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Metastatic phenotype of a new stable human melanoma substrain (Mel-7m) is described. After grafting into beige/nude mice, these cells spontaneously metastasize into the lungs in 84.2% of cases. The strain can be used in experimental oncology.

By 1994, the number of cases of prostate cancer diagnosed will increase by 50% over a 2-year period. Treatment of all cases diagnosed decrease prostate cancer mortality; however, not all of these cases are destined to cause symptoms or impact adversely on the quality of life of the patient. For the latter patients, a deferred approach is appropriate. Selecting which patient requires treatment, and if so, which treatment to use remains an area of increasing controversy. For those with tumors outside of the gland, improving local control rates and defining metastatic risk are of importance. In this regard, significant advances in our understanding of the biology of prostatic cancers have been made, and some of the specific genetic abnormalities associated with the metastatic phenotype defined. For those with established metastases, improving the results with standard hormonal therapies is an area of active investigation. Selected patients who progress while using androgen-ablation remain sensitive to second-line therapies such as flutamide withdrawal or other hormonal treatments. A new definition of so-called hormone-independent disease is defined. Ultimately, more effective therapies aimed at hormone-refractory cells will be required to improve survival.

Parallel in vitro exposure to 5-azacytidine or to ethyl methanesulfonate either significantly reduced or had no major impact on the experimental metastasis capacity of different clones of the human prostatic carcinoma subline 1-LN-PC-3-1A. The impact of these agents on metastatic capacity varied (1) among different clones and (2) between clones and the uncloned parental line. Analysis of the process by which these agents modify the metastatic phenotype of human tumor cells may offer insight into the process of phenotypic diversification.

We have studied several metastatic variant cell lines derived from a common clonal origin and their transformed and untransformed parental cell lines. A number of in vitro characteristics were examined for each tumor line and these properties were correlated with the ability of the tumor cells to form pulmonary nodules in an experimental metastasis assay. Direct correlations with metastatic behavior in the lung colony assay were found to exist with the amount of cell-bound Concanavalin A and the procoagulant activities of cell lysates. In vitro parameters that did not correlate with the metastatic phenotype were: population doubling times in culture, saturation density achieved in culture, the number of colony-forming cells shed from confluent cultures, rates of cellular attachment to homotypic or heterotypic cell monolayers, plasminogen-activator production and procoagulant activity produced in serum-free conditioned medium.

SUMMARY Abercrombie and his colleagues have accumulated evidence that changes in the heterotypic contact-inhibition response are largely responsible for the invasiveness of cells, at least in culture. We have identified a 37 000 Mr protein on the surface of mouse fibrosarcoma cells that is involved in their in vitro invasion. Blocking this protein with specific antibodies inhibits the invasion of chicken heart fibroblasts by the tumour cells and normal heterotypic contact inhibition is restored. These results are presented in the general framework of metastatic mechanisms and we review a selection of more recent studies aimed at describing the metastatic phenotype in molecular terms.

Identification of the cellular and subcellular alterations responsible for the metastatic behavior of malignant tumor cells and development of reliable screening programs for detecting new therapeutic agents for improved treatment of metastatic disease both depend crucially on the availability of experimental systems that can serve as relevant models of human cancer. Recent advances in our understanding of the pathogenesis of cancer metastasis have raised serious doubts about the usefulness of many of the experimental approaches that have long been used in the study of metastasis. Recent findings showing that metastases are caused by specific subpopulations of metastatic tumor cells, and that not all cells in a malignant primary tumor possess metastatic properties, are of profound importance for experimental efforts to understand the mechanism of metastasis and to develop effective therapies for metastatic disease. Heterogeneity in the expression of the metastatic phenotype among cells from the same tumor means that the traditional, and widely used, approach of analyzing primary tumors and cultured cell lines containing multiple, phenotypically heterogeneous, subpopulations of cells may provide little or no insight into the properties of the metastatic subpopulations, particularly if they represent only a minor fraction of the entire population. Similarly, the practice of screening potential therapeutic modalities for their ability to reduce the mass and/or growth rate of a primary tumor may be inadequate in predicting the responsiveness of metastatic lesions. Solution of these problems requires that new methods must be devised to isolate and characterize the specific subpopulations of tumor cells endowed with metastatic potential. In addition, knowledge of how the extraordinary phenotypic diversity found in tumor cell subpopulations from the same tumor is generated and how subpopulation diversity is regulated during progressive growth of both the primary tumor and its metastases are of fundamental importance if we are to design meaningful experimental systems for studying the metastatic process. This article reviews our current understanding of these complex issues and their implications for the experimental analysis of the malignant phenotype. The merits and shortcomings of different experimental systems are discussed in detail together with the identification of areas in which new experimental strategies and models are now needed.

