Contact Interactions Between Cells That Suppress Neoplastic Development: Can They Also Explain Metastatic Dormancy?

Contact Interactions Between Cells That Suppress Neoplastic Development: Can They Also Explain Metastatic Dormancy? Harry Rubin Department of Molecular and Cell Biology, Life Sciences Addition, University of California, Berkeley, CA 94720‐3200

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

XII. XIII. XIV.

Introduction Suppression of Transformation Among Fibroblasts Suppression of Transformation Among Epithelial Cells Is GJC Required in Cell–Cell Suppression of Tumor Development? The Role of Plasma Membrane Activity in Regulation of Cell Growth Suppressive Effects of Mesenchymal Tissue on Normal and Neoplastic Epithelial Proliferation The Prototype of Progression to Metastasis as seen in Human Malignant Melanoma Characteristics of Cultured Human Melanocytes Isolated from Different Stages of Melanoma Progression Is there a Relationship Between the Cell Contact Interactions that Suppress Neoplastic Development and the Phenomenon of Metastatic Dormancy? Characteristics of Metastatic Dormancy Tumor Cell Adhesion to Cells in Distant Organs A. Endothelial Cells B. Parenchymal Cells Possible Alternative Explanations of Metastatic Dormancy Molecular Basis of Cell–Cell Adhesion Conclusions References

A comprehensive listing with accompanying discussions is given for established cases of interactions between normal and neoplastic cells of the same histotype that suppress neoplastic development. General principles that apply to the process are: (a) the requirement for a large excess of normal cells in direct contact with the neoplastic cells; (b) the effectiveness of suppression decreases with the degree of malignant progression of the neoplastic cells; and (c) the transformability of normal cells decreases under long‐term negative selection, which also increases their contact suppression of neoplastic cells. Although suppression requires adhesive contact, it does not require gap junction communication, and it represents the first line of defense against tumor development. In contrast, potentially metastatic cells released from primary carcinomas into the circulation are activated to multiply when they form heterotypic adhesions with

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0065-230X/08 $35.00 DOI: 10.1016/S0065-230X(08)00006-7

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endothelial and parenchymal cells of distant organs. The great majority of the disseminated cancer cells (DCCs) fail to develop heterotypic cells adhesions in distant organs, and remain metastatically dormant as single cells. The lack of growth factors for the DCC in foreign territory also contributes to metastatic dormancy. The major insights about suppression of neoplastic development by homotypic contact arose from strictly operational experiments with living cells in culture. Molecular characterization of the cell–cell adhesions that underlie neoplastic suppression and metastasis activation has had only limited success, probably because of the complex variety of molecules involved. Hence, a program is outlined for further operational experiments on cell–cell interactions in tumor suppression to deepen our understanding of the neoplastic process, and provide possible avenues for its control. # 2008 Elsevier Inc.

I. INTRODUCTION The concepts of initiation and promotion were adopted to characterize the observations that the single application of a chemical carcinogen to the skin of mice produced no neoplastic lesion, but the repeated application to the initiated area of a promoting agent any time thereafter resulted in the appearance of multiple papillomas of epidermal origin, some of which progressed to carcinomas (reviewed in Rubin, 2001, 2003). Related observations have been made in a wide variety of organs in experimental animals and in the development of human neoplasms (Pitot, 2002). The generally accepted implication of these observations is that the initiating treatment induces tumor‐related mutations in epidermal cells that retain their normal phenotype until their microenvironment was perturbed by promoters, which are themselves nonmutagenic. With the advent of molecular genetics it was shown that tumor‐related mutations occur even in normal untreated tissues (Cha et al., 1994; Kasami et al., 1997; Tsai et al., 1996). The first hint of the microenvironmental relations that might underlie the suppression of initiated cells came from studies of fibroblasts in culture which maintained their normal morphology and growth behavior despite infection with tumor‐inducing viruses (Rubin, 1960a,b; Stoker, 1964; Stoker et al., 1966). Those studies showed that contact of solitary infected cells with confluent, contact‐inhibited cultures of normal fibroblasts prevents, and even reverses, the transformation of the infected cells. There followed many other examples of suppression of transformation in fibroblasts, but the full significance of these observations was not appreciated until the same kind of contact suppression was reported with initiated epidermal cells surrounded by normal keratinocytes (Hennings et al., 1990; Strickland et al., 1992), which provided an explanation for the ubiquitous occurrence of initiated cells in diverse epithelial tissues (Rubin, 2006, 2007). Recently, additional, important examples of contact‐related suppression of neoplasia by normal homotypic cells have come to light, which are included in the comprehensive review of the field that forms the first part of the present article.

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The foregoing results raised the question whether contact suppression of the neoplastic state might play a role in the phenomenon of metastatic dormancy. In metastatic dormancy, cells released into the circulation from primary tumors lodge in distant organs, but fail to form metastatic tumors there even though a very small fraction of cells from the same clonal population might manage to do so. There are, of course, differences from the contact suppression of initiated cells. Cells released into the circulation from primary tumors have not only lost their capacity to be regulated by homotypic normal cells, but are no longer retained even within their homotypic primary tumor. Any suppression provided by contact with normal cells in distant tumors would have to be not only heterotypic, but diversely heterotypic to be effective in every tissue the disseminated tumor cells encounter. Despite these reservations, I made a literature search of the conditions that underlie metastatic dormancy, which are discussed in the second part of this article. Not surprisingly, the metastatic literature is mainly concerned with the local conditions that enhance rather than suppress metastatic growth, but absence of, or failure to respond to those conditions might help to understand the origins of metastatic dormancy. The findings indicate that adhesive interactions of circulating tumor cells with heterotypic cells of distant organs promote metastasis formation, which is the opposite of the homotypic cell interactions that suppress primary tumor development. In addition to the adhesive interactions, the absence of appropriate soluble growth factors in distant organs could contribute to metastatic dormancy. The basic biological principle of order in the large over heterogeneity in the small which governs primary tumor development (Rubin, 2006, 2007) appears to be inoperative in the development of metastases. Further exploration of the principle of ordered heterogeneity in both situations is suggested to clarify their operational relationships.

II. SUPPRESSION OF TRANSFORMATION AMONG FIBROBLASTS The first indication of the nature of tumor suppression came from studies on the interactions between normal and neoplastic cells of fibroblastic origin (Table I). Chick embryo fibroblasts (CEFs), infected with a low dose of the Bryan strain of Rous sarcoma virus (RSV) within 24 h after seeding of the cells, would undergo neoplastic transformation resulting in their continued proliferation to form discrete, multilayered foci after the surrounding noninfected cells had stopped proliferating. The number of transformed cells in a culture could be assayed by their capacity to initiate foci when mixed in suspension with a large excess of uninfected cells (Rubin, 1960a).

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162 Table I

Suppression of Transformation Among Fibroblasts

Source of neoplastic cells RSV‐infected 1
CEF

Polyoma virus‐infected line of hamster fibroblasts Chemically transformed line of 10T1/2 mouse fibroblasts 5 sublines of an REF line, each transformed by a different oncogene Spontaneously transformed NIH 3T3 line of mouse fibroblasts Mouse glial cells transformed by transfected viral oncogene Spontaneously transformed Balb/c 3T3 mouse fibroblasts Spontaneously transformed Balb/c 3T3 mouse fibroblasts

Normalizing conditions

References

Normal CEF Normal CEF in high [CS] or moderate [FBS] Normal hamster or mouse fibroblasts Non‐transformed 10T1/2 line of 10T1/2 mouse fibroblasts

Rubin (1960a,b)

Early passage REF and an established REF line

Martin et al. (1991)

Sublines of NIH 3T3 mouse fibroblasts

Rubin (1994)

Non‐transformed glial cells

Alexander et al. (2004)

Self‐normalization of the transformed cells at very high density Lowered Mg2þ concentration in medium

Rubin and Chu (1982)

Stoker (1964, 1967) Mehta et al. (1986)

Rubin (1982)

CEF, chick embryo fibroblasts; CS, calf serum; FBS, fetal bovine serum; REF, rat embryo fibroblasts.

If, however, a culture of noninfected CEF was allowed to grow to confluence and undergo contact inhibition before adding cells already transformed at 3 days after RSV infection, no transformed foci appeared. The results suggested that the transformed cells were normalized and their growth suppressed by contact with the growth‐inhibited, confluent monolayer of normal cells. This conclusion was supplemented by the observation that the development of transformed foci was suppressed when RSV was added at the time of seeding the normal cells in the standard transformation assay for RSV, if the medium contained unusually high concentrations of calf serum (15–20%, v/v) or conventional concentrations of fetal bovine serum (5–10%, v/v) (Rubin, 1960b). Suppression of transformation occurred only when the concentrations of RSV were low enough to form discrete, countable foci surrounded by normal cells. If high enough concentrations of RSV were added to infect most of the cells, the entire culture underwent transformation regardless of how high the serum concentration was. Growth curves of

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the number of infected cells assayed from serum‐suppressed cultures revealed that they assumed the regulatory behavior of normal uninfected cells since they stopped proliferating within a day after the culture as a whole did. In contrast, the infected cells in permissively low concentrations of serum showed no such constraint at confluence. The regulated growth of infected cells in the first case was attributed to direct interaction between them and the surrounding normal cells, which was enhanced by high concentrations of serum. The serum effect was later interpreted as inhibition of proteases released at the cell surface (Rubin, 1970). This interpretation was supported by the digestion of fibrin by Rous sarcomas in plasma clots (Fischer, 1946), the identification of a surface protease of RSV‐ transformed cells in monolayer culture (Unkeless et al., 1973), and the capacity of proteases to stimulate the mitotic cycle of CEFs (Sefton and Rubin, 1970). These findings were extended to mammalian cells in experiments using a clone of established hamster fibroblasts transformed by infection with polyoma virus (Stoker, 1964). Colonies of the clone exhibited random orientation and unrestricted growth when in contact with one another on a bare surface, or when growing amid a low density of normal hamster or mouse fibroblasts. They made no visible colonies or foci, however, when seeded on a contact‐inhibited layer of normal fibroblasts (Stoker et al., 1966). There was transfer of hypoxanthine from a dense sheet of normal mouse embryo cells to polyoma‐transformed hamster cells, raising the possibility that there was also transfer of growth‐regulatory molecules (Stoker, 1967). That possibility seemed to gain support from experiments mainly with the 10T1/2 line of mouse fibroblasts transformed by methylcholanthrene, which were suppressed after seeding on top of a confluent layer of the original nontransformed 101/2 line (Mehta et al., 1986). The suppression was correlated with gap junction communication (GJC) as probed by dye transfer between the normal and transformed cells. A variety of treatments were applied to enhance or inhibit GJC among the cell combinations. It was concluded that heterologous GJC among the cells is required to suppress growth of the transformed cells. The generality of this conclusion was questioned in studies with five sublines of an immortal line of rat fibroblasts, each transformed by a different oncogene and cocultured with either the original nontransformed line, or early passages of normal rat embryo fibroblasts (REFs) (Martin et al., 1991). There was no correlation of transformed cell suppression with GJC. Furthermore, total inhibition of fluorescent dye transfer between normal and transformed cells failed to relieve the growth suppression of any of the transformed cell populations in combination with either of the two nontransformed populations. The results are obviously inconsistent with those of the previous paragraph (Mehta et al., 1986).

