management of cancer and the molecular mechanisms involved represent some of the most intriguing and challenging questions in m o d e r n tumour biology. Metastasis is a unique behaviour pattern quite distinct from general tumorigenicity, as can be shown by the inability of some undeniably tumorigenic cell populations [2,3] or cell lines [4-6] to form tumour colonies after blood-borne dissemination even if the cells are inoculated directly into blood vessels. This is corroborated separately by numerous investigations [7-10] which have now shown convincingly that there is considerable di-

0304-419X/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

228 versity between different cloned tumour cell populations even within a single neoplasm with regard to metastatic capability, some being totally ineffective, and that similar diversity exists among populations of naturally occurring tumours of a given organ [2,3,11-13]. There is therefore little room for doubt that metastatic capability is determined by intrinsic properties of the tumour cell, separate and additional to those responsible for its tumorigenicity. Non-neoplastic cells do .,not make progressively growing colonies after subcutaneous or intravenous inoculation [3], so the process of metastasis requires an initial tumourigenic capability. This is not to say that non-neoplastic cells cannot, in some circumstances, disseminate and survive in ectopic sites. Lymphocytes, polymorphs and monocytes/macrophages for instance, after dispersal in the blood, enter or travel through many tissues. Endometriosis, a condition in which endometrium seeds the pelvic cavity [14,15], and the presence of trophoblastic cells in the blood in pregnancy [16] are other possible examples, but in metastasis the end result is unlimited growth and destruction of adjacent normal tissue by multiple colonies of disorganised cells. Thus, some cell traffic may occur in normal subjects, but go largely unrecognised because the elements concerned do not multiply to form focal aggregates, where they come to rest (see Tarin [17] for further discussion). The process of metastasis involves local invasion and destruction of intercellular matrix including collagen, intravasation into blood vessels, lymphatics or other channels of transport (for example, the subarachnoid space), survival in the jetstream of the blood and of impaction in the next capillary network, extravasation out of the vessel, growth in the new location and destruction of indigenous cells as well as coercion ~)f the local tissues to provide a fibro-vascular stroma. Ability to survive in the alien metabolic environment of a different organ is also an essential requirement. Success in the metastatic process requires tumour cells to be able to accomplish all these steps in the right seque~ace and, as cellular behaviour is governed by the prevailing pattern of g ene expression, the coordinate or at least the concomitant malfunction of genes giving a selective advantage in each step of the metastatic process, is required. Failure to achieve any of these steps renders the

cells generally unfit to metastasise and aborts the whole sequence. II. Review of data from human autopsy studies

The foregoing account has been concerned with the tumour cell itself but metastasis, being a kinetic event in a living organism, must also involve interplay between the tumour cells and the anatomical and physiological constitution of the host. Soon after it became recognised that malignant tumours could generate secondary tumour growths in distant sites, observers began to consider whether dissemination was achieved by the release of a soluble agent or an infectious organism in the body or by migration of cells composing the tumour. This issue was decisively settled when advances in optical physics provided reliable compound microscopes. These revealed tumour cells free in the blood and lymph and impacted in the capillaries of various organs as well as in the early stages of exit from the vessels and of growth in the solid tissue of various organs. Thus, local vascular anatomy was quickly recognised as one important host factor affecting metastatic tumour spread, but others soon followed: the histological resemblance of such deposits to the tumour from which they originated is often used in clinical practice to deduce the site of the occult primary lesion. The distribution of these secondary deposits in the body is also sometimes helpful in such circumstances because it is well known from numerous autopsy series that metastases are not randomly scattered and that patterns of organ colonisation bear a strong relationship to the site and histological type of the original tumour. For example, Paget [18] in 1889 reported, on the basis of a study of 735 autopsies of patients with carcinoma of the breast, that secondary deposits of this tumour type occur predominantly in the bones, lungs, brain and liver and rarely in other sites. Subsequent autopsy studies have repeatedly corroborated these observations and extended the concept of patterns of metastatic spread to a variety of other tumour types [17,19,20]. Paget [18] reasoned from his observations that while 'tumour cells were scattered in all directions', like seeds on the wind, they could only grow to form secondary tumours if they came to rest in congenial sites.

