D O R M A N T M E T A S T A S E S - S T U D I E S I N E X P E R I M E N T A L A N I M A L S

PETER ALEXANDER C.R.C. Medical Oncology Unit, University of Southampton, Southampton General Hospital,

Southampton SO9 4 X Y

SUMMARY. Several experimental systems related to tumour cell dormancy are outlined together with some of the possible mechanisms by which the host might maintain tumour foci in such a quiescent state.

T H E recurrence of tumours at distant sites many years following successful removal of the primary lesion has been reported for many types of tumour, being most frequent in patients with breast carcinomas and malignant melanomas (review by Wheelock and Brodovsky, 1982). There are now opportunities to gain knowledge of the mechanisms involved from animal models.

The hypothesis that late recurrences follow on the progressive and relentless growth of a few residual cells, which have either a long mitotic interval or a high loss rate, does not explain adequately much of the human experience and is also not supported by observations in animal models. An alternative point of view is that the dormant lesions are in a stationary state and expansion takes place only after there has been some change which allows net growth. The dormant lesion could be made up of either a cluster of dividing cells in which the rate of cell death equals the rate of proliferation or of non-dividing cells which retain viability for long periods. The precipitating factor which causes a clinically apparent metastasis could, in theory, be either a somatic transformation of the malignant cells or a change in their environment.

In experimental models. the latter mechanism has been reported. I n all of the experimental systems in which dormancy has been established, host factors have been implicated in restraining the residual tumour cells.

T W O TYPES OF ANIMAL MODELS

Dormancy has most frequently been investigated by creating the conditions under which locally implanted tumour cells do not grow. The key experiments in this field are shown in table I.

We have studied dormant metastases (as opposed to locally dormant implants, table I) with transplanted chemically-induced sarcomas and lymphomas using a protocol in which the primary implant and its local draining nodes are surgically removed and the animals kept for a year to detect distant metastases. Many immunogenic sarcomas and lymphomas show a low and frequently zero incidence of metastases in this test system, but. in an

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TABLE I

E.xamples of tumour dormant implants

Induction of dormant state 1. Oestrogen-dependent carcinoma remains latent when

inoculated into normal host but grows on adminis- tration of oestrogen. Latency lasts at least one year (Nobel and Hoover. 1975)

intraperitoneally into immunised recipients. Progressively growing ascitic growth occurs as mice age (Wheelock, 1979)

3. Low numbers of carcinoma cells injected into portal vein; growth in the liver occurs only after repeated laparotomies. (Larger number of tumour cells grow immediately) (Fisher and Fisher, 1959)

2. Many immunogenic leukaemia cells inoculated

Probable mechanism Cells do not divide but remain viable in

absence of hormonal stimulus

Detectable number of dividing leukaemic cells can be found in the peritoneal cavity but there is an equilibrium between proliferation and killing by mononuclear cells

interfered with by trauma State of cells unknown. Restrained growth is

TABLE I1

Post-excision treatment and effect on metastases

Post-excision treatment Effect on metastases None Low incidence of metastases Immunosuppress by:

(i) Prolonged thoracic duct drainage (effect reversed

(ii) Total body irradiation (iii) Cyclosporin A

50-100%) develop distant lymph node and by return of lymphocytes) lung metastases

TABLE 111

Bioassajys for dormant turnour cells

Bioassay Effect Transplant cells from lung or distant lymph nodes intra- Recipients grow tumours at an incidence

peritoneally into immune suppressed syngeneic recipients of 50-100%

immunosuppressed host, many metastasise (Eccles and Alexander, 1974; Eccles, Heckford and Alexander, 1980; Eccles, 1982). The presence of metastases which remain dormant was demonstrated with such immunogenic tumours either by applying immunosuppressive regimens following removal of the primary tumour (Eccles and Alexander, 1975; Eccles et al., table 11) or by a direct bioassay (table 111). In the latter, lungs and distant lymph nodes from rats and mice in which the primary tumour had been removed were transplanted into immunosuppressed recipients in which they gave rise to tumours. In this way the presence of tumour cells was demonstrated in lung and lymph nodes that would have remained tumour free in the original host. Such experiments pursued the following protocol. Tumours were implanted into syngeneic rats or mice. After 2-3 weeks, the primary tumour was surgically excised. Then after a further 1-12 weeks, the animals are immunosuppressed and observed over a further 12 months. The various regimens and results are depicted in tables I1 and 111.