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Spontaneous transformation occurs in the NIH 3T3 line of mouse fibroblasts by selection for overgrowth in serial rounds of prolonged confluence (Rubin, 1994). In contrast, negative selection apparently occurred in about 330 short‐ term, serial passages of the original NIH 3T3 cells at very low density and gave rise to a subline that resisted transformation. Moreover, confluent cultures of the subline suppressed the proliferation, and normalized the morphology of transformed cells derived from another subline. In contrast, susceptible sublines that had undergone only 30–60 low‐density passages permitted continuous expansion of transformed foci. The suppression by the higher passage cells did not assert itself until several days after they became confluent, indicating that the establishment of strong contact inhibition of those cells with each other was required before they could inhibit the transformed cells. When that happened, the small colonies of transformed cells that had already formed blended in with the nontransformed background, indicating that the cells had taken on a normal fibroblastic appearance, and were themselves inhibited from further proliferation. However, they resumed their transformed phenotype when assayed on a permissive background. Fibroblasts, presumably of glial origin, were obtained from the brains of mice that had a homozygous null connexin 43 knockout, which substantially reduced GJC (Alexander et al., 2004). A group of these cells was transformed by transfection with the src kinase gene. Growth and transformed morphology of the transformed cells were suppressed by contact with confluent monolayers of the nontransformed fibroblasts even when the already low GJC was further reduced by an inhibitor. Not only was there no dye transfer between cells, but electrical conductance between most of the heterologous pairs was also eliminated. The results indicated that significant GJC was not required for contact normalization, and that other intercellular junctions mediated this growth‐regulatory process. Pure cultures of transformed mouse fibroblasts rapidly deplete their medium when grown at high density with the conventional ratio of cells to medium, even when the medium is changed daily. However, if the transformed cells were restricted to the area of a small coverslip in a large culture dish with more than ample medium, they multiplied to a 10 times higher saturation density than do nontransformed cells, and they became contact‐ inhibited in the absence of nontransformed cells (Rubin and Chu, 1982). In doing so, the cells took on the appearance of a multilayered culture of normal cells, and displayed a markedly reduced rate of DNA synthesis. They had a greatly reduced capacity to produce colonies when subcultured in agar, and retained the flattened appearance of normal cells for about 1 day when reseeded at low density on plastic before resuming their transformed appearance. This homologous contact normalization indicates that there is no need for the transfer of regulatory molecules from normal cells for the transformed cells to assume a normal phenotype.

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Among other changes in the normalized cells was a reduction in the intracellular content of Mg2þ. Indeed, normalization could be brought about in less crowded cultures of the transformed cells by drastically lowering the Mg2þ concentration of the medium with a consequent reduction of the Mg2þ content of the cells (Rubin, 1982). The combined results were consistent with a central role of Mg2þ in the coordinate control of cell proliferation (Rubin, 1975, 2005).

III. SUPPRESSION OF TRANSFORMATION AMONG EPITHELIAL CELLS Cancers of epithelial origin or carcinomas constitute over 90% of human neoplasia (Cairns, 1978). The first indication of tumor suppression among epithelial cells came from studies of the comparative growth of hyperplastic alveolar nodules (HANs), which are precursors of mammary cancer in mice, after injection into intact mammary gland fat pads versus injection into fat pads that had been cleared of mammary epithelium (Table II; Faulkin and

Table II

Suppression of Transformation Among Epithelial Cells

Source of neoplastic cells

Normalizing cells

Hyperplastic mouse mammary alveolar epithelium

Normal mammary epithelium

Preneoplastic and neoplastic rat tracheal epithelium Neoplastic mouse keratinocytes Neoplastic human keratinocytes

Normal tracheal epithelium Normal keratinocytes

a

Human melanoma cells

Normal human keratinocytes

Rat hepatocarcinoma cells

Normal human keratinocytes Rat liver in vivo

Neoplastic imaginal disc epithelium of Drosophila

Normal imaginal disc epithelium

References DeOme et al. (1978), Faulkin and DeOme (1960), Medina et al. (1978) Gillett et al. (1989), Terzaghi‐Howe (1987) Hennings et al. (1990, 1992), Strickland et al. (1992) Alt‐Holland et al. (2005), Javaherian et al. (1998), Mudgil et al. (2003) Hsu et al. (2000b) Coleman et al. (1993), McCullough et al. (1998) Bilder (2004), Brumby and Richardson (2003)

aTransfected with and overexpressing E‐cadherin. Although melanocytes originate in the neural ridge and migrate to the epidermis during development, they and their neoplastic derivatives are considered here along with epithelial cells because they are mainly situated in the basal layer of the epidermis in humans, and are regulated by contact with keratinocytes.

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DeOme, 1960). The transplanted HAN grew much more rapidly in the gland‐free fat pads than in the intact fat pads, indicating that normal mammary epithelium suppressed growth of the hyperplastic tissue. Unlike the suppression of neoplastic growth among fibroblasts (Table I) which required direct contact between cells, the suppression of the HANs occurred at a distance, suggesting the release of inhibitory substances from the normal mammary epithelium. There was similar evidence for the inhibition of mammary carcinoma growth by diffusible substances from normal epithelium but it was less effective than the inhibition of HAN growth. Such diffusible inhibitors are also responsible for maintaining the distance between normal mammary tubules (Faulkin and DeOme, 1960) and between branches of tubules (Nelson et al., 2006). The cells of HANs are also subject to growth inhibition by contact with normal mammary epithelium. The mixture of enzymatically dissociated HAN cells with an excess of dissociated normal mammary epithelial cells from virgin, pregnant, or lactating mice significantly decreased the tumorigenicity of the HAN cells upon injection into the gland‐free fat pad (Medina et al., 1978). Further evidence of contact inhibition of hyperactive growth came from experiments in which mammary epithelial cells from mammary cancer‐prone mice were removed, dissociated, and injected into the gland‐ free fat pads of young mice. HANs were produced within 2–3 months in the injected mice whereas 8–9 months were required for their appearance in the undisturbed host (DeOme et al., 1978). If the mammary epithelium was transplanted as intact pieces, each containing many cells, the onset of HANs was delayed over that produced by the dissociated cells (Medina et al., 1978), supporting a suppressive role of direct cell–cell interaction. Studies were later done on the suppressive effect of normal tracheal epithelium on carcinogen‐altered rat tracheal epithelial cells (Terzaghi‐ Howe, 1987). Ten thousand or more normal tracheal epithelial cells were inoculated with a 2‐ to 100‐fold excess of a carcinogen‐exposed aneuploid neoplastic cell line into tracheas in which the resident epithelia had been destroyed, and the tracheas were then transplanted subcutaneously into syngeneic hosts. The regenerated epithelium comprised normal diploid epithelial cells (almost entirely), and no tumors developed. Substitution of esophageal epithelial cells for normal tracheal cells also suppressed growth of the neoplastic tracheal cells. In contrast, tumors developed quickly upon adding neoplastic cells alone to deepithelialized tracheas, or to a small scratched area of otherwise intact tracheal epithelium, so the neoplastic cells in both cases occupied a contiguous surface of submucosa. The suppressive effect of the normal epithelial cells therefore appeared to require contact with the neoplastic line. The results differed from those of the aforementioned mixing experiments in which suppression was obtained with a minority of normal cells rather than an excess, which suggested that

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the normal cells attached more firmly to the denuded tracheal submucosa and displaced adjacent neoplastic cells. Preferential attachment might also explain the heterotypic suppression of tracheal neoplasia by a minority of esophageal cells. Mixing experiments were done with the same deepithelialized tracheal technique, but using different lines of preneoplastic and neoplastic tracheal cells (Gillett et al., 1989). The results were complex and irregular, but significant suppression occurred only with 100‐fold or greater excesses of normal to neoplastic cells, and suppression was uniformly successful only with the preneoplastic cells. The variable extent of suppression among the cell lines suggested that they represented different stages of neoplasia. It was surmised that progression of the neoplastic state decreases the susceptibility of the neoplastic cells to suppression by normal cells. Studies of the mixtures in cell cultures rather than the deepithelialized trachea identified neoplastic cells that persisted even where no lesions could be seen in the trachea. This suggested that, unlike the earlier experiments in suppression of tracheal neoplasia (Terzaghi‐Howe, 1987), the neoplastic cells were not eliminated by the normal cells, but were kept in a dormant state. Some of the most clearly defined and informative experiments on suppression of epithelial tumor development by normal epithelial cells were done on epidermal neoplasia, in part because they involved questions generated by the exhaustively studied in vivo system of initiation and promotion. Mouse keratinocytes derived from untreated skin terminally differentiate in cell culture without feeder layers in medium containing more than 0.1 mM calcium, but evade differentiation and multiply rapidly in lower concentrations (Hennings et al., 1980). Keratinocytes cultured from skin exposed to carcinogens in vivo yield occasional foci of proliferating cells that are resistant to calcium‐induced terminal differentiation (Kulesz‐Martin et al., 1980). These calcium‐resistant foci were not tumorigenic when first formed, but some cell lines derived from them progressed to tumor formation (Kulesz‐Martin et al., 1983). Colony formation in calcium by such cells was markedly delayed when they were cocultured with a large excess of normal keratinocytes, but the delay was shortened in the continuous presence of a promoting agent (Hennings et al., 1990). In contrast, there was no inhibition of colony formation from the initiated cells by adding a large excess of fibroblasts, or growing them in medium that had been conditioned on keratinocyte cultures. Similarly, only normal keratinocytes inhibited tumor formation on mouse skin by grafts of cells from papillomas that had been induced in the initiation– promotion procedure (Strickland et al., 1992). However, the keratinocytes failed to inhibit the growth of malignant carcinoma cells when the two cell types were mixed in skin grafts. A line of initiated keratinocytes that multiplied in high calcium, but formed normal epidermis when grafted in vivo, had lost