229 Restated in modern terms, Paget's conclusions were that for metastasis to occur it is necessary for the tumour to contain cells which have the intrinsic properties to accomplish the process and for such cells to land in organs with suitable microenvironmerits permitting their growth. This interpretation came to be known as the 'seed and soil' hypothesis and considerable evidence now exists from the autopsy studies (referred to above) and experimental investigations (see below) to support it. The hypothesis was, however, contested by Ewing [21], Coman [22] and others [23-26] who contended that the major factor determining the acknowledged patterning of metastatic distribution was the pathway of drainage of blood and lymph from the site of the primary tumour (the so-called 'mechanical' theory). Implicit in this interpretation was the belief that the tumour calls did not form seedling metastases everywhere because they never reached the systemic arterial circulation on account of being trapped in local venous plexuses and capillary networks. As will be shown below, cells from many types of tumours can and do enter the systemic circulation soon after being released into the blood and lymphatics and, in any case, recent studies involving direct release of tumour cells into the aorta [27] or into vessels supplying individual organs [12,13,28] have demonstrated that cells from a tumour known to be capable of haematogenous metastasis do not form secondary tumours everywhere they lodge. The theory attributing distribution of metastases to pathways of venous drainage also fails to account satisfactorily for several known patterns of spread such as the predeliction of carcinomas of the bronchus to form metastases in the adrenals and in the cerebellum. Efforts to accommodate these observations within a unified explanation based on venous or lymphatic drainage have included invoking the suggestion that, under certain circumstances, retrograde flow of blood or lymph carries the tumour cells to the si.te in ques: tion [23,29]. Although this might apply in certain specific circumstances such as the spread of prostatic carcinoma to the vertebrae via Batson's venous plexus, even slight acquaintance with anatomy makes it clear that this explanation is implausible for explaining most selective patterns of spread such as, for instance, those of bronchial

carcinomas to the brain, especially the cerebellum, or of ocular melanomas to the liver and lungs. Recently, a modification of the proximal capillary network entrapment (i.e., 'mechanical') theory has been proposed. This postulates that tumour cells carried by the venous blood or the lymphatics, after being trapped in the capillaries of the next organ they encounter (usually the lungs, liver or vertebrae), grow there to form secondary turnouts and that this organ then acts as a 'generalising site' for further dissemination of the tumour (Refs. 24,25,30; see also Willis [19] p. 182). Although it cannot be denied that such 'metastasis from metastases' can occur (see Ref. 31), both personal experience and the study of published autopsy series indicates that the 'generalising site' hypothesis does not provide a satisfactory unifying explanation for many of the observed patterns of metastatic spread. For example, autopsies on patients with breast carcinoma not infrequently reveal diffuse skeletal metastases without any detectable pulmonary or hepatic metastases. Even among patients who have pulmonary metastases from breast carcinoma, the distribution of metastases in other organs is not random or ubiquitous, bone being most commonly involved and many organs being consistently spared. As the bone metastases result from seeding via the arterial circulation other organs must also have been seeded and failure of cells to grow there must be due to additional factors. IlL Conclusions from human autopsy data

Evaluation of the information from the numerous series of autopsies on patients with disseminated cancer indicates that tumours do have preferential patterns of metastatic colonisation related to their organ of origin and histological type. It also suggests that none of the above hypotheses accounting for these preferential patterns of colonisation satisfactorily accounts for all the observations. The frequency of metastasis in the lungs, liver and vertebral colunm suggests that sieving of tumour cells in organs which drain large venous catchment areas can influence metastasis distribution patterns of certain types of tumours, but mechanical entrapment alone does not satisfactorily explain all metastasis localisation phe-