The mechanisms by which activated Ras accelerates malignant transformation of normal cells are not fully understood. Here, we characterized the role and molecular mechanism of γ-catenin in regulating the malignant phenotype of Rat2 cells induced by codon 12-mutant K-Ras (K-Ras12V). Suppression of γ-catenin signaling by K-Ras12V was an early event and played a crucial role in promoting the acquisition of a highly metastatic phenotype of Rat2 cells. Notably, the gene encoding histone deacetylase 4 (HDAC4) was identified as a target of γ-catenin during this process. The transcription factor, lymphoid enhancer-binding factor-1 (Lef1), was involved in the modulation of HDAC4 transcription, and disruption of this pathway was a key event in promoting the invasion and migration of K-Ras12V-transduced Rat2 cells. Thus, our findings extend the range of targets for the development of new drugs for the therapy of oncogenic K-Ras-driven cancer.

Metastasis is one of the major problems if not the major problem in the diagnosis, staging and treatment of cancer. Although over the last few years, there have been major advances in our understanding of cancer, conceptual advances have often led to the recognition of many problems, where previously only comparatively few were recognized. This is particularly true for metastasis, where we are currently at the stage of recognizing at least some of the basic problems, and hopefully can begin to arrive at some of their solutions.

The acquisition of the ability to metastasize is the ultimate and most deadly level of the tumorigenic progression. Metastatic cells are able to dissociate from the primary tumor, invade through the surrounding tissue and endothelial layer into the bloodstream, migrate through the bloodstream to the site of metastasis and then reinvade and proliferate to generate a new tumor lesion (Poste and Fidler 1980). Throughout this process the metastatic cell assumes a number of different morphological configurations varying from densely packed within the tumor mass, deformed and elongated in invasion, and free-floating in the bloodstream. During each of these stages it must acquire distinct functional capabilities requiring the expression of distinct molecular entities.

Many interesting results were presented. A very interesting observation was reported by Eisenbach et al. (96), who found that metastatic mouse tumor cells express little or no major histocompatibility complex (MHC) class I H-2K molecules. This may allow these cells to avoid the expression of a transplantation antigen that could induce the rejection of metastatic cells. In the course of studying this phenomenon, they made the remarkable observation that the fos oncogene promotes the expression of H-2K.

The cell biology of cancer metastasis has been studied in considerable detail over the past 10 years, yielding many important new insights into the patho genesis of this important and life-threatening disease (reviewed by Poste and Fidler, 1980; Poste, 1982; Nicolson and Milas, 1984). By contrast, efforts at understanding the underlying biochemistry and molecular biology of this process have been limited and largely unsuccessful. A much greater understanding of the molecular mechanisms responsible for the expression of the metastatic phenotype will be required if we are to develop novel and productive approaches to the therapy of metastatic disease by any approach other than random and semiempirical screening, which hitherto has made little impact in developing anticancer drugs against the more common solid malignancies of man. In the absence of any obvious biochemical target to guide the direction of such an endeavor, it is becoming increasingly clear that the process of identifying molecular properties that correlate either qualitatively or quantitatively with metastatic potential will be a prolonged and challenging task and with no guarantee that any correlation, once identified, will be causal or will offer a suitably “unique” and pharmacologically exploitable target for the design of new therapeutic agents directed against it.

Metastasis is an apparently complex process in which cells are required to perform a number of functions as they move from a primary tumor to establish new tumors at distant sites. One of the current challenges to our understanding of this process is to determine how molecular alterations (either genetic or epigenetic) in tumor cells contribute to the apparently separate functions performed by metastatic cells. The difficulty with this approach has been to find assays for metastatic properties that are appropriate to molecular studies. We have developed an assay in the embryonic chick that lends itself readily to molecular genetic analyses of metastatic properties of cells (1). In this review, this assay and the advantages that it offers to molecular studies will be discussed. We have used this assay to begin an examination of genes that can contribute to the metastatic phenotype; some of these studies will also be described here.