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the capacity to inhibit tumor development by papilloma cells. This loss of inhibitory capacity for papilloma development is potentially of great significance because it suggests that the apparently normal epidermal cells that constitute the cellular microenvironment surrounding erstwhile tumorigenic cells in carcinogen‐initiated skin may be more permissive for tumor growth than are keratinocytes from untreated skin. It is noteworthy that there was no GJC between normal keratinocytes and the papilloma cells whose growth they inhibited (Hennings et al., 1992), indicating that contact between the normal and benign tumor cells is sufficient to suppress growth of the latter. An organotypic reconstruction of human skin in culture has been used to study the effect of normal keratinocytes on the growth of low‐grade malignant epidermal cells (Javaherian et al., 1998). Ratios of normal to malignant cells of 4:1 or higher eliminated the malignant cells from the organotypic cultures by displacing them from the basal layer and leading them to terminal differentiation. In a 1:1 ratio, the malignant cells persisted in expanding foci from which they eventually invaded the collagen substratum. The elimination of the malignant cells in the excess of normal keratinocytes, however, is not a model for the indefinite persistence of initiated cells in the skin, nor does it correspond to the failure of keratinocytes to suppress carcinoma cell proliferation in vivo (Strickland et al., 1992). Indeed, when the collagen substratum for the epidermis in organotypic cultures was covered with human basement membrane proteins, the low‐grade malignant cells were not eliminated, but invaded the substratum (Alt‐Holland et al., 2005). The results indicated that the normal keratinocytes outcompeted the neoplastic cell for attachment to collagen but not to basement membrane. The situation resembles that of the tracheal carcinoma cells that were suppressed by even a small minority of normal tracheal epithelium as a result of competition for attachment to the mesenchyme of deepithelialized tracheas (Terzaghi‐Howe, 1987). Epidermal tumors mainly induced by ultraviolet light are the most common form of neoplasia in humans (Ziegler et al., 1993). Clones of keratinocytes carrying mutations in the p53 tumor suppressor gene (TSG) occur at high frequency in the skin and can involve as much as 4% of the sun‐exposed epidermis of humans (Jonason et al., 1996). Sunlight acts both as a tumorigenic mutagen and a tumor promoter which favors clonal expansion of p53‐ mutated cells. Stem cell components act as physical barriers to clonal expansion of the p53 mutant keratinocyte (Zhang et al., 2001). Sustained ultraviolet irradiation enables the p53 mutant keratinocyte to expand to adjacent stem cell compartments without incurring an additional mutation. Studies in mice indicate that apoptosis of normal keratinocytes in the stem cell compartment will drive clonal expansion of the p53‐mutated clones (Zhang et al., 2005). Mixtures of p53‐mutated intraepithelial human tumor cells and normal keratinocytes were used to fabricate organotypic

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skin cultures, and then exposed to ultraviolet light (Mudgil et al., 2003). The ultraviolet treatment induced apoptosis in the normal keratinocytes but not in the tumor cells, which allowed expansion of the tumor cell colonies, thereby confirming the role of apoptosis in the expansion of p53 tumor clones found in mice. Melanomas are neoplasms that arise from melanocytes, which are interspersed as single cells in human skin among basal keratinocytes of the epidermis. Undifferentiated, but not differentiated, keratinocytes control growth, morphology, and antigen expression through direct cell–cell contact (Valyi‐Nagy et al., 1993). One of the most significant features accompanying melanoma development and progression is the expression of cell surface antigens not expressed in melanocytes. The expression of melanoma antigens is also induced in melanocytes after their isolation from skin and subsequent rapid division in culture (Herlyn et al., 1987), but expression is downregulated when the melanocytes are cocultured with keratinocytes (Shih et al., 1994). Neither fibroblasts nor medium from keratinocytes exert these effects on cultured melanocytes. Unlike the melanocytes, melanoma cells from primary and metastatic lesions continue to express those antigens constitutively in the presence of keratinocytes. The loss of regulatory dominance by keratinocytes occurs with downregulation of E‐cadherin expression and replacement by N‐cadherin in melanoma cells, resulting in loss of their adhesion to keratinocytes (Hsu et al., 2000b). Transduction of E‐cadherin expression in the melanoma cells leads to their adhesion to keratinocytes, thereby rendering them susceptible to keratinocyte‐mediated control. E‐cadherin expression in the presence of keratinocytes also inhibits invasion of the melanoma cells into the dermis by downregulating invasion‐related adhesion receptors, and inducing apoptosis. Normal melanocytes, but not melanoma cells, establish GJC with keratinocytes (Hsu et al., 2000a). However, the melanoma cells establish GJC among themselves and with fibroblasts. Although the loss of E‐cadherin is associated with malignancy, the increase of another cell adhesion protein, melanoma cell adhesion molecule (MCAM), correlates with malignancy (Li et al., 2004). Ectopic expression of E‐cadherin or the E‐cadherin alpha catenin fusion protein in melanoma cells restores their adhesion to keratinocytes. Deletion of the E‐cadherin cytoplasmic domain blocks restoration of that adhesion. Although GJC is associated with the normalization by E‐cadherin of melanoma cells when they come in contact with undifferentiated keratinocytes, it is noteworthy that the expression of E‐cadherin induces adhesion to keratinocytes (Hsu et al., 2000a; Kanno et al., 1984). Therefore, it is consistent with other findings that involve regulatory interactions between neoplastic and normal cells (Alexander et al., 2004; Hennings et al., 1992; Martin et al., 1991) that adhesion per se between the plasma membranes of E‐cadherin‐expressing melanoma cells and

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keratinocytes is sufficient to normalize the phenotype of the melanoma cells. (Note: In mice, melanocytes are in the dermis, and a whole different set of regulatory relations must hold (Fukunaga‐Kalabis et al., 2006).) Another informative series of experiments about the modulating effect of normal tissues on the expression of neoplastic cells arose from transplanting rat hepatocarcinoma cells derived from stem‐like liver cell cultures into the liver or the subcutaneous tissue of normal rats. Some of the transplanted hepatocarcinoma cells of either a moderately aggressive or a highly aggressive line migrated into the hepatic plates (Coleman et al., 1993). Solitary cells in the hepatic plates from the moderately aggressive hepatocarcinoma line took on the appearance and regulated growth behavior of normal mature hepatocytes in young rats. However, cells from the more aggressive line multiplied in the hepatic plates, albeit slowly, and largely maintained their neoplastic morphology. Both cell lines multiplied rapidly into tumors after transplantation into subcutaneous tissue, where they assumed a bipolar mesenchymal cell appearance. Solitary cells from the less aggressive line multiplied into small foci when transplanted in the liver of old (18–24 months) rats (McCullough et al., 1998). The initially normalized hepatocarcinoma cells in the young rat retained that phenotype until they began to multiply at 14 months after their transplantation, forming small foci in the liver. The overall results of these experiments showed that the intact liver of young rats has the capacity to normalize hepatocarcinoma cells originating from stem‐like normal liver cells but that subcutaneous connective tissue has no such capacity. The liver loses the normalizing capacity as the rat ages, which suggests that the large increase of incidence in cancer with age results, at least in part, from the decrease in the normalizing capacity of the immediate epithelial cell microenvironment with age. The capacity of the young intact liver to normalize solitary hepatocarcinoma cells contrasts with the failure of normal keratinocytes to suppress the growth of epidermal carcinoma cells. The liver is also capable of maintaining metastasizing mammary cancer cells in a dormant state for extended periods of time (Naumov et al., 2002), although it is not known whether this capacity declines with age. It would appear that mixing keratinocytes with epidermal carcinoma cells in skin grafts is a less effective procedure for normalizing cancer cells than is the intact liver with its large masses of essentially pure hepatocytes. Drosophilae are ideal organisms for the study of tumor genetics because their genomes are so fully defined, and readily manipulated. Recessive‐lethal mutations that cause tumors in Drosophila larvae were first reviewed in 1978 (Gateff, 1978) and their study along molecular lines later elaborated (Bilder, 2004). Zygotic mutations in any of about 15 genes cause tumors in all proliferating tissues of larvae, but have been most extensively studied in epithelial organs called imaginal discs that serve as primordia for most adult structures. Unlike vertebrate tumors, which require combinations of

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genetic changes for their full development, a single zygotic mutation of the normal allele in flies carrying a heterozygous TSG is sufficient for neoplastic growth of the cells throughout all the imaginal discs. The biallelic TSG mutations have a very high penetrance, resulting in manifold increases in size of the imaginal discs and death of the larvae. Since all the cells carry the mutation, there are no wild‐type epithelial cells to modulate their neoplastic proliferation. The three most carefully studied TSGs encode cytoplasmic proteins found at the cell membrane, leading to a loss of ability to organize an epithelial monolayer. More recently, it has been found that mutations of the components of the endocytic machinery, which is required to internalize cell surface proteins, also produce larval tumors (Bilder, personal communication). Mutated TSG clones can be made in an otherwise wild‐type imaginal disc through mitotic recombination (Bilder, 2004). These clones grow more slowly than their wild‐type neighbors and are eliminated by the process of cell competition (Bilder, 2004). The elimination accounts for the extreme rarity of spontaneous tumors in adult Drosophila, and introduces the question of how the cells carrying the biallelic mutations are eliminated. The TSG scribble mutant clones do not overgrow because of cell death mediated by the Jun N‐terminal signaling pathway and the surrounding wild‐type tissue (Brumby and Richardson, 2003). In contrast, when oncogenic Ras or Notch is expressed within the scribble clones, cell death is prevented, and tumors develop. The mechanism of the wild‐type tissue in causing apoptosis of scribble clones is unknown, except to note that it differs from most cases of the suppression of tumor development in vertebrate organs in which the potential tumor cells persist indefinitely. It is also noteworthy that the imaginal discs are pure epithelium so the suppressive effects of the wild‐type tissue on TSG mutant clones are not complicated by possible mesenchymal effects, e.g., integrin mutations produce no tumors (Bilder, 2004).