230 nomena; nor does the possibility of 'generalisation' from metastases in an organ draining blood from the affected primary site, although it is highly likely that this mechanism is contributory to further dissemination in many circumstances. Equally, the evidence demonstrating that the microenvironments in different organ types influence metastatic distribution (see below) does not refute the possibility that mechanical factors can exert an effect on distribution patterns of metastases fl;om some tumour types. On the basis of pure observation alone, it is not possible to determine the relative importance of these three real or potential influences on distribution which are not necessarily mutually exclusive. Therefore, controversy has continued between those who consider that the human autopsy data support the mechanical entrapment viewpoint of Ewing [1], Coman [22] and, more recently, Weiss [26], and those who interpret them as more satisfyingly explained by the 'seed and soil' hypothesis of Paget. The issue is not trivial because understanding of the mechanisms restricting metastasis formation in some organs in individuals in whom metastasis-competent cells are known to have entered the arterial circulation is likely to have longrange implications for the understanding and medical management of the metastatic process in general. Such further understanding can only be achieved by experimental analysis, the current status of which is reviewed below.

IV. Experimental analysis of mechanisms influencing distribution of tumour metastases IV-A. Distribution of tumour cells released intravascularly The first reliable studies on this topic were performed by Fidler [32] when he introduced the use of the isotopicaUy-labeUed thymidine analogue 125I-Urd for labelling tumour cells in tracking experiments. As the analogue is not conserved or reutilised after degradation of D N A following cell death, it provides more accurate information on cell distribution after inoculation than previous studies with other isotopes. Using B16 melanoma cells it was found that after 24 h only about 1.5% of the inoculum had survived but that bound label

(i.e., cells) entered the systemic circulation and could be detected in all organs within minutes after injection. In addition, Potter et al. [33] using primary murine mammary tumour cells labelled with fluorescein isothiocyanate demonstrated, by direct visualisation in frozen sections examined in a UV microscope, that whole viable tumour cells were present in all organs within 15 min of intravenous inoculation into mice. The same was confirmed with ceils from Lucke carcinomas injected into their natural hosts, Leopard frogs (Juacaba et al., unpublished data). Although it cannot be proved that tumour cells shed into the blood always enter the arterial circulation and reach all organs, these findings with ceils from naturally occurring and serially propagated tumours in widely different types of animal studied by various independent research groups indicate that transit of tumour cells through the lungs to recirculate is not uncommon, at least in the species studied. Moreover, the work of Juacaba et al. [27] demonstrated that arterially inoculated mammary tumour cells could traverse peripheral capillary beds and form metastases downstream in the lungs, indicating that they were not damaged in passing through the capillary network. It is possible that some tumours may be composed of cells which are not easily deformed and therefore lodge predominantly in the first capillary network encountered. Zeidman [34] observed partial retention of V2 carcinoma cells in capillary networks in time-lapse cinematographic studies, and this could obviously significantly affect the distribution of their metastases. However, from the studies already reviewed above it can be seen that even tumours composed of cells which can traverse capillary beds have selective patterns of colonisation. Once it is accepted that mixed populations of metastasiscompetent and incompetent cells from a heterogeneous tumour are scattered more or less evenly throughout the body, after mixing in the bloodstream, it follows that in.dividual organs may be able to inhibit metastasis formation by competent cells lodging within them (see below). I V-B. Observations on patients infused intravenously with malignant cells Some patients with inoperable abdominal cancer develop recurrent effusions in the peritoneal cavity