Despite the evidence that neoplasms begin with the oncogenic transformation of a single normal cell they virtually always become genotypically and phenotypically heterogeneous by the time they are clinically detectable (Nowell 1976). This is thought to occur through the continuous emergence of new variant subclones as a result of random or induced genetic mutations combined with selection of the more aggressive of these new subpopulations (Nowell 1976). As a result, tumor progression can be viewed as a Darwinian-like microevolutionary process. There is evidence that variant cell subpopulations having competence for metastasis or manifesting certain kinds of drug resistance arise as a result of this process (Nowell 1976). With respect to the metastatic phenotype, a prediction of this model is that cells recovered from metastases would on average manifest a greater metastatic ability than tumor cells recovered from the corresponding primary tumor. This prediction was fulfilled, as first reported by Fidler (1973). As a result of this pioneering study it has become routine to compare cells of primary tumors and their metastases as a means of determining the cellular factors which govern expression of metastatic behavior.

Summary We have reported that the synthesis of tropomyosin isoform 2 (TM2) is significantly suppressed in high-metastatic Lewis lung carcinoma cells compared with that in low-metastatic cells. In order to examine whether the change is also observed in other high-metastatic tumor cells, we compared the pattern of expressions of TM isoforms between NIH3T3 cells and v-Ha- ras -transformed NIH3T3 (pH1–3) cells, the latter of which was highly metastatic when injected into BALB/c nude mice. The results showed that the expression of TM2 was less in pH1–3 cells than in NIH3T3 cells, suggesting that the suppression of TM2 synthesis is involved in the expression of metastatic phenotype.

The issue of whether metastases result from the random survival of cells released from a primary tumor or from the selective growth of specialized tumor subpopulations endowed with metastatic properties is important to our understanding of the metastatic process and to the development of therapeutic modalities against metastatic disease. We have found that the tumor cells populating spontaneous metastases are more metastatic than the cells populating the parent neoplasm, clearly indicating that metastasis is selective and not random. The selective nature of metastasis is a consistent observation, however, only when tumor cells are obtained from spontaneous metastases from mice bearing heterogenous, poorly metastatic tumors. Tumor cells from spontaneous metastases from mice bearing tumors that have been selected for metastatic potential or that are homogeneous (cloned) do not differ significantly in metastatic potential from tumor cells populating the parent tumor. Thus, under some conditions the process of metastasis can appear random. Although tumor cells from different individual metastases may be homogeneous with regard to a metastatic phenotype, they may be heterogeneous with regard to their sensitivity to chemotherapeutic agents. Thus, although metastasis selects for metastatic variants, resulting in the population of metastatic foci with tumor cells endowed with metastatic properties, it does not appear to select for phenotypes irrelevant to the process of metastasis such as sensitivity to therapeutic agents.

The human prostate tumor subline 1-LN-PC-3-1A (1-LN) is reproducibly metastatic in adult athymic nude mice. Cells surviving a brief in vitro exposure to ethyl methanesulfonate (EMS) exhibited a profound decrease in capacity for experimental lung metastasis in nude mice. Thirty days after EMS treatment, 1 X 10(6) uncloned EMS-treated 1-LN cells (1-LN-EMS-10) were injected IV into groups of 6 to 8-week-old male athymic nude mice (BALB/cAnBOM). A median of 8.5 colonies/lung was observed among 20 1-LN-EMS-10-injected mice, which was significantly different from the median of 51 colonies/lung produced among 14 1-LN-injected mice (P = 0.0002). This altered phenotype remained stable during 150 days of continuous culture. However, the 1-LN-EMS-10 cells were tumorigenic in 10/10 nude mice injected SC. Single lung tumor colonies recovered from 1-LN-EMS-10-injected mice and reinjected IV into nude mice produced medians of 32-63 colonies/lung. The altered metastatic phenotype resulting from treatment of 1-LN with EMS was reversed by exposure to a noncytotoxic dose of 5-azacytidine, but unaffected by a second exposure to EMS. Collectively these data demonstrate that the metastatic phenotype of these human tumor cells in athymic nude mice can be heritably altered by in vitro exposure to EMS and 5-azacytidine. Analysis of the mechanisms underlying these phenotypic changes may provide insight into parts of the complex process of tumor cell evolution.