IV. IS GJC REQUIRED IN CELL–CELL SUPPRESSION OF TUMOR DEVELOPMENT? It was apparent from the earliest studies among fibroblasts and their transformed counterparts that (a) contact between the two cell types is required for suppression of neoplastic development and (b) the normal cells had to be contact‐inhibited among themselves for the neoplastic suppression of transformed cells to be effective (Rubin, 1960a; Stoker, 1964). The discovery that there was exchange of molecules in the normal and neoplastic cells raised the question whether such GJC is necessary for suppression of neoplastic development (Stoker, 1967). Experiments directed

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toward this question reported a correlation between GJC and suppression of fibroblast transformation, and suggested that cyclic AMP is the regulatory molecule (Mehta et al., 1986). However, subsequent experiments with rat fibroblasts transformed by five different oncogenes found no correlation of suppression with GJC, and that total inhibition of dye transfer did not prevent the suppression (Martin et al., 1991). Shortly thereafter it was found that there was no GJC between mouse papilloma cells and the normal keratinocytes that suppressed their proliferation (Hennings et al., 1992). In a related matter consistent with the lack of a role for GJC in suppression, TPA relieved the suppression of papilloma growth by keratinocytes but had no effect on homologous GJC among either the papilloma cells themselves or among the keratinocytes (Hennings et al., 1992). More recently, the modern techniques of molecular genetics have been combined with the earlier physiological methods to distinguish between the roles of contact per se and of GJC (Alexander et al., 2004). Brain fibroblasts, presumably glial cells which lacked GJC, were taken by Caesarean section from connexin‐knockout mice, and transformed by transfection with the v‐Src kinase. Direct contact between transformed and nontransformed cells was required for growth suppression of the former without the need for dye transfer between the cells. There was also evidence against the requirement of electrical communication by small ion transfer between the cells. In addition, there was an increase in expression of serum deprivation response protein, consistent with contact inhibition of membrane activity as the suppressive mechanism of transformation. Further evidence against the requirement for GJC in suppression of transformation came from the observation that suppression can be induced among the transformed cells themselves, without intervention by nontransformed cells. As noted earlier, spontaneously transformed mouse fibroblasts undergo contact inhibition when grown to very high density on a small coverslip in a large volume of medium to avoid depletion of the medium (Rubin and Chu, 1982). The cells take on the appearance and other growth characteristics of nontransformed cells, and these are maintained up to 1 day after subculture at low density. This result shows there is no need for transfer of regulatory molecules from nontransformed molecules from nontransformed cells, or stimulatory molecules from the transformed cells to effect their regulation. With the exception of one paper reporting correlation between GJC and suppression (Mehta et al., 1986), the overall results are consistent with contact inhibition of plasma membrane activity as the underlying mechanism of suppressing the transformed phenotype of fibroblasts and epithelial cells. It should be noted that GJC itself requires contact between cells, and the reported correlation of GJC with suppression (Mehta et al., 1986) could be an indicator of the extent of adhesion between the normal and the transformed cells.

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V. THE ROLE OF PLASMA MEMBRANE ACTIVITY IN REGULATION OF CELL GROWTH It is well established that protein growth factors such as insulin initiate their stimulatory action on cells by attaching to specific receptors on the plasma membrane (Cuatrecasas, 1969). It has also been shown that trypsin immobilized on polystyrene beads initiates division of chicken embryo fibroblasts without entering the cell or being released to the medium (Carney and Cunningham, 1977). Calcium pyrophosphate stimulates the division of an established line of mouse fibroblasts but only when insoluble floccules are formed by the agent (Bowen‐Pope and Rubin, 1983). These floccules attach to the cell surface but can be completely recovered by briefly lowering the pH, thereby blocking further action on the cells. Further evidence of the role of plasma membrane activity in regulating the proliferation of cells comes from the relation between population density of fibroblasts and their growth rate. The fibroblasts proliferate at a maximal rate at relatively low population densities at which they move over the surface of the culture dish while exhibiting an actively ruffling membrane (Abercrombie and Heaysman, 1954). The ruffling stops when the membrane forms an adhesion with another fibroblast (Abercrombie and Ambrose, 1958). The cell may break its adhesive contact with the other cell and move away, thereby maintaining a high growth rate. When the population density becomes high enough to form a confluent sheet, all ruffling and independent movement of the cells are inhibited, and the rate of proliferation of the population decreases markedly in the phenomenon of contact inhibition of proliferation (Todaro et al., 1965). Epithelial cells also exhibit contact inhibition (Castor, 1968; Eagle and Levine, 1967) but they form tighter adhesions with one another than do fibroblasts, and do not break contacts once they are established (Middleton, 1973). Proliferation is therefore maximized at the free edge of a coherent sheet or of a colony of epithelial cells. Epithelial cells neither inhibit fibroblasts, nor are they inhibited by them (Eagle and Levine, 1967). Less is known about whether epithelial cells from different tissues inhibit each other except that esophageal epithelia can replace tracheal epithelia in competing with a line of tracheal carcinoma cells for attachment to tracheal mesenchyme (Terzaghi‐Howe, 1987). When fibroblasts are transformed into sarcoma cells they assume a rounded morphology in culture and do not form ruffled membranes (Abercrombie and Ambrose, 1958). The sarcoma cells do have a very actively moving surface in the form of fine, rather spiky processes extending and retracting around the periphery. There is little or no mutual adhesion or reduction in

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surface activity or locomotion when one sarcoma cell meets another or meets a fibroblast, nor do these changes occur in a fibroblast when it encounters a sarcoma cell. The proliferation of sarcoma cells continues after they contact one another or fibroblasts, and form multilayered colonies of transformed foci on a confluent sheet of fibroblasts, which are confined to a monolayer. As noted earlier, if the fibroblasts had themselves formed a fully contact‐inhibited sheet before adding the dissociated sarcoma cells, the latter in some combinations assume the morphology of fibroblasts and stop proliferating. As also noted, sarcoma cells can be forced to undergo contact inhibition at densities an order of magnitude higher than fibroblasts do, and normalize their appearance (Rubin and Chu, 1982). The overall results of these cell interactions indicate that normal homophilic cells inhibit their proliferation by forming adhesions between their surface membranes which immobilize the membranes, thereby accounting for the inhibition. Transformation to malignancy markedly reduces the homophilic adhesions and permits continuing proliferation. The role of the plasma membrane in regulating cell proliferation was directly demonstrated by adding cell‐free membrane preparations to multiplying cells. Membrane preparations from 3T3 fibroblasts inhibited DNA synthesis of intact 3T3 fibroblasts, but not that of transformed cells (Wittenberger and Glaser, 1977). Inhibitory effects on sparsely seeded human lung fibroblasts were obtained by adding membranes isolated from confluent sheets of the same cells (Wieser et al., 1985). Removing the oligosaccharides of glycoproteins from the lung fibroblast membranes inactivated their growth‐inhibiting effect, indicating those components mediate the contact inhibition of the cells. Purified rat liver plasma membranes inhibited growth of rat hepatocytes at low density (Nakamura et al., 1983). These results strongly support the sufficiency of direct cell–cell membrane interactions in regulating cell proliferation among normal cells, and between normal and neoplastic cells to regulate the latter. It should be borne in mind, however, that the susceptibility of neoplastic cells to regulation by normal cells decreases with increase in their malignancy. This was most clearly evident in the epidermal cell system in which keratinocytes suppress the proliferation of papilloma cells, but not of carcinoma cells (Hennings et al., 1990; Strickland et al., 1992). This finding is correlated with the observation that papilloma cells have almost as firm adhesion to one another as normal cells do, but squamous carcinoma cells have much lower mutual adhesiveness (Coman, 1944). It was also seen in the transplantation of rat hepatocarcinoma stem cells into the normal rat liver where cells of the moderately malignant line assumed the phenotype of a normal hepatocyte, but those of the highly malignant line formed slow‐growing tumors (Coleman et al., 1993). Similar but less striking results were obtained with preneoplastic and neoplastic tracheal epithelium transplanted to the trachea

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(Gillett et al., 1989). While those correlations are consistent when the phenotypes of adhesiveness or malignancy of living cells are considered, they are less consistent when correlations are sought between identified adhesive molecules, and the extent of cell–cell adhesion or other neoplastic characters (Foty and Steinberg, 2004; Rubin, 2007).

VI. SUPPRESSIVE EFFECTS OF MESENCHYMAL TISSUE ON NORMAL AND NEOPLASTIC EPITHELIAL PROLIFERATION It is commonly thought that the basement membrane of epithelial tissue prevents invasion of the underlying connective tissue by normal and benign epithelium (Weinberg, 2007). However, the basement membrane is a thin, fragile structure that, in the case of the epidermis, is breached every time there is a bleeding wound, yet there is no invasive growth of the epidermis into the connective tissue. More than half the injections by a needle through the skin carry fragments of epidermis into the subcutaneous tissue, where most fail to survive, although some produce epidermoid cysts (Gibson and Norris, 1958). Benign papillomatous growth, by definition, is noninvasive; growth of the papillomas is entirely in an upward direction (Cramer and Stowell, 1942) even when papilloma cells are grafted in suspension along with an excess of fibroblasts in the absence of basement membrane (Strickland et al., 1992). It would therefore appear that a combination of strong homotypic adhesion among epidermal cells, and the alien environment of the underlying connective tissue maintain the noninvasive architecture of the normal epidermis and of papilloma cells. About half of the fragments of mouse mammary epithelium that are transplanted into subcutaneous tissue retain their viability for many months, most of the others regress (DeOme et al., 1978). In some instances, the transplants develop into HANs, but do not progress to invasive tumors. Most HANs maintain their viability after transplantation into the subcutis, but a significant minority regress, and very few progress to invasive tumors. In contrast, about 10% of normal mammary epithelia injected into mammary fat pads that had been cleared from a resident epithelium developed into invasive tumors, while almost 50% of transplanted HANs did so. The results indicate that connective tissue is a more hostile place for growth of mammary epithelium and its development into invasive tumors than is the adipose tissue of the mammary fat pads that forms the normal surrounding of the mammary ducts. The development of a malignant epithelial tumor involves the capacity to flourish in a previously restricted environment, presumably by genetic or

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genomic changes in the tumor cells. It does not require any permissive change in the connective tissue itself, since the tumor can now be transplanted into the subcutaneous tissue of normal isogenic animals. Invasiveness is facilitated not only by the capacity to proliferate in connective tissue but also by a decrease in the adhesiveness of the tumor cells to one another (Coman, 1944; McCutcheon et al., 1948) and to the nontumor epithelial cells surrounding them (Coman, 1953; Mareel et al., 1990). There is a further reduction in adhesiveness associated with metastasis, which allows separation of cells from the invasive tumor mass and entry into the circulation, as well as a capacity to proliferate in a different organ. Taken to an extreme, ascites tumor cells which multiply while suspended as individuals in peritoneal fluid, can be developed by selection from solid tumors (Klein, 1951). There is a consistent difference in the rate of conversion to the ascites form between different tumors, indicating an underlying genetic variation. In the selective process, the tumor cells exhibit increasing negative charge as they increase their tendency to multiply in the ascites form (Purdom and Ambrose, 1958). At the same time there is an increase in the capacity of the cells for metastasis (Ringertz et al., 1957), indicating a negative correlation between adhesiveness and metastasis.