231 which cause distension and pain. Traditionally, this was treated by removal of the fluid through the abdominal wall with a needle (a procedure known as paracentesis) but this caused severe depletion of proteins and electrolytes resulting in rapid debilitation and cachexia. Recently however, it has been found that several such patients substantially benefit from transferring the fluid back to the circulation via a plastic tube, inserted subcutaneously, connecting the peritoneal cavity to the jugular vein (a peritoneo-venous shunt) [35]. The technique allows discomfort to be relieved without causing metabolic disturbances. However, since no filter can be interposed without rapidly blocking the flow, large numbers of tumour cells are necessarily infused directly into the circulation. Initially, there was considerable anxiety over whether this might result in massive and overwhelming metastatic turnout growth leading to accelerated decline and death of the patients. Clinical observations on such patients did not substantiate these fears and later autopsy studies confirmed that metastasis is not an inevitable consequence of discharging large numbers of tumour cells into the blood nor a ubiquitous occurrence in the body even when colonies form in some organs [12,13,36]. In a detailed autopsy study of 18 patients treated by this technique, only half were found to have developed haematogenous metastases, the remainder being completely free of such lesions, some despite long survival. Even when metastases did form they were small and clinically asymptomatic and the technique is therefore beneficial without being hazardous. It also has the important byproduct that it provides opportunities of unrivalled power and relevance for the study of mechanisms underlying tumour metastasis in humans. In the nine patients with evidence of haematogenous spread, it was found that metastases were forming in some organs but not in others. Indeed, in some the lungs were not colonised, even though they are the first site where sieving effects due to capillary beds are encountered by tumour cells released intravenously, and it can therefore be concluded that tumours cannot colonise all sites in which substantial numbers of viable tumour cells lodge. This experimentally confirms, in living human subjects, Paget's deduction, based on autopsy

data, and the findings demolish the contention of Ewing [21] (cited in Ref. 22) and others, that the distribution of metastases is solely determined by the anatomy of vascular and lymphatic drainage from the site of the primary tumour. Even so, it remains necessary to realise that the sieving effects of capillary beds can reduce the tumour cell burden entering the arterial circulation and thus have some influence on whether metastases form downstream. Among patients with shunts, some had no pulmonary deposits [12] yet still demonstrated preferential colonisation of some organs via the systemic arterial pathway and hence failure to colonise others. These patterns of metastasis were not relatable to blood flow through the organs concerned nor to proportions of tumour cells arriving in various sites (which can be deduced from the proportion of cardiac output distribution to each organ) and varied with different tumour types and between different individuals with the same types of tumours. IV-C. Studies in animals and in vitro

The findings in humans are therefore in direct agreement with our previous studies on naturally occurring murine mammary tumours [28] and those of other workers such as Hart and Fidler [37] on the B16 melanoma, in which the mode of inoculation of the tumour cells intravenously was directly homologous. In the experiments of Tarin and Price [28] cells from each of a panel of 23 separate spontaneous murine mammary tumours were inoculated by each of four different routes into separate batches of animals and it was found that the individual primary tumours differed in their capability to colonise the lungs, liver, peritoneal cavity or the subcutaneous tissue. Moreover, several tumours could colonise one or more sites but consistently failed to colonise others. Such failure was not the result of non-specific hostility of a site to mammary tumour cells because other tumours in this batch succeeded in growing in that location. It is concluded that colonisation capability after intravascular or intracoelomic dispersal of tumour calls is not the same as general turnout transplantability and that the local microenvironment in the site of tumour call lodgement can modulate the intrinsic colomsation capability of tumour cells.

