Successful removal of the primary tumor therefore does not necessarily mean the patient is cured. If and when the occult tumors become clinically detect-.
COMMENTARY New insights into the evolutionary growth of tumors revealed by Southern gel analysis of tumors genetically tagged with plasmid or proviral DNA insertions
R. S. KERBEL*, I. CORNIL and B. KORCZAK Division of Cancer and Cell Biology, Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto, Ontario M5C 1X5 and Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada * Author for correspondence
Introduction: the evolutionary growth of tumors and development of metastatic disease Despite the enormous efforts and advances made in understanding the biology of cancer, few significant advances have been in the cure of the disease (Bailor and Smith, 1986; Cohen and Diamond, 1986). While there are many factors involved in helping to explain the slow pace of progress in clinical treatment, two stand out: the ability of tumors to metastasize and their relative resistance to toxic agents and drugs (Nowell, 1976, 1989). Thus, when a patient first presents with a clinically detectable 'primary' tumor, e.g. a breast cancer, the individual may already have clinically occult (micrometastatic) disease in other organs, such as the bones, brain, or lungs. Successful removal of the primary tumor therefore does not necessarily mean the patient is cured. If and when the occult tumors become clinically detectable some time later, they may be impossible to remove by surgical means. As a result, other treatment protocols may then be initiated such as chemotherapy or hormonal therapy. But these too may ultimately fail, because the tumors are intrinsically resistant to these therapies, or, alternatively the tumors may initially respond because they are partially sensitive, only to reappear later in a much more resistant form. This latter process is thought to be a consequence of acquired drug resistance manifested by the outgrowth of a genetically mutant subpopulation of cells (Skipper, 1983). How and why do these processes come about? A good part of the answer lies in the evolutionary development of tumors - a process known as 'tumor progression'. It is generally acknowledged that most types of tumor, whether of human or experimental animal origin, arise from the neoplastic transformation of a single altered cell (Fialkow, 1979; Woodruff, 1988). The progenitor cell, because it possesses some kind of selective growth advantage, gives rise to a neoplastic clone that cannot be clinically detected before it reaches a mass of about 109 Journal of Cell Science 94, 381-387 (1989) Printed in Great Britain © The Company of Biologists Limited 1989
cells, or about 1 cm in diameter. As this clone expands it gives rise to genetically mutated variant subpopulations in a sequential manner; some of these subclones or subpopulations may overgrow and displace their predecessors. Others may be able to metastasize to distant organs via the body's vasculature and thereby establish secondary satellite tumor growths in certain vital organs or other sites. With respect to the process of metastasis it is often noted in the literature that the metastatically competent subclones that arise during the progressive expansion of the primary tumor mass remain there as a cryptic, minority population (see Kerbel et al. 1988, for a review). Therefore, it has been reasoned that the existence of genes whose expression (or loss of expression) influence metastatic competence, can be revealed by simply comparing 'primary' tumors with their respective distant metastases. This type of approach, however, has frequently failed to reveal such genetic differences, whereas in other cases it has (Kerbel et al. 1988). Such discrepancies could be partly resolved if it could be shown that metastatically competent variant subpopulations have a growth advantage, so that over time they come to dominate the primary tumor itself. Demonstrating this would require experimental approaches to identify and follow the fate of metastatically competent subclones during their progressive growth in vivo. In this article, we shall briefly review the development and application of such an approach and show how it can be used to study a variety of issues relevant to malignant tumor progression, cell-cell interactions, and cell lineage relationships of tumors.