VII. THE PROTOTYPE OF PROGRESSION TO METASTASIS AS SEEN IN HUMAN MALIGNANT MELANOMA Perhaps the most thoroughly studied human progression models, extending from normal cells through benign and malignant neoplasia to metastasis, is the melanoma model. The visibility of these tumors in the skin and their pigmentation make them an ideal subject for continuous observation. The Pigmented Lesion Clinic started at the Massachusetts General Hospital by Wallace Clark, continued in Philadelphia at Temple University, and succeeded by the Pigmented Lesion Group at the University of Pennsylvania, recorded hundreds of clinical and pathology attributes in over 1300 patients (Clark, 1994, and personal communication). One of the earlier reports from this group involved 261 patients with primary melanoma and followed for a minimum of 5½ years (Clark et al., 1984). Later on, there were over 1000 new patients with normal and abnormal nevi or melanomas who were observed for shorter times (Clark et al., 1986). These studies led to classifying melanoma progression into five stages. The first of these is the common acquired melanocytic nevus or mole, in which there is focal proliferation of benign cells. The second stage is a melanocytic nevus with aberrant differentiation and an aberrant form of melanocytic hyperplasia. The third stage

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exhibits nuclear atypia, i.e., dysplasia. The fourth stage is known as the radial growth phase (RGP) and is the first sign of primary melanoma. It is characterized by net enlargement of the tumor at its periphery, but is not associated with metastasis. The fifth stage is the vertical growth phase (VGP) which appears as spheroidal nodules that extend into the dermis. The cells of this phase are those that give rise to the sixth phase of metastasis or the ability to maintain autonomous growth that is discontinuous from the primary site. To elaborate on these stages, the first evident focal proliferation of melanocytes in humans is the common acquired melanocytic nevus (Clark et al., 1984). It is also the initial proliferative lesion seen in response to the carcinogen DMBA in certain guinea pig strains (Pawlowski et al., 1976). It therefore represents the first stage of melanocytic tumor progression. After a few years, many of the nevi undergo a pathway of differentiation into Schwann cells. At the same time the nevi may extend into the dermis, extinguishing the basement membrane boundary between epidermis and dermis. However, the lesion is considered a compound melanocytic nevus, and rather than being invasive, Schwannian differentiation may occur in the dermis, followed by disappearance of the lesion. Another pathway followed by the nevi is hyperplasia of cells in the periphery of the nevus (Clark et al., 1984). The proliferation occurs in the basilar intraepidermal melanocytes, and there is no differentiation. Atypical melanocytes may appear in two forms in the area of melanocytic growth at the shoulder of the nevus, which constitute melanocytic dysplasia. It identifies patients at heightened risk for the development of malignant melanoma. The chief marker of dysplasia is nuclear atypia. It is often followed by the RGP of malignant melanoma, which shows partial growth autonomy and is considered the first phase of a primary neoplasm. If untreated such lesions are quite likely to progress further to the next step of the VGP. Prior to the autonomous RGP, progression of individual lesions from one class of lesion to another is a very rare event. However, from the RGP of autonomous growth, forward progression is the rule not the exception (Clark et al., 1984). Progression from melanocytic dysplasia to the RGP of superficial spreading melanoma is focal, qualitative, and has the characteristics of a mutational event. Generally there is an associated broad dense plaque of lymphocytes that underlies invasive melanoma cells. This pattern of invasiveness, however, does not lead directly to metastasis. The VGP starts as the focal appearance within the RGP of a new population of cells that tend to grow as an expanding spheroidal nodule similar to the growth of a metastasis. The cellular aggregates of the VGP are larger than the clusters of cells that form the RGP, and grow at a faster rate. They extend more deeply than those of the radial phase, but the local lymphocytic immune response disappears (Clark et al., 1986).

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It was assumed by many in the 1960s that malignant melanoma is the most malignant of cancers, but the actual fatality rate is 50–60% (Clark, 1994). It does have the capacity to metastasize to a wide variety of organs throughout the body (Rusciano and Burger, 1992). Unlike most other cutaneous cancers, tumor progression in melanocyte neoplasia commonly leads to metastasis (Clark et al., 1984). The pigmentation and surface location allows identification of early lesions when only 0.1–0.2 mm diameter and the progression from such small benign lesions to metastatic malignancy allows systematic study of the full range of lesional entities. Such studies have taken melanocytic neoplasia from one of the less understood forms of malignant progression to probably the most completely described form. One particularly significant distinction made in these studies is that metastases only occur when cells acquire the competence to proliferate in the dermis as seen in the VGP, as opposed to their simple extension into the dermis, which can occur even in the nonneoplastic stages.

VIII. CHARACTERISTICS OF CULTURED HUMAN MELANOCYTES ISOLATED FROM DIFFERENT STAGES OF MELANOMA PROGRESSION Melanocytic cells from normal skin, common acquired congenital nevi, radial (RGP) and vertical (VGP) growth phases of primary melanoma, and from metastatic melanoma were reported to have properties in cell culture that reflect their original state of tumor progression in vivo (Herlyn et al., 1985b). The relative phenotypic stability of cultured melanocytic cells can be explained by their karyotypic stability because they maintain the same chromosomal abnormalities in freshly isolated melanomas and both short‐ and long‐term cultured cell lines derived from the same lesions. The growth characteristics of cells from various stages of progression are shown in Table III. It can be seen in Table III that the normal melanocytes from newborn foreskins and from the nonmalignant nevi assume a spindle shape in cell culture, but so do cells from the single RGP primary tumor, and 4 of the 7 VGP tumors, the other three being epithelioid. Cells from the two metastatic tumors were epithelioid. The normal melanocytes and those from the nevi had a limited lifespan in culture, whereas the cells from the primary tumors and from metastases grew indefinitely. It should be noted, however, that indefinite growth may arise from a minority of cells in the original tumors, especially in the case of the RGP, which has a very low capacity for colony formation in agar and produce no tumors in nude mice, nor do they lead directly to metastases (Clark et al., 1984, 1986). In contrast, there is significant colony formation over a wide range in agar by cells from the VGP that

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Table III Growth of Cultured Melanocytic Cells Isolated from Newborn Skin, Nevus, and Primary and Metastatic Melanoma

Origin

Morphology

Normal melanocytes from Newborn Spindle foreskin (2) Common Spindle acquired nevus (2) Congenital Spindle nevus (2)

Indefinite growth in vitro

% colony formers in agar

Tumor formation in nude mice

No

0.012–0.024

No

No

0.014–1.03

No

No

0.47–0.84

No

RGP Early primary melanoma (1)

Spindle

Yes

<0.001

No

VGP Late primary melanoma (7) Metastatic melanoma (2)

3 spindle and 4 epithelioid Epithelioid

Yes

3.7–19.1

Yes

Yes

22.7–31.8

Yes

Values in parentheses indicate number of original samples. Abstracted from Herlyn 1985b.

is exceeded by the cells from the two metastases, and all the tumors in these two categories produced tumors in nude mice. Chromosome studies were done on direct preparations, early and late passage cell cultures from the various stages of melanoma progression (Balaban et al., 1984). Nevus cells had a normal karyotype whereas the early passage tumor of the RGP was pseudodiploid with an extra copy of chromosome 6p translocated onto chromosome 22, as well as other abnormalities. The VGP‐advanced primary and metastatic melanomas were aneuploid with multiple aberrations and nonrandom abnormalities of chromosomes 1, 6, and 7. The overall similarity of karyotype between VGP with its growth in the dermis and metastasis is consistent with the observation that metastases arise only from cells of the VGP, although metastatic spread from this phase is not the rule (Clark, 1994). As noted earlier, it is only these two stages that have a high efficiency of colony formation in soft agar and are tumorigenic in nude mice (Herlyn et al., 1985b). Their cells had similar morphology and plating efficiency in cell culture (Herlyn et al., 1985a). However, the metastatic cells generally had a shorter population‐doubling time, growth to a higher cell density, higher tyrosinase activity, and more pigmentation than do cells of the VGP.

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The colony‐forming efficiency of the metastatic cells in agar was also somewhat higher than that of the VGP cells, and they could be found growing in peritoneal exudates, indicative of a loose relationship to one another. On the one hand, monoclonal antibodies to VGP and metastatic cells bound poorly to nevus cells. On the other hand, antinevus antibodies bound to melanocytes, nevus cells and RGP primary melanoma cells but not to VGP or metastatic cells (Herlyn et al., 1985b).

IX. IS THERE A RELATIONSHIP BETWEEN THE CELL CONTACT INTERACTIONS THAT SUPPRESS NEOPLASTIC DEVELOPMENT AND THE PHENOMENON OF METASTATIC DORMANCY? The major characteristics of the suppression of the neoplastic phenotype can be summarized as follows. Studies with fibroblasts in culture revealed that cells transformed by viruses (Rubin, 1960a; Stoker, 1964), carcinogenic chemicals (Mehta et al., 1986), or spontaneously (Rubin, 1994) assume the morphology and growth‐regulatory behavior of normal cells when surrounded by, and in full contact with an excess of the normal fibroblasts. The latter had to be confluent, and under contact inhibition themselves before the suppression could take hold (Rubin, 1960a, 1994). The suppression is brought about by contact between the plasma membranes of the normal and transformed cells in the same manner that the normal cells induce contact inhibition among themselves, by restricting the membrane activity of the cells. Although no specific attempt was made to determine whether epithelial cells could suppress transformation in fibroblasts, it seems unlikely since there is no contact inhibition between normal cells of the two cell types (Eagle and Levine, 1967). Basically similar results are found when neoplastic epithelial cells are brought in contact with homotypic normal cells. Papilloma cells are suppressed in contact with an excess of normal keratinocytes both in vitro and in vivo, but not by fibroblasts (Hennings et al., 1990; Strickland et al., 1992). However, epidermal carcinoma cells are not suppressed by the keratinocytes (Strickland et al., 1992). Rat hepatocarcinoma cells of stem cell origin take on the phenotype of normal hepatocytes when transplanted into the normal liver as solitary cells which come into contact with hepatic plates (McCullough et al., 1998). Human melanoma cells that are metastasis‐ competent in severely immunodeficient mice have obviously escaped contact with and regulation by keratinocytes, but they resume that contact and are normalized when transfected to overexpress the E‐cadherin gene (Hsu et al., 2000b). Such observations raise the question whether metastatic

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dormancy results from contact of the neoplastic cells with the heterotypic cells of the organ in which they are lodged.