We have previously demonstrated that rat embryo cells transformed by the ras oncogene alone are both tumorigenic and highly metastatic when injected into nude mice. In contrast, rat embryo cells transformed with the ras oncogene and the adenovirus 2 (Ad2) Ela gene are tumorigenic but either fail to metastasize, or exhibit a very low metastatic potential. Here we demonstrate that transfection of the Ad2 Ela gene into several of the ras transformed rat embryo cell lines results in a dramatic reduction in metastatic potential relative to the parental cell line. Transfection of cDNAs for the 12S and 13S Ela transcripts showed that both gene products are capable of reducing the metastatic potential of the ras transformed cell lines, however the 12S cDNA was more effective. This effect is specific to the Ad2 Ela gene as ras transformed cell lines expressing the Ad12 Ela gene or the human N-myc gene maintained their high metastatic potential. We hypothesize that the Ad2 Ela gene may regulate the expression of one or more cellular genes that contribute to the metastatic phenotype.

Altered control of cellular proliferation and altered differentiation represent two important pathways determining the phenotypic properties of tumour cells. Indeed, induction of differentiation as a means of reducing tumour proliferative capability and directing the development of benign from malignant cells has long held promise as an alternative to conventional chemotherapy as a therapeutic approach to neoplastic diseases (Pierce 1967). The induction of differentiation as a means of reducing malignancy and improving survival has been studied most extensively using haematological tumours, particularly the myeloid and erythroid leukaemias of mice, but the phenomenon can be duplicated in some common solid neoplasms (Bennett et al. 1986).

By the time of diagnosis, most malignant neoplasms are heterogeneous and contain numerous subpopulations of cells with diverse biologic characteristics, which include the ability to invade and produce metastasis. The metastatic process is not random. Rather, metastases result from the survival and proliferation of a few specialized metastatic cells that preexist within parent neoplasms. Diversity for the metastatic phenotype may be a consequence of the multicellular origin of a neoplasm, or it may be the result of continuous evolution and progression of tumors either unicellular or multicellular in origin. Metastatic variants, in general, are less phenotypically stable than benign or nonmetastatic clones, and they also exhibit higher rates of spontaneous mutations. Although metastases may have a clonal-unicellular origin, the genetic instability of these cells coupled with host selection pressure can rapidly generate biologic diversity among and within metastases. These data support the concept that tumor evolution and progression toward increased malignancy could be regulated by genetic mechanisms.

It is now a well-worn adage, almost a cliche in cancer research circles, that the process of cancer metastasis is an inefficient process. Primary tumours may release millions of cells into the vascular system and yet a very small proportion, estimated at about 0.01%, can successfully form distant metastases. There has been some debate as to whether cells with metasta-sising ability pre-exist in a neoplasm and hence their dissemination to distant sites is a non-random process or whether, as Weiss (1983) has argued, most cancer cells have the ability to metastasise and that they randomly enter a ‘transient metastatic’ compartment which increases their chances of negotiating the multiple hurdles of the metastatic cascade.

To produce metastasis, malignant tumor cells must be able to complete a sequence of many steps that depend not only on tumor cell properties but also on ability of the tumor cells to interact effectively with host homeostatic mechanisms to avoid destruction. Therefore, it should be possible to isolate clonal populations non- or low metastatic. In a study of K-1735 clones introduced into normal syngeneic hosts, the reasons for the lack of or low ability of metastasis production did indeed differ among different clones. Some clones were identified that were low metastatic in syngeneic C3H/HeN mice because of antigenic characteristics. Others failed to give rise to metastases because they could not survive and grow once arrested in the lung parenchyma. These data suggested that the success of future studies dealing with genetic analysis of the metastatic phenotype could depend on the use of appropriate tumor cell populations.

The cell biology of cancer metastasis has been studied in considerable detail over the last ten years yielding many important new insights into the pathogenesis of this important and life-threatening disease which have been well reviewed in recent years 1–3. In contrast, efforts at understanding the underlying biochemistry and molecular biology of this process have been limited, and largely unsuccessful. A much greater understanding of the molecular mechanisms responsible for the expression of the metastatic phenotype will be required if we are to develop novel and productive approaches to the therapy of metastatic disease by any approach other than random and semi-empirical screening, which hitherto has made little impact in developing anticancer drugs against the more common solid malignancies of man. In the absence of any obvious biochemical target to guide the direction of such an endeavor it is becoming increasingly clear that the process of identifying molecular properties that correlate either qualitatively or quantitatively with metastatic potential will be a lengthy and challenging task and with no guarantee that any correlation, once identified, will be causal or will offer a suitably “unique” and pharmacologically exploitable target for the design of new therapeutic agents directed against it.