Genetic and biochemical markers of tumor cell clonality A variety of genetic and phenotypic markers have been used to establish the clonal nature of tumors and metastases and to study clonal evolution of tumor growth (see
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Woodruff, 1988, for a review; and Fialkow, 1979; Kerbel et al. 1988). They include chromosome/cytogenetic markers, enzyme polymorphisms, immunoglobulin markers and drug-resistance markers. More recently, endogenous molecular genetic markers (Vogelstein et al. 1989; Kern et al. 1989) such as restriction fragment length polymorphisms (RFLPs) have been employed. A few years ago we (and independently, Talmadge et al. 1987) decided to apply a method that had already been used with considerable success to study cell lineage and cell fate in early and later embryonic development, in haematopoiesis, and in the nervous system (see Price, 1987, for review). The method exploits the random integration of transferred foreign DNA molecules into the genomes of recipient cells as a way of generating large numbers of cells containing unique and identifiable genetic markers, which are identified by Southern blotting. The principles involved have been explained in detail by Price (1987) and ourselves (Kerbel et al. 1988). In brief, because transfected plasmid DNA, or proviral DNA copied from the RNA of retrovirus vectors, integrates in a more or less random fashion, digestion of genomic DNA with, say, a restriction enzyme that cuts outside the integrated DNA will generate unique-sized fragments of DNA incorporating the foreign DNA and host-flanking 5' and 3' DNA in any given transfectant or infectant. This is so because the distance of the nearest upstream or downstream relevant restriction sites flanking the inserted DNA recognized by the restriction endonuclease used to digest the DNA will vary from one cell (transfectant or infectant) to another. Assuming a single copy of the plasmid or proviral DNA is inserted, each clone will contain a unique DNA marker (i.e. restriction fragment of variable length) detectable by Southern blotting using an appropriate hybridization probe. The manner in which we have exploited this to study cell lineages in tumors and the clonal evolution of tumor growth in vivo is schematically summarized in Fig. 1. A plasmid (or retroviral vector) containing a dominant selectable drug resistance marker (e.g. the neo gene) is used to transfect or infect a mouse or human tumor cell population so that a large number of independent clones is obtained. Every clone will have its own unique genetic marker, detectable by Southern blotting using, for example, the neo gene or a fragment of it, as a hybridization probe. If a large number of the clones are pooled, the DNA obtained from such a mixture will present as a faint, broad smear on a Southern blot, since no given clone will exist in a high enough proportion to enable its unique 'clonotypic' genetic signature to be seen. Suppose that such a mixture is then injected into a mouse; the relative clonal composition of primary tumors and any metastases that subsequently emerge in the animal can be determined by Southern blot analysis. For example, if the 'primary' tumor (i.e. the tumor removed from the site of injection) has been overgrown by the progeny of a small number of clones this would be easily detected. Similarly, if metastases are derived from the seeding of organs by single tumor cells, i.e. are clonal growths, as previously shown or suggested by cytogenetic analysis 382
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(Talmadge et al. 1982; Fidler and Talmadge, 1986), this could also be easily determined. Moreover, the lineage relationship of different metastases (located in the same or in different organs) to each other, or to the primary tumor, would be established (Kerbel et al. 1987; Talmadge and Zbar, 1987; Korczaketa/. 1988). So too could the relative rate and extent of clonal selection. In short, the dynamics or developmental nature of clonal evolution. As will be summarized below, we have applied this approach to study lineage and clonal evolution in a mouse mammary carcinoma (called SP1) in syngeneic CBA/j mice, and more recently, a human malignant melanoma (called MeWo) grown in nude mice. The results have revealed new and potentially important insights into tumor progression. In particular a new aspect of metastatic tumor growth — 'clonal dominance' of primary tumours by metastatically competent tumor cell variants TUMOR CELL POPULATION
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Southern Blot Analysis Fig. 1. Schematic representation of the procedure used to study cell lineages during in vivo growth of primary tumors and metastases. A mouse or human tumor cell line is genetically tagged in vitro by random integrations of foreign DNA so that a large number of individual clones is isolated, each bearing its own unique genetic signature (which can be visualized by Southern blotting using an appropriate hybridization probe). The foreign DNA is transferred either by plasmid transfection or retrovirus vector infection. A large number of clones is then pooled, which results in the disappearance of the unique signature associated with any given clone: DNA from such a pooled population will instead present as a faint smear on the gel. The pooled population is used as an inoculum to inject syngeneic or nude mice. Some time later the primary tumors and metastases are removed and analyzed by Southern blotting for their relative clonal compositions and clonal identities. In the scheme shown here, each clone is associated with a single, unique-sized fragment capable of hybridizing with a ;;eo-specific hybridization probe. This assumes a single insertion of a single copy of the «eo-containing plasmid or proviral DNA, and that a restriction enzyme that does not cut within the foreign integrated DNA is used for digestion of genomic DNA. See text for more details.