X. CHARACTERISTICS OF METASTATIC DORMANCY Evidence for metastatic dormancy arose when rats were injected intraportally with as few as 50 carcinosarcoma cells but failed to develop liver tumors by 20 weeks unless they were subjected to weekly laparotomy and hepatic manipulation, beginning at 12–13 weeks (Fisher and Fisher, 1959). The 100% incidence of liver tumors in the treated animals indicated the neoplastic cells remained dormant, yet retained all of their former capacity to proliferate. Dormancy was recognized clinically in human cancer with the occurrence of metastases years after removal of the primary tumor (Demicheli et al., 1996). However, the status of the cancer between the time of the primary treatment and metastatic recurrence was unknown. Several factors were suggested as possible contributors to metastatic dormancy, one of which was the survival in tissue of solitary cancer cells that are neither proliferating nor undergoing apoptosis. Concrete evidence for such solitary cells came from experiments in which melanoma cells labeled with fluorescent nanospheres were injected into the mesenteric vein of mice to target the liver (Luzzi et al., 1998). Eighty percent of the injected cells survived in the liver microcirculation and extravasated by day 3, but that number decreased to 36% at 13 days (Fig. 1). Only 1 in 40 of the extravasated cells formed micrometastases (4–16 cells) by day 3, and only 1 in 100 of these

Time (p.i.)

Solitary cells

Micrometastases Tumors (4–16 cells)

Total loss (%)

0

100%

Inject 3⫻105 B16F1 cells

0

90 min

87.4

3d

81.4

2.04

13 d

36.1

0.07

12.6

16.6

0.02

63.8

Fig. 1 Flow chart summarizing survival data in the liver of mouse melanoma cells injected intraportally to target mouse liver. See text for details. From Luzzi et al. (1998).

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progressed to macroscopic tumors by day 13. In contrast to the solitary cells and the micrometastases, more than 90% of the cells in the macroscopic tumors were proliferating. Thus, in this model, metastatic inefficiency was principally determined by failure of solitary cells to initiate growth, and failure of early micrometastases to continue growth into microscopic tumors. The results agree with the earlier finding that virtually all melanoma cells injected in the chick embryo chorioallantoic membrane survive in the microcirculation and successfully extravasate by 24 h (Koop et al., 1995). In the chick embryo model, extravasation is independent of metastatic ability, and occurs even when normal mouse embryo fibroblasts are injected (Koop et al., 1996). The implication of these studies is that the primary determinants of metastatic inefficiency are the postextravasation survival and growth of cells. The results indicate that early cell destruction in the microcirculation, an inability of cells to extravasate and failure of angiogenesis are not major contributors to metastatic inefficiency. Two lines of mouse mammary cancer cells, one that was highly metastatic and the other poorly metastatic for the liver, produced tumors after injection into the mammary fat pad (Naumov et al., 2002). Solitary cells from both cell lines were recognizable in the liver after appearance of the primary tumors, indicating that the cells had carried out the first steps of metastasis. To study the kinetics of solitary cell survival, the cells were labeled with nanospheres and injected intraportally. By 3 days, all observed cancer cells of both types had extravasated (Fig. 2). There was a small loss of the poorly metastatic cells shortly after injection, but there was no further loss of cells up to 3 weeks, and about 80% of them remained as undivided cells.

% Solitary cell survival

100 80 60 40 20 0 90 min

3

10

14

18

21

77

Time post-injection (days)

Fig. 2 Long‐term survival in the liver of poorly metastatic solitary mammary carcinoma cells injected intraportally to target the mouse liver. See text for details. From Naumov et al. (2002).

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Even after 11 weeks, about 50% of the poorly metastatic cells that were injected remained as solitary cells, with low levels of both apoptosis and proliferation. In the case of the highly metastatic line, all cells had extravasated by 3 days, and most of them (64%) remained as solitary, undivided cells at 10 days. A small population (0.6%) had begun to form metastases by 10 days, but their number decreased 100‐fold by 14 days. By contrast, the number of solitary cells fell by only a factor of 3, indicating that the overall rate of loss of early metastases was almost 2 orders of magnitude greater than that for solitary cells. Although the tumor burden increased to 70% of the liver in 21 days, surprisingly large numbers of the dormant solitary cells could still be seen, and they were similar in appearance to those of the poorly metastatic line. The proportion of remaining solitary cells of the highly metastatic line could not be estimated beyond 21 days because of the large tumor burden in the liver, but those of the poorly metastatic line remained fully viable in the liver parenchyma through the last point taken at 11 weeks. They could therefore account for the occasional metastases in the liver in some mice after long latency periods. The line of melanoma cells used to study the fate of disseminated cells in the liver was used for the same purpose after targeting to the lung (Cameron et al., 2000). As in the liver, a large proportion of melanoma cells extravasated and solitary cells showed an initially slow decline to 74% by day 3, a rapid decline to 25% by day 4, followed by a steady decline to 3.5% by days 12–14. This contrasts with 36% solitary cells at 13 days in the liver (Luzzi et al., 1998). However, the percentage of multicellular foci at 13–14 days in the lung was much higher than those in the liver at the same time, and the overall metastatic efficiency was about 10% in the lung as compared with 0.002% in the liver. The results indicate a sharp difference in growth response of the melanoma cells in the microenvironment of the two organs. The above experiments were initiated by intravenous injection of mouse cancer cells targeted to the liver or lung, but a different set of experiments began with human mammary cancer cells that were inoculated into the mammary glands of nude mice to form primary tumors there (Goodison et al., 2003). After tumors appeared in the mammary gland from isogenic, nonmetastatic, and metastatic lines, cells that escaped spontaneously from both cell lines were seen in the lungs. No metastases were seen in the lungs by 6 months with the nonmetastatic line despite the continuing presence of scattered cancer cells. These self‐disseminated human tumor cells were retrievable from the tissues long after resection of the primary tumor, as manifested by indefinite proliferation in vitro and local tumorigenicity in the mammary gland. Scattered tumor cells from the lung were still immortal but were rendered indefinitely quiescent by the microenvironment conditions in the lung tissue. In contrast, many cells from the metastatic line grew into metastases in the lungs, but others remained solitary and quiescent.

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Therefore, even in a clonally‐derived cell population with metastatic properties, many cells do not mobilize the organ‐specific growth properties needed to generate metastases. The studies with the human mammary cancer cells were extended to include a variety of organs in which no spontaneous metastases were formed from primary tumors, in addition to the lungs and lymph nodes where the metastatic line usually produced growth (Suzuki et al., 2006). Dormant cells were recovered from metastasis‐free organs 3 months after injection into mammary glands, including cells in those lungs and lymph nodes that had no metastases. Cells that grew indefinitely in culture and could induce primary mammary tumors were retrieved from blood, lungs, lymph nodes, spleen, bone marrow, liver, and kidneys from mice in which the primary tumor was not resected, but not from the blood when the tumors had been resected. Cells recovered from the high metastatic line also produced metastases in the lung and lymph nodes. Although the resolution of light microscopy was not always sufficient to establish unequivocally whether the spontaneously metastasized labeled cells were intra‐ or extravascular, there was convincing evidence in some instances that the tumor cell had departed from the blood and entered the tissue of the host organ. Further evidence for extravasation and entry into the tissue was the failure to detect the cancer cells in blood cultures of the mice 1–4 weeks after resection of the primary tumors. The results showed that the growth of disseminated cancer cells in all the organs tested can be suspended for 6 months or longer by the microenvironmental conditions, although growth was still active in other organs of the same host.

XI. TUMOR CELL ADHESION TO CELLS IN DISTANT ORGANS A. Endothelial Cells The question to be addressed at this point is whether metastatic dormancy has any relation to the suppression of tumor development by cell–cell interactions between preneoplastic and neoplastic cells with an excess of their normal homotypic counterparts. Because the best established cases of homotypic suppression of tumor development result from direct contact between the normal and neoplastic cells, evidence was first sought for heterotypic contact interactions between disseminated cancer cells (DCC) and the cells of their host organs that might account for metastatic dormancy. As might be expected, the studies of adhesion in metastasis have focused upon its role in the promotion rather than suppression of metastatic lesions. However, the major features of the metastatic cell adhesion literature will be examined to

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learn whether it reveals hidden evidence of suppression. The first tissue encountered between DCC and an organ in which they may lodge is, of course, the endothelial cell surface of the organ’s vasculature. It has long been considered that adhesion of the DCC to specific organ microvessel endothelial cells is an important event in determining organ‐specific metastatic growth (Nicolson, 1988). B16a melanoma cells injected into the tail vein of mice appeared in the lung and were arrested there by contact with the endothelial plasma membrane (Crissman et al., 1985). The endothelial cells were gradually displaced by tumor cells which achieved contact with the vascular basement membrane at 4 h. Mitotic figures were evident by 24 h, and the tumor appeared to proliferate intravascularly along the basement membrane. Extravasation occurred through tumor cell proliferation and destruction of the vascular basement membrane. In the case of B16F10 melanoma cells, there was a partial retraction of the endothelial cells following attachment of the tumor cells, which then attached to the basement membrane and the basolateral endothelial cells (Lapis et al., 1988). The endothelial cells extended to cover the tumor cells, which proliferated to fill the lumen. The endothelial layer became mechanically disrupted and the tumor cells extravasated. In both cases contact of the tumor cells with endothelial cells led to proliferation of tumor cell growth. The specificity of adhesion between murine tumor cells and capillary epithelium was addressed with a panel of eight different histological types of tumor cells interacting with endothelial cell monolayers from four different organs (Auerbach et al., 1987). The tumor cells differed in adhesive propensity for different endothelial cells. Some, but not all, of the adhesive preferences correlated with the in vivo metastatic behavior of the tumors, indicating that endothelial cell surface‐associated specificities may play a significant role in determining the pattern of metastases. Two sublines of a large cell lymphoma line were selected for enhanced liver and lung colonization and tested for adhesion to mouse liver sinusoidal endothelium, lung microvessel endothelium and bovine aortic epithelium (Belloni et al., 1986). Only the selected lung‐colonizing melanoma cells adhered preferentially to lung endothelium, but both the lung and liver‐ colonizing lines adhered to hepatic sinus endothelium significantly more than the parental line did. These and other results suggested that organ specificity of metastasis may be determined in part by specific tumor cell‐endothelial cell interactions (Nicolson, 1988). Human fibrosarcoma cells injected intravenously into rodents attached to the endothelia of pulmonary precapillary arterioles and capillaries (Al‐Mehdi et al., 2000). Proliferation of the tumor cells in this case originated intravascularly, and was restricted to those attached to the endothelium. The intravascular origin of the proliferation was made especially clear by the technique of labeling the vascular surface of the endothelial cells with

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acetylated low‐density lipoprotein, and labeling the tumor cells with green fluorescent protein. The accumulated results show that attachment of DCC to endothelial cells results in their proliferation rather than dormancy.