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Similar conclusions flow from Hart and Fidler's [37] findings, that the predeliction of B16 melanoma cells to form deposits in the lungs and the ovaries after intravenous inoculation is retained, even when these tissues are transplanted to abnormal sites. Verification of this interpretation, that the patterns of metastatic tumour colonisation are at least partially determined by microenvironmental conditions in various organs, requires demonstration of the mechanisms by which such effects could be mediated and recent experiments in this laboratory have been directed towards this aim. Colonies of C 3 H / A ~ mice carrying the mammary tumour virus have a high incidence of mammary tumours, 30% of which spontaneously metastasise to the lungs [3]. About half of the tumours can establish secondary deposits after intravenous injection and again such seedlings are found almost exclusively in the lungs. If injected via the aorta, the tumour ceils still show a strong predeliction for forming pulmonary deposits even after traversing the peripheral capillary beds and a few tumours can additionally colonise the adrenals, ovaries, kidneys and skeletal muscle but it is extremely rare to find deposits anywhere else [27]. As mentioned above, Potter et al. [33] found that fluorescein isothiocyanate-labelled cells from these same tumours could be detected in quantity in all organs examined within 15 rain, regardless of the route of intravascular inoculation. These tumours were therefore selected as suitable for investigation of the mechanisms by which microenvironmental effects of various organs on metastasis formation could be mediated. It was found [38] that culture of lung fragments with mammary tumour cells could promote the attachment and increase the proportion of cells surviving in primary culture from some of the mammary turnoUts. Further experiments on a panel of 52 separate naturally occurring tumours showed that the effects could be exerted by cell-free culture medium conditioned by lung fragments and that medium conditioned by liver and other organs rarely colonised by mouse mammary tumours consistently diminished survival of the tumour cells in the flask. Conditioned media from kidneys and ovaries, which occasionally harbour tumours after arterial inoculation of cell suspen-

sions, were found to promote survival and attachment of cells from some of the tumours. In contrast, media conditioned by normal organs inhibited or killed normal lactating mammary epithelial cells and a variety of other non-neoplastic cell types so far tested. When lung fragments were reimplanted in vivo after co-culture with mammary tumour cells, a high proportion formed tumours which subsequently metastasised, whereas co-cultured fragments from other organs rarely resulted in implant-derived tumours [39]. The findings are therefore compatible with the hypothesis that normal organs can usually suppress the formation of tumour metastases and that tumours which succeed in establishing metastases have evolved means of escaping the inhibitory effects of organs in which the deposits are found. There is need for caution, however, in interpreting the results because they relate only to a single type of spontaneous tumour tested with conditioned media from a limited range of organs. If confirmed in separate experiments with other tumour types, these conclusions have the implication that t u m o u r cells, even of high metastatic potential in some sites, are not invincible everywhere and remain vulnerable to as yet unknown control mechanisms naturally operating in the body. The fact that soluble substances of so far unknown composition diffusing out of organs in vitro have been demonstrated to have some effects on cells does not necessarily indicate that this is the natural or only mode of interaction between the tumour cells and their environment and other mechanisms such as direct cell-to-cell contact will need to be investigated. Interactions between different cell populations cooperating in the formation of organs in embryonic life and in the maintenance of their histological organisation in adults have been shown by experiment to be mediated by a variety of mechanisms depending on the circumstances and on the organs or species involved [40-42]. Some interactions require the transfer of soluble molecules between the interacting populations, whereas others require direct cell-to-cell contact. It would therefore not be surprising if wide generalisations and unifying explanations do not apply to the mechanisms underlying the disorganisation and disorderly behaviour which are characteristic of turnouts. It has, for instance, re-

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cently been suggested [43,44] that organ-associated N K cell activity could ~ccount for the success or failure of metastasis and even the distribution of deposits formed by B16 melanoma cells or Lewis lung carcinoma cells injected intravenously. Also, Nicolson et al. [45] have reported data indicating that circulating tumour cells may preferentially adhere to the endothelium of certain organs and that this may be a further factor influencing patterns of metastasis. In their experiments, antibodies to cell surface constituents blocked hepatic localisation of intravenously inoculated RAW 117 turnout cells and of metastases in this organ. V. Selectivity and stability of the metastatic process Separate studies of the behaviour of these naturally occurring mammary tumours in vivo have recently [11] provided a different perspective on the metastatic process. When taken in conjunction with similar information from patients treated with peritoneo-venous shunts, they provide an overview of metastasis as a stepwise phenomenon in which events influencing distribution of deposits constitute later acting elements. Price et al. [11] compared the lung colonisation capability of each of 77 autochthonous murine mammary tumours with its own spontaneous metastatic behaviour. 24 of the neoplasms had spontaneously metastasised to the lungs of the tumour bearer. It was found that there was no general correspondence between spontaneous and induced (experimental) metastatic capability, indicating that ability to exit from the turnout does not guarantee ability to colonise and that the reverse also cannot be taken for granted. Interpretation of the findings in terms of ancillary dose-response studies reported in the same paper indicated that escape from the primary tumour is an active process effected by a select population with special properties. Blood bioassay and time-cour~ studies on pulmonary deposit formation in these animals indicated that shedding of metastatic cells occurred early in mammary tumour development and, surprisingly, that large tumours were apparently no longer shedding. It was also found by transplantation of puln~onary metastases back into the mammary fatpads of fresh recipients that the constituent cells were nearly always tumourigenic (95%) but not necessarily able to