- has been uncovered, the implications of which are discussed herein, and elsewhere (Kerbel et al. 1989). Clonal dominance of primary tumors by metastatically competent cells: analysis using plasmid-marked tumor cells The SP1 mammary tumor does not normally metastasize from a subcutaneous implantation site (Kerbel et al. 1987). However, the process of calcium phosphatemediated transfection results in as many as 10-20 % of the clones acquiring this phenotype (Kerbel et al. 1987). The metastases are normally found in the lungs. Thus when the plasmid pSV2«eo is used to tag genetically SP1 cells, approximately one out of every 10 or 20 clones may be competent for metastasis. When a population of between 50 and 100 pSV2«eo-transfected SP1 clones was selected in G418 and pooled, the DNA extracted from the cells was found, as expected, to present as a faint smear on Southern blotting after being probed with pSVzweo (Kerbel et al. 1987). This cell mixture was injected into syngeneic CBA/j mice and the primary tumors were removed about 6-7 weeks later along with solitary lung metastases from each animal. When these tumors were analyzed by Southern blotting all were found to essentially comprise the progeny of a single clone (Kerbel et al. 1987); moreover, the identity of the clone was the same from one animal to another, whether it was a primary tumor or a metastasis. Further analysis showed this was not due to an inability of the other injected clones to form tumors. Thus if primary tumors were removed at earlier time points, e.g. 3 weeks after injection, the tumors were found to be populated by a large number of the injected clones (Waghorne et al. 1988). But, remarkably, by 4-5 weeks after injection just a week or two later - dominance of the tumors by the single clone (called neo5) became readily apparent (Waghorne et al. 1988). The results therefore seemed to indicate that a single clone, initially present in the mixture in a ratio as low as l/50 to 1/100 of the cells, came to dominate primary tumors in an exponential-like manner. Furthermore this clone was metastatically competent. Is this dominance a feature of other metastatic clones? Reconstitution experiments in which another genetically marked metastatically competent clone was mixed with an excess of the parental non-metastatic (and non-tagged) SP1 tumor cells showed this was the case (Waghorne et al. 1988). The degree of enrichment of such clones was calculated to be of the order of 5- to 50-fold over a 6- to 7-week period. Interestingly, this dominance was not due to inherently shorter population doubling times of metastatic cells, since their growth rates were shown to be exactly the same in vivo as the parental SP1 tumor (Waghorne et al. 1988). This implies that an interaction between a metastatic clone and a large number of nonmetastatic clones may bring about the eventual dominance of the former. This could in theory occur by release of growth factors or cell contact via gap junctions, which is something we are studying now. It is of some interest to
consider that these results reflect recent interesting publications demonstrating a variety of phenotypes, for example metastasis, protease production and drug resistance, can be influenced by similar cell-cell interclonal interactions (Poste et al. 1981; Tofilon et al. 1984; Heppner, 1989; Lyons et al. 1989). Clonal interactions can also induce genomic alterations such as rearrangements or amplifications of transfected genes in tumor cell populations (Itaya et al. 1989). The application of the genetic technology described here to isolate and track the fate of large numbers of clones in vivo should help facilitate studies in the inherently complex area of investigation of 'tumor cell societies' (Heppner, 1989). The results summarized above using plasmid-transfected/marked SP1 cells show that late-stage ('advanced') primary tumors can be clonally dominated, i.e. overgrown, by metastatically competent subpopulations. Phenotypic analysis of primary human cancers, e.g. colorectal carcinomas and malignant melanomas, also suggests this type of metastatic cell dominance (see Kerbel et al. 1988, for review). This dominance effect reaffirms the notion of the metastatic cell being the 'apotheosis of cancer' (Hart et al. 1989). It also shows the results of comparative phenotypic or genotypic analyses between primary tumors and metastases can be heavily influenced when primary tumors are removed and analyzed: late-stage advanced tumors that are dominated by metastatically competent cells may appear similar or even indistinguishable from distant metastases, whereas earlier-stage primary tumors may be quite different. This simple idea could help resolve many of the discrepancies in the literature involving studies designed to uncover tumor cell features that influence metastasis. It also can explain one major source of genotypic and phenotypic variability observed when different primary tumors of similar histological origin are studied: if the phenotype or gene being studied - say, response to a particular growth factor - varies with the degree of metastatic competence, then the behaviour of a given primary tumor