B. Parenchymal Cells An alternative to the origin of metastatic dormancy of DCC by adhesion to endothelial cells is their adhesion to the parenchymal cells of the host organs after extravasation. Experiments suggest that endothelial cells express the same or nearly the same organ‐specific molecules as do parenchymal cells from the same organ (Nicholson, 1988). Cell adhesion studies have been conducted with malignant cells and parenchymal cells from their target and nontarget organs. Sublines of B16 mouse melanoma cells were developed that had undergone increasing numbers of selective subcutaneous or intravenous passages for capacity to produce increasing metastatic growths in the lungs of mice (Nicolson and Winkelhake, 1975). Cells from the lung metastases were suspended and mixed with cells from different organs prepared to exclude membrane debris, erythrocytes, platelets, and cell aggregates. The degree of aggregation between the melanoma cells and lung cells increased with increasing rounds of selection of the melanoma cells, which also produced increasing lung metastases. In contrast, there was no correlation of aggregation with metastasis in mixtures of the lung‐ colonizing melanoma cell lines with cells from liver, spleen, and kidney. Furthermore, the maximum binding of the melanoma cells to any of these organs was much lower than it was to lung cells. Many other studies indicate that highly metastatic cells adhere at greater rates or more extensively to parenchymal cells of target rather than nontarget organs. For example, liver‐ metastasizing lymphoma cells bound to isolated liver parenchymal cells in proportion to their capacity to colonize the liver in vivo (McGuire et al., 1984; Schirrmacher et al., 1980). Sublines of large cell lymphoma cells were selected by in vivo colonization of the liver and produced extensive metastasis to the liver. They also bound much more selectively to aggregates (McGuire et al., 1984) or frozen sections (Kieran and Longenecker, 1983) of liver cells than to nontarget organ cells or tissues. Spleen‐metastasizing leukemia cells bound to isolated spleen cells but not to lung cells (Phondke et al., 1981). That such preferential adhesion to target organs is mediated by specific cell receptors was shown by blocking adhesion to liver cells in vitro and liver metastasis in vivo with antibodies to cell surface determinants on lymphoma cells (McGuire et al., 1984). In conclusion, tumor cell adhesion mechanisms for endothelial and parenchymal cells are mediated by different multiple adhesion molecules operating in parallel, and they commonly use normal cell adhesion processes

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(Belloni and Nicolson, 1988; Nicolson, 1988). Since the adhesion of DCC to heterotypic cells of distant organs enhances the proliferation of the tumor cells in metastasis formation, it stands in sharp contrast to the suppression of the tumor development by adhesion between early stage neoplastic cells and an excess of homotypic normal cells, as most clearly illustrated in the interactions between initiated and normal epidermal cells (Hennings et al., 1990; Strickland et al., 1992).

XII. POSSIBLE ALTERNATIVE EXPLANATIONS OF METASTATIC DORMANCY A hint of a possible corollary to metastatic dormancy may be found in the failure of epidermal cells to invade the underlying dermis despite frequent breaches in the basement membrane (Tarin, 1972). It is also seen in the failure of epidermal cells to grow in the underlying mesenchyme when commonly deposited there by ordinary needle injections through skin (Gibson and Norris, 1958). A closer example to metastatic dormancy comes from experiments in which enzymatically dissociated normal thyroid cells were intravenously injected into mice, and lodged in the lung (Taptiklis, 1968). Continuous ingestion of methylthiourea, which inhibited the production of thyroxin by the host thyroid gland in the experimental mice, prevented the feedback inhibition of thyroid‐stimulating hormone. The resulting overproduction of thyroid‐stimulating hormone led to proliferation and follicle formation of the thyroid cells that had lain dormant in the lung for as long as one year. The evidence indicated that the normal thyroid cells penetrated the endothelial barrier and remained dormant in the interstitial tissue of the lung. Subsequent experiments with intravenous injection of dissociated normal, hyperplastic, and neoplastic thyroid cells into methylthiourea‐ingesting mice revealed a common ability to penetrate the endothelium by intravascular proliferation, and migration of the thyroid cells to an extravascular position where they proliferated (Taptiklis, 1969). The vascular penetration appears to occur by unstimulated cells of all three types, and was later also found to occur in unstimulated chick embryos inoculated intravenously with normal and transformed mouse fibroblasts (Koop et al., 1996). Even the neoplastic thyroid cells remained dormant in the absence of thyroid‐stimulating hormone, indicating their hormone dependence. In the presence of the hormone, the neoplastic thyroid cells lost polarity, grew much more rapidly than either the normal or the hyperplastic cells, invaded the bronchi, and completely obliterated the acinar spaces. The overall results are consistent with the idea that metastatic dormancy arises from the paucity of growth‐stimulatory

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factors in host organs, rather than any suppressive effect of heterotypic cell–cell interactions. Further evidence for a role of growth factor deficiency in maintaining metastatic dormancy comes from experiments on the invasiveness of human epidermoid carcinoma, which is enhanced by the binding of urokinase plasminogen activator to its receptor on the cancer cells (Yu et al., 1997). If the expression of the receptor is blocked, the carcinoma cells enter a state of dormancy, which resembles that observed with human cancer metastasis. Although the dormancy was protracted, the cells eventually emerged to initiate progressive growth and form metastases, indicating that other factors can compensate for the lack of a full complement of surface receptors. High expression of the receptors and binding of the plasminogen activator entrains signaling pathways which increase the pro‐ proliferative balance of a high ratio of extracellular signal‐regulated kinase ERK to p38 (Aguirre‐Ghiso et al., 2003). The organ specificity of metastasis development has been associated with receptors for soluble growth factors in the vasculature of target organs (Pasqualini and Ruoslahti, 1996). Endothelial cells appear to express phosphorylated receptors for platelet‐derived growth factor when they are exposed to tumor cells that produce the growth factor (Uehara et al., 2003). Hence, such tumor cells that are carried to an organ that does not have the receptors may remain dormant. A discontinuous 70 cM (centimorgan) region of human chromosome 17 contributes to the dormancy of a rat line of prostatic cancer cells (Chekmareva et al., 1998). The growth inhibition seems to result from an effect of one or more genes at the metastatic site, and not from a circulating angiogenesis inhibitor. However, angiogenesis plays a limiting role in the growth of selected model systems at the primary site of cell inoculation (Naumov et al., 2006), and might play a similar role in limiting the size of metastases. It is unlikely to be the limiting factor in the dormancy of solitary disseminated cells which feature so prominently in models of metastatic dormancy (Luzzi et al., 1998; Naumov et al., 2002), since they have the same nutritional support as the surrounding normal cells. In any case, the role of angiogenesis in limiting tumor size in the decades‐ long development of some human cancers is less apparent than it is in experimental model systems since most of the cell growth in these tumors occurs at their periphery, where they are fed by diffusion from preexisting normal capillaries (Caspersson and Santesson, 1942); see also Sardari‐Nia et al. (2007) and Pezzella et al. (1997). As a result, the general biosynthetic activity and viability of cells in primary human tumors decreases with distance from the tumor periphery whereas cell size increases followed by necrosis near the center. However, small proliferating cells with high biosynthetic activity can be found more deeply within the human tumors in close proximity to the occasional capillaries that penetrate the tumors.

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There is evidence that soluble factors released within organs may play a role in determining the organ specificity of metastatic growth. Medium conditioned by the growth of each of a variety of mouse organ cultures was used to affect the survival and attachment to the culture dish of cells from 52 spontaneous mammary carcinomas (Horak et al., 1986). Medium conditioned by lung fragments was most successful in supporting the overall survival and attachment of the mammary carcinoma cells, followed by ovary and kidney conditioned media. Liver and thyroid conditioned media had negative effects on almost all the mammary carcinomas. Since mouse mammary tumors develop metastases mainly in the lungs and occasionally in the ovaries or kidneys (if inoculated via the aorta), the effects of these organs on the tumor cells were considered in good agreement with the in vivo observations. Using B16 melanoma cells, it was found that cells with high lung‐ colonizing capacity were growth stimulated by lung‐conditioned medium significantly more than by conditioned medium from other tissues (Nicolson and Dulski, 1986). Similar results were obtained with high ovary‐colonizing capacity B16 cells stimulated by ovary‐conditioned medium. In contrast, however, the growth of brain‐colonizing B16 cells was not stimulated by factors released from brain tissue. Hence, it appears that metastasis of B16 melanoma cells to specific organ sites is dependent not only on preferential target–organ adhesion but may be supplemented by soluble organ‐derived factors.

XIII. MOLECULAR BASIS OF CELL–CELL ADHESION Cell–cell adhesion plays a central role in developmental biology, normal cell function, and neoplasia, and efforts to identify the underlying molecular components of this adhesion have been made in all these fields. Conspicuous efforts were made to identify the membrane proteins involved in the specific sorting out of cells from different tissues during embryonic development, and were recently reviewed (Rubin, 2007). Emphasis was placed on the role of cadherins (Gumbiner and Yamada, 1993; Takeichi, 1991), but the number of molecules of this type is large, and cross adherence among them is so common that distinctive association with particular cell–cell associations could not be achieved (Foty and Steinberg, 2004). As a result, it was recommended that the understanding of adhesive relationships would be better served by focusing on functional measurements of differential cohesive and adhesive relationships of living cells in cell sorting and tumor development (Foty and Steinberg, 2004). This conclusion is reinforced by the large number of other classes of adhesion molecules, which along with

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cadherins, are decreased in some tumors (Mareel et al., 1992) and increased in others (Auersperg et al., 1999; Bindels et al., 2000). Somewhat surprisingly in view of the loss of adherence between cells in human colon cancer (McCutcheon et al., 1948), there is a typical increase in the adhesion molecule carcinoembryonic antigen (CEA) in these cases (Shuster et al., 1980). This increase has been attributed to the shifting of the small amounts of CEA in the luminal surface of normal columnar colorectal epithelium to large amount along the borders between the cells in cancers where they are thought to displace other adhesion molecules (Benchimol et al., 1989). As noted earlier, there is a downregulation of E‐cadherin during the transformation of melanocytes into malignant melanoma cells and its replacement by N‐cadherin (Hsu et al., 2000b). The decrease in E‐cadherin is accompanied by a loss of adhesion to keratinocytes which regulate the growth of melanocytes, but adhesion and regulation can be restored by transducing and overexpressing E‐cadherin in the melanoma cells. It should be noted, however, that the loss of E‐cadherin is considered but one of the mechanisms for the malignant behavior of melanoma cells (Hsu et al., 2000b), since many other surface antigens are lost during transformation (Herlyn et al., 1987). In the case of metastatic cell adhesion to endothelial receptors, at least seven major luminal glycoproteins common to microvessels derived from six different organs have to be taken into account (Fig. 3; Belloni and Nicolson, 1988). Five of the glycoproteins appeared to be expressed differentially in particular organs. Nicolson concluded (Nicolson, 1988) that “tumor cell adhesion mechanisms (for endothelial cells, basement membranes, parenchymal cells, platelets, etc.) are mediated by different multiple adhesion molecules, operating in parallel, and many if not all tumor cells probably use normal cell adhesion mechanisms. In addition, different adhesion systems are involved in tumor cell adhesion to different structures (endothelium versus basement membrane, etc.). Thus organ specificity is determined at one or more levels of tumor interaction such as at the level of microvessel endothelial cell, basement membrane, parenchymal cell and so on.” He also concluded that differential adhesion properties alone are probably insufficient for determining the organ specificity of metastasis, and other mechanisms such as tumor autocrine and paracrine growth factors are likely to be involved. Nicolson’s observations on the number of plasma membrane components likely to be involved in metastatic formation are similar to those made about the multiplicity of cadherins in the homophilic adhesion in the specific sorting out of cells in embryonic development (Foty and Steinberg, 2004). Integrins are also involved in metastatic adhesion, as in the case of 31 integrin interaction with laminin 5 of the vascular basement membrane (Wang et al., 2004). There again the integrin–laminin binding is only part