recapitulate the metastatic process (35%). This suggests that the progeny of cells which have already shown their fitness for the metastatic process do not necessarily all inherit or retain this capability, although repeated selection of metastases and reinoculation of their cells [46] can eventually lead to the development of cell lines which have high metastatic potency. Recent observations on serially passaged cloned cell lines from transplantable tumours also indicate that metastatic capability is inherently unstable and that the instability is greater in populations of high metastatic capability than in those with low metastatic performance. Currently availa, hie evidence therefore suggests that the pathway to metastatic behaviour is a 'two-way street', some cells becoming more potent and others less so. The basis for this instability has been explored by Cifone and Fidler [47] who reported evidence that the genetic mutation frequency with respect to dominant drug resistance markers was higher amongst clones of high metastatic capability than among o n e s with low capability. Ling and his associates [48] also provided evidence based on genetic fluctuation analysis experiments favouring a mechanism based on mutation for the genesis of metastatic heterogeneity in neoplastic cell populations. Recently, however, Frost and co-workers [6,49] proposed that heritable phenotypic diversity within malignant tumours can arise from epigenetic mechanisms acting via effects on DNA methylation. Olsson and Forchhammer [50] have provided evidence that exposure to the demethylation agent 5-azacytidine can lead to modulation of metastatic capability. They reported changes induced by this agent from non-metastatic to metastatic phenotype and also changes in the reverse direction in some cell lines which had previously been stable for up to 2 years passaging in vitro. All of these research groups agree that genetic and epigenetic mechanisms for generation of instability are not necessarily mutually exclusive. Evidence obtained by Poste and colleagues [10,51] working with the B16 melanoma indicates that this instability in metastatic potency increases if individual cells are cloned and isolated from others within the same turnout and is stabilised by recombination of isolated clones. This suggests that short range cell interactions in the original

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tumour limit the generation of new phenotypes but that as the cells shower out from their primary site their progeny in metastases generate new diversity with regard to many properties including metastatic behaviour [51] and probably drug resistance and radiosensitivity. It can therefore easily be appreciated that both the task of therapy and of correlating individual metabolic properties of tumour cells such as their surface glycoprotein composition [52,53] or enzyme secretion patterns [54-57] with metastatic performance is extremely complex because of the intrinsic heterogeneity of the sample population and its ever-changing nature. On reflection, it seems likely that individual cellular biochemical properties of tumour cells are the end-product of the prevailing pattern of gene activation and are, in this sense, more epiphenomena than directly causal elements. It therefore seems logical that the molecular mechanisms underlying the multifactorial process of metastasis are driven from deranged activity within the cell nucleus, which appears to be influenced by microenvironments in various organs, and current developments in research on gene regulation have provided the means with which to direct our attention to the centre of this extremely interesting and complex field.

Acknowledgements This work was supported by the Cancer Research Campaign of Great Britain. I wish to thank all clinical and scientific colleagues who have collaborated with me in conducting this work. It is also a pleasure to thank Mrs. P. Messer for help in coordinating clinical and scientific elements of the work and in preparing the manuscript.

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