Considerable controversy exists as to whether the metastatic phenotype is selective or random in nature, and if selective, whether it is always inherited and expressed in a stable, or unstable, manner. Which of the alternatives is correct has important implications and ramifications for virtually every aspect of the experimental study of metastasis. Based on analogous experimental situations (e.g., drug resistance due to gene amplification) and results from two separate experimental systems obtained in our laboratories, we conclude that the metastatic phenotype is indeed selective in nature, i.e., there are genotypically distinct subpopulations of metastatic variants. However, the ways in which the genetic information relevant to the metastatic phenotype is regulated and expressed will determine the relative stability of the phenotype: both unstable and stable variants can co-exist. We hypothesize that changes in DNA methylation (i.e., of 5-methylcytosine) is one of several possible mechanisms which can account for an unstably expressed phenotype. We also argue that the selection pressure required to isolate the stable variety of variant may be much stronger than what many investigators have previously used and can account for some of the discrepancies in the experimental metastasis literature.

The issue of whether metastases result from the random survival of cells released from the primary tumour or from the selective growth of specialized subpopulations of cells having properties that allow them to complete the metastatic process is important to our understanding of tumour biology1,2. Previous studies have provided indirect evidence that the process of metastasis favours the survival of cells having a metastatic phenotype(s)3–8. Direct evidence that the process is selective would be provided by the demonstration that cells populating spontaneous metastases are, in general, more metastatic than most of the parent tumour cells; some9–11, but not all12,13, previous reports have failed to provide such evidence, suggesting that the process is random. These discrepancies could be due to differences in the biological characteristics of the various tumour systems studied and in the experimental conditions14. In the present study, these variables were minimized by using three metastatic variant cell lines of the B16 melanoma. We report that, even in controlled conditions, the process of metastasis can appear as either selective or random depending on the nature of the initial population. Specifically, metastasis by the unselected, poorly metastatic parent B16 tumour was indeed selective. In contrast, metastasis by previously selected B16 lines appeared to be random.

Background Aside from the stepwise genetic alterations known to underlie cancer cell creation, the microenvironment is known to profoundly influence subsequent tumor development, morphology and metastasis. Invasive cluster formation has been assumed to be dependent on directed migration and a heterogeneous environment - a conclusion derived from complex models of tumor-environment interaction. At the same time, these models have not included the prospect, now supported by a preponderance of evidence, that only a minority of cancer cells may have stem cell capacity. This proves to weigh heavily on the microenvironmental requirements for the display of characteristic tumor growth phenotypes. We show using agent-based modeling that some defining features of tumor growth ascribed to directed migration might also be realized under random migration, and discuss broader implications for cause-and-effect determination in general. Results Considering only the properties of random migration in tumors composed of stem cells and committed cells, we are able to recapitulate a characteristic clustering feature of invasive tumor growth, a property we attribute to "self-metastatic" growth. When the additional influence of directed migrations under chemotactic environments are considered, we find that tumor growth and invasive morphology are supported while the tumor is distant from the source, but are progressively discouraged as the tumor converges about that source. Conclusions We show that invasive clustering can derive from basic kinetic assumptions often neglected in more complex models. While higher-order mechanisms, e.g. directed migration upon chemotactic stimuli, may result in clustering growth morphologies, exclusive attributions of this phenotype to this or other structured microenvironments would be inappropriate, in light of our finding these features are observable in a homogeneous environment. Furthermore, directed migration will result in loss of the invasive phenotype as the tumor approaches the attractor source. Reviewers: This article was reviewed by Mark Little and Glen Webb.

We have developed a simple and reliable method to determine the chemical potential of cholesterol in plasma membranes of living cells in vitro. For a variety of cultured cells, including non-metastatic breast cancer cells, the chemical potential of cholesterol is maintained at a level of about −1.9 kBT per moleculerelative to crystalline cholesterol. But for a metastatic breast cancer cell line, this chemical potential is appreciably greater, at about −0.7 kBT per molecule. In light of these observations and recent reports of cholesterol's role in breast cancer progression we have developed a method to alter and maintain the chemical potential of cholesterol in plasma membranes of cultured cells at any desired level. Through this technique, we lowered the chemical potential of cholesterol in the metastatic cell line to that of the non-metastatic line and found that this significantly reduced the expression levels of several key proteins implicated in breast cancer progression and metastatic spread.