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700

600

Kidney

500

Intensity

Spleen 400 Liver 300 Lung 200 Heart 100 Brain 0 200

116 97.5

68

43

Apparent Mr

Fig. 3 Densitometric scan of iodinated microvascular endothelial cell surface proteins from various mouse organs. Arrows indicate differentially‐expressed surface proteins. From Belloni and Nicolson (1988) and Nicolson (1988).

of the story since anti‐integrin antibody only partially reduced pulmonary arrest of metastatic cells. The presence of multiple adhesion molecules on the surface of parenchymal cells would buffer the cells of intact tissues against mutations in any one of them, and help to explain the maintenance of their normal phenotype in the face of myriad genetic changes found in every cell (Rubin, 2006).

XIV. CONCLUSIONS 1. Virtually all the examples of suppression of the neoplastic phenotype in Tables I and II involve contact between an excess of normal regulated cells in contact with solitary homotypic cells of neoplastic potential. The basic principles of cell contact suppression were first adduced from studies of

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3.

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fibroblast cocultures, but since there were no different degrees of transformation in the fibroblasts, the examples shed no light on the relative susceptibility to suppression at progressive stages of transformation. The most informative observations on the latter point came from studies on normal keratinocytes mixed in excess with initiated papilloma cells or with carcinoma cells. There was strong suppression of the papilloma cells by normal keratinocytes but no suppression of the epidermal carcinoma cells (Strickland et al., 1992). Cell contact was required for suppression of the papilloma cells but GJC was not (Hennings et al., 1992). Hepatocarcinoma cells of moderate malignancy were fully normalized when transplanted as solitary cells in the liver plates, but a more malignant line of hepatocarcinoma cells was only partly modulated in the same conditions (Coleman et al., 1993; McCullough et al., 1998). The implication is that a large intact organ predominantly composed of a single type of parenchymal cells is a more effective modulator of neoplastic behavior than mixtures made in 2‐dimensional cell cultures like those of keratinocytes and neoplastic epidermal cells. There is broad tissue specificity in the suppression of papilloma cells, since fibroblasts do not suppress them (Hennings et al., 1990; Strickland et al., 1992). Similarly, the hepatocarcinoma cells proliferated at a maximal rate when transplanted subcutaneously or leaked from the liver into the peritoneal cavity (Coleman et al., 1993; McCullough et al., 1998). However, in neither case was any attempt made to ascertain the effect of epithelial cells other than those of the same tissue origin as the neoplastic cells. In one atypical case, esophageal cells inhibited tracheal carcinoma cells, but only a small minority of the normal cells was required and most likely involved competition for attachment to the tracheal mesenchyme (Terzaghi‐Howe, 1987). The normalizing effect of liver on the hepatocarcinoma cells declines as the organism ages. This suggests that the ordering capacity of normal cells decreases with age, which may contribute to the widely observed increased incidence of solid epithelial cancers with age. Epidermal cells in an early stage of initiation allows them, unlike normal keratinocytes, to multiply in vitro in adequate calcium, but to make normal epidermis rather than papillomas in grafts. Despite their normal appearance and function, they have lost the capacity to suppress papilloma growth. This also suggests that the immediate cellular microenvironment in chemical carcinogenesis plays a dynamic role in determining the development of tumors. It has, for example, been shown that sustained exposure of skin to ultraviolet light results in the expansion of intraepithelial neoplastic clones, mainly by damaging the suppressive normal epidermis rather than mutagenizing the clones (Mudgil et al., 2003; Zhang et al., 2001).

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6. Little work has been done to identify the adhesive molecules in contact suppression of neoplasia by normal cells. Analysis of adhesion‐driven sorting out of embryonic cells from different tissues indicates that the large number of cadherin subtypes and their overlapping function are too dynamic and complex to evaluate the contribution of each type to the process (Foty and Steinberg, 2004). The authors propose that quantitative, functional measurements of cell–cell adhesion provide a more reliable predictor of tissue‐specific sorting out than does quantitation of various adhesion molecules. The same conclusion may be applicable in tumor suppression in light of the inconsistency of relating particular adhesion molecules to malignant transformation (Rubin, 2007). 7. Almost all the major insights on cell–cell interaction in tumor suppression in Tables I and II came from operational experiments without regard to molecular reduction. This type of operational experiment seems to have largely disappeared in recent years, but its usefulness has hardly been exhausted. There are a number of operational experiments that can be done to further evaluate the role of the tumor suppressive activity of cell contact interactions in neoplastic development. Some such experiments are listed as follows in terms of epidermal carcinogenesis in which the in vivo dynamics of initiation and promotion are best understood, but the basic design can be adapted to other neoplastic cell systems. They include: (a) the effect of age on suppression of papilloma cells by normal keratinocytes; (b) the capacity of other epithelial cells such as those from the esophagus to suppress epidermal papilloma cells; (c) comparison of the suppressive capacity of keratinocytes from sensitive and resistant mouse strains; and (d) the effect of carcinogen treatment on the suppressive capacity of keratinocytes. Given the insights already obtained from the normalization of hepatocarcinoma cells transplanted into the liver, the development of a cell culture model of this system would permit a range of quantitative, functional experiments that were not possible to do in vivo. 8. The tumor suppressive effects of a large number of contacting homotypic normal cells may be subsumed under the basic biological principle of “order in the large over heterogeneity in the small” (Elsasser, 1998; Rubin, 2006, 2007). The great heterogeneity of cancer cells has been demonstrated for many behavioral characteristics (Fidler, 1978; Heppner, 1984), and more recently for mRNA sequences (Brulliard et al., 2007). Cellular (Fry et al., 1966) and molecular genetic (Bahar et al., 2006) heterogeneity are also characteristic of aging. Such age‐ related increase in the heterogeneity of cells could account for the reduced capacity of aging organs to normalize the behavior of neoplastic cells (Coleman et al., 1993; McCullough et al., 1998). 9. There is no credible evidence that heterotypic cell–cell adhesion induces metastatic dormancy. Rather, the adhesion of DCC to endothelial or

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parenchymal cells activates metastatic growth, but fortunately involves only a small fraction of the disseminated cells (Luzzi et al., 1998; Naumov et al., 2002; Suzuki et al., 2006). Significant factors in metastatic dormancy are the lack of capacity of most cancer cells to adhere to heterotypic receptors of host organ cells, and the lack of specific soluble growth factors at the site. The requirement for adhesion in metastasis may be related to the requirement for cells to attach to and spread on a solid substratum in culture in order to proliferate. 10. Efforts have been made to characterize the cell surface molecules of endothelial cells that might bind DCC to initiate metastases (McGuire et al., 1984; Nicolson and Winkelhake, 1975). A large number of such molecular species are common to many different organs but vary in quantity; there are however few that are specific to individual organs (Fig. 3; Belloni and Nicolson, 1988; Nicolson, 1988). Antibody to a particular endothelial surface integrin only partially reduced the frequency of metastases to the lung of fibrosarcoma cells (Wang et al., 2004). As was the case with cadherins in cell sorting, the number of molecules that bind metastatic cells is likely to be large and cross‐ reactive, so a full accounting of their role in promoting adhesion will be complex. Some idea of the complexity of the factors involved in metastasis is seen in Fig. 4 (Nicolson, 2002), which was later considered

Tumor and host microenvironment Platelets Growth factors Coagulation factors Enzymes

T cells Cytokines Cytolytic factors Degradative enzymes Macrophages Cytolytic factors Cytostatic factors Mutagenic factors Growth factors

Matrix

Tumor cells Cytolic factors Chemotactic factors Degradative enzymes Procoagulants, MIFs Secreted antigens Parenchymal cells Growth factors Growth inhibitors Nutritional factors Hormones, proteins

Endothelial cells Growth factors Growth inhibitors Fibrinolytic factors Enzymes, nutrients

NK & LAK Cells Cytokines Cytolytic factors Degradative enzymes

Mast cells Growth factors Glycosaminoglycans Extracellular matrix Cytokines, histamine Degradative enzymes

Fibroblasts Growth factors Differentiation factors Inductive factors Other cytokines Extracellular matrix Degradative enzymes Enzyme inhibitors

G. L. Nicolson 8/92

Fig. 4 Tumor and host microenvironmental factors involved in metastasis. From Nicolson (2002).

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to represent an underestimate of the number of molecules involved (Mareel, 2004). Physical measurements of the strength of adhesions among living cells, e.g., (Coman, 1944; Foty et al., 1996; McCutcheon et al., 1948) could be a more productive approach to their meaning for metastasis. Gene expression profiles characteristic of the metastatic process indicate a formidable degree of complexity. About 75 host‐regulated genes were found to differ in expression between highly and poorly metastatic clones from the same human breast carcinoma (Montel et al., 2006). An expression pattern of 128 genes is required to distinguish primary and metastatic carcinomas (Ramaswamy et al., 2003). No doubt others will be found in other metastatic situations. The problem falls into Elsasser’s rule that “causal chains [in organisms] cannot be traced beyond a terminal point because they are lost in unfathomable complexity. . .” (Elsasser, 1998). Therefore, the pervasive preoccupation with searching for causal chains in neoplasia at the molecular level should be complemented by operational studies on cell–cell interactions to understand the primary microenvironmental aspects of tumor development. The importance of an overarching biological theory (Elsasser, 1998) for structuring and unifying the diversity of observations that are sure to follow cannot be overemphasized.

ACKNOWLEDGMENTS I am grateful for the manuscript preparation and editing by Dorothy M. Rubin. Correspondence with Ann Chambers, Ruth Muschel, Garth Nicolson and David Tarin provided useful insights on modern views of tumor metastasis. Discussion with David Bilder clarified points about Drosophila tumors. George Klein made helpful suggestions about the manuscript.

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