D OR M A N T M E T A S TA S ES 38 1

“ DORMANT ” METASTASES ARE NOT DERIVED FROM A SUB-POPULATION

Eccles et al. (1980) showed using five different tumour lines that the cancer cells in the metastases that had been induced by immunosuppression had the same biological proporties as the cancer cells from the local tumours which grew following an intramuscular implant. The cells derived from the metastases which had been dormant until the animals had been treated with cyclosporin A were indistinguishable from those of the local tumour by the following criteria: (1) Number of cells required for a ‘‘ take ” intramuscularly or subcutaneously (TD50); (2) Growth rate; (3) Immunogenicity; (4) Macrophage content; and ( 5 ) Capacity to metastasise spontaneously in normal (i.e. not immunosuppressed) hosts.

In this connection it is worthwhile noting that similar comparisons were made between tumour cells from the primary and distant metastases that had arisen without immunosuppression (i.e. in normal hosts) (Alexander, 198 1, 1982). In no case did we find any evidence that the metastases were derived from sub-populations of cells that were inherently more metastatic. In general, the metastatic potential, assessed by implanting tumours intramuscularly, of cells derived from metastases was the same as that of the parent tumour. There is no controversy that cancer cell populations can be heterogeneous or that variants with different metastatic properties can be induced. This, however, plays only a minor role in the genesis of metastases. The success of shed cells to grow into metastases is a stochastic, and not a selective process (Alexander, 198 1). It must be emphasised that all other investigators who have approached this question by the same procedure (i.e. comparing directly the cancer cells from a metastases with those from a primary) have reached the same conclusion (Giavazzi et af., 1980; Pang et af., 1981; Milas and Peters, 1983; Weiss, Holmes and Ward, 1983). The only apparent exception is a study by Talmadge and Fidler (1982), but these authors introduced a confounding extraneous factor by testing the cells from metastases after these had been cultured in tlitro.

SHOWING A HERITABLE PHENOTYPE FOR DORMANCY

How DOES THE HOST RESTRAIN THE DORMANT METASTASES?

The possibility cannot be definitely excluded that, in the system studied by us: the dormant metastases are microlesions in which cell proliferation is counterbalanced by cell loss and that immunosuppression perturbs this equilibrium. However, the available evidence favours mechanisms where host factors impose restraint on cell division such that the tumour cell is truly latent. The dormant metastases are clearly refractory to being killed by immunocytes since, after surgical excision of the primary implant, the animals have acquired specific immunity which insures that between lo4 to lo6 tumour cells (the number depending on the tumour used) are killed after being inoculated intramuscularly, subcutaneously or intravenously. Yet the residual tumour cells which lie dormant are clearly not killed by the immunocytes present in rats

382 PETER A L E X A N D E R

immunised against the tumour which causes the dormant cells to be refractory to the immune processes evoked in the host. This may be due to their being in an unusual anatomical situation or to protective processes such as coating by blood clots. It is possible that the factors which render these cells mitotically quiescent also confer resistance to lysis by immunocytes.

A state of reversible quiescence may also be brought about by maturation and, in dormancy, the degree and/or extent of differentiation of the malignant cells may be greater than that which occurs in growing lesions. Escape from dormancy can then be viewed either as a change in the environment which hinders differentiation, and thus reduces cell loss by maturation, or a change which induces true differentiation resulting in progressive growth. Preliminary data on the growth of human acute myeloid leukaemic cells in culture and as xenografts (Palu et al., 1979) has led us to explore the concept that, in some instances, dedifferentiation may be prevented by a diffusible tumour product. As a result, the tumour cells within small lesions differentiate (because the local concentration of the tumour-produced inhibitor of differentiation is too low) whereas in large lesions, differentiation is prevented and rapid tumour growth occurs. Such a hypothesis is operationally indistinguishable from that in which the tumour produces a growth factor in the absence of which the tumour cells do not proliferate. Again, small lesions can be quiescent because the local concentration of a diffusible factor will be too low to initiate mitosis.

ACKNOWLEGEMENTS This investigation was supported by a grant from the Cancer Research Campaign.

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