could clearly depend upon the extent to which it was clonally dominated by metastatically competent cells. Many studies using early versus late-stage primary malignant melanomas that vary in their competence for metastasis support this idea (Herlyn et al. 1987; Holzmann et al. 1987). Tumor cell lineage analysis using retrovirus vector-infected cell populations Virtually all studies exploiting random integration of foreign DNA to study cell lineage in normal cell systems have utilized retroviral vectors as the means of tagging cells genetically (Price, 1987). The advantages of this technology are considerable, including its very high efficiency as a method of gene transfer, relative stability of the proviral insertions, the low number (one or two) of insertions per infectant, and lack of toxicity of the infection process (Dick et al. 1986; Price, 1987). As a result, very large numbers of stably tagged cells can be obtained in a single-step selection. We have used a replication-deficient Moloney leukemia virus-based vecEvolutionary growth of tumors
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tor called AeApMoTN for our studies (Korczak et al. 1988). The retrovirus construct contained the dominant selectable neo gene enabling infectants to be isolated in medium containing the antibiotic G418. The vector was found to infect SPl cells at a frequency of about 1 in 5000 cells (Korczak et al. 1988). This enabled us to pool 104 or 10s G418-resistant SPl clones after a single infection and selection. One of the more striking findings we noted after injecting such a large mixture of marked cell clones into syngeneic CBA/J mice was the extent of clonal selection: thus the resultant tumors were found to be dominated by less than 10 clones! An example of this is shown in Fig. 2. In this experiment the SPl cells, after being tagged with the retrovirus vector, were exposed in vitro to an ionophore (A23187) or phorbol 12-myristate 13-acetate (PMA), which induced the cells to express metastatic potential (Korczak et al. 1989). It will be noted that the individual primary tumors are dominated by a small number of clones, but in this case the nature and number of the dominant clones seems to vary from one tumor to another, with some exceptions. It will also be noted that the lung metastases in a given animal are sometimes derived from one of the dominant clones present in the primary tumor obtained from the same animal (e.g. A3, PI, M2). The results are different in some respects from those described using the plasmid-transfected tumor cells described above, where the same clone always dominated every tumor analyzed. However, this may be due to the
A1
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ratio of metastatic to non-metastatic cells present in the inoculum as well as to the number of clones injected. Thus, in the aforementioned (plasmid-transfection) experiments the ratio was in the range of 1/10 to 1/100, which presumably allowed dominance of the same single clone in each primary tumor. In contrast, the experiment described in the legend to Fig. 2 utilized a population of SPl cells many or most of which were competent for metastasis because of the ionophore or drug treatments. Thus the circumstances did not exist to allow dominance of the same metastatic clone in every tumor. The results also show that, whereas the primary tumors may comprise more than one dominant clone, the metastases are usually monoclonal, attesting to their clonal nature at the time of analysis and their possible origin from single tumor cells seeded into the lungs — in agreement with previous results prescribed by us (Korczak et al. 1988) and other groups using cytogenetic (Talmadge et al. 1982) or isoenzyme (Oostsuyama et al. 1987) markers. It would thus appear that metastasis is nature's way of 'cloning' a tumor cell population in vivo (see Fidler and Talmadge, 1986). The 'clonal dominance' effect was recently confirmed by Thompson et al. (1989), who injected mice with mouse prostrate glandular cells that had been transformed in vitro with a retrovirus vector containing the ras and myc oncogenes. An estimated number of between 20 and 100 epithelial cells containing unique proviral integrations were injected into mice. Nevertheless, the tumors that arose were found to be
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Fig. 2. Analysis of cell-virus DNA junction fragments in primary tumors and individual metastases obtained from mice given an injection of a pooled mixture of 10s different G418-resistant SPl clones ('SPl-weo' clones). The tagged tumor cells were obtained by infection of the SPl mouse mammary adenocarcinoma with a retrovirus vector, AeApMoTN (Korczak et al. 1988), that contains the selectable neo gene. The pooled cells were then treated with ionophore A23187 or PMA to induce metastatic competence (Korczak et al. 1989). Individual CBA/j mice were then injected with a total of 10s cells into the subcutis. The lanes are as follows: inoculum (i); uninfected control SPl cells (0); metastases (a,b,c,d) and primary tumors (P) isolated from individual animals after injection with SPl-«eo cells were treated for 2h with 2f
