Cytotechnology 0 1990 Kluwer

3: 117-122, 1990. Academic

Publishers.

Printed

in the Netherlands.

Review

Tissue culture in the study of cancer

Salvatore

Casillo’,

Albino

La Peral and Luigi Frati*

‘Istituto Tecnologie Biomediche, ‘La Sapienza’ Roma, Italia

CNR, Roma, Italia;

2Dipartimento

Medicina

Sperimentale,

Universitci

Received 1 September 1989

The goal of cell biologists who study cancer in tissue culture is to understand the behaviour of cancer cells in the intact organism. Since the in situ model in experimental animals is very complex, culture techniques have been developed to study populations of cells in a relatively simplified environment. The metabolic activities of pure populations of cells can be studied. The mechanisms and the regulation of numerous cell functions including growth and migration can be examined directly and quantitated relatively easily. Microenvironments can be created as models of specific in vivo conditions that are likely to occur during cancer development. Last but not least, in many instances information can be obtained more rapidly and much more economically than by the use of tumors in experimental animals. The question that is forever present is whether phenomena observed in culture actually do occur in vivo, especially in the pathogenesis of a disease. For instance, human cancers vary, in a exceedingly complex fashion, in their tendency to invade adjacent tissues and to metastasize to distant sites. At the same time, it is relatively simple to isolate, in culture, populations of cancer cells that vary in their tendency to migrate and to mix with other cell types and to study mechanisms which underlie this variation (Nicholson, 1984; Raz et al., 1986). However, any hypotheses concerning a causal relationship between the invasive behaviour of the cancer cells in the intact organism and mechanisms which underlie their

migratory pattern in culture must be tested in cultured cells that have been reintroduced into systems of varying complexity, including, preferably, experimental animals. Several other points must be kept in mind. When establishing cultures of tumors that arose in viva, isolation techniques should not be such that we are selecting cells out of the total in situ population. In many cases we want a heterogeneous population of cells which reflects the cellular heterogeneity of a malignant tumor (Happner, 1984). Techniques are now available, however, to clone cells as well, so that relatively homogeneous populations can be utilized to study specific questions. When culturing cells under standard conditions on plastic surfaces, we lose the three-dimensional-geometry and the special interrelationship between cells and tissues which exist in the intact organism. These interrelationship control cell proliferation, differentiation and metabolism in normal tissues. Therefore, explantation into two dimensional cultures will have profound effects on normal cells. In cancer cells the responsiveness to environmental controls is disturbed. It follows that the effects of explantation into cultures will differ between normal and cancer cells from a given tissue. Therefore, extrapolation of observations made in culture to the intact organism must be made with great care, and attempts should be made to reproduce in culture the essential conditions present in vivo (Leighton and Tchao, 1984).

118 With these speculations in mind, tissue culture is invaluable for studying the natural history, variability causation and treatment of cancer without the complexities of tumor/host interactions .

Cancer

as a clinical

entity

In spite of the immense amount of thought and research that has gone into the study of cancer, no satisfactory definition of this disease at the cellular level exists and no specific cellular change has been identified that is both consistently present and essential for the malignant state. As pointed out by Foulds (1959), ‘malignancy is essentially an abstract concept based on medical practice; it is not a biological entity’. In medical practice, a cancer can be defined as a destructive mass of abnormal cells which proliferate inappropriately and progressively spread to sites beyond the confines of the normal tissue of origin and, if not treated, will eventually kill their host. The development of cancer, after the initial action of carcinogen(s), is a complex, slow process which, may take many years. In the course of this process, cells undergo gradual, stepwise changes at the genetic and epidegenetic levels. These are inherited from one cell generation to the next and irreversible alter many phenotypic cellular characteristics. In the course of this progression the cells populations proceed from the initiation phase where the carcinogen effects are latent (phenotypically undetectable), to a stage where the cells show limited morphologic and functional alterations but still obey most regulatory mechanisms. Clinically such cell population are recognized as premalignant lesions. These are characteristically limited in location to the site of a normal tissue of origin, and are usually curable by surgical removal. Subsequently the cells become increasingly autonomous (independent) from homeostatic control mechanisms, including those that regulate cell proliferation and cell migration. As a result, in the fully malignant state, cancer cells replicate persistently and spread into adjacent tissues (local invasion) and

to distant sites (metastasis). Tissue destruction by invasion and metastasis is the most common direct cause of death due to cancer. Another important characteristic of malignant neoplasms, which is related to the development of autonomy, is their phenotypic and genetic instability and variability (Heppner, 1984). As a result, cancer cells have a remarkable capacity to adapt to environmental changes and to overcome harmful influences. Such adaptation occur both by the selection of genetically altered subpopulations and by hereditary epigenetic changes. It is this characteristic heterogeneity of cancer cells that poses the greatest barrier to consistently successful therapy. The basis for phenotypic variability and for phenotypic stability changes in the course of carcinogenesis: variability becomes determined to an increasing degree by hereditary genetic or epigenetic abnormalities acquired by the cells with malignancy, while at the same time the cells become refractory to extracellular influences. Cell functions thus become controlled autonomously, i.e. independently of the environment (autocrine stimulation, Laug et al., 1985) and a result, the stability of the malignant phenotype relative to host influences is increased beyond the normal level (Auersperg and Finnegan, 1974). Clinically, the most significant consequence of autonomy is uncontrolled growth in terms of cell replication and cell movement. However, the functional and structural diversity among tumor of the same histologic origin similarly appears to be the result of a loss of responsiveness to both the developmental and the environmental cues that control normal tissues, and the substitution, for these cues, of abnormal regulative influences inherent in the malignant cells. This substitution accounts not only for hystologic variation among tumors, but also for the increased capacity of neoplastic cells to maintain similar characteristics under varying physiologic and anatomic conditions within the organism, as observed, for example, in the histologic resemblance between primary tumors and their metastases.

119 Cancer

cells in culture

The phenotypic cellular changes which accompany transformation to malignancy are too numerous and complex to review here. Among those which are responsible for the characteristics of cancer cells in tissue culture are, (a) reduced requirements for anchorage (attachment to a solid surface) (Pollack et al., 1984) and for complex culture media, growth factors and sera to support proliferative activity; (b) increased cloning efficiency (the capacity to form colonies from single cells), indicative of increased independence from environmental influences; (c) alterations of the cell surface and of the cytoskeleton, increased protease secretion. and changes in the production of the requirements for extracellular matrix components; all of these contribute to abnormalities in cellular morphology, intercellular associations and cell-cell communication (Liotta et al., 1986; Raz et al., 1986). As a consequence, regulation of cell proliferation and movement is disturbed and results in the abnormal growth patterns which distinguish cultures of malignant cells (Pollack et al., 1984). Interestingly, there is increasing evidence that malignant cells overcome the breakdown in the normal interrelationships with their environment by substituting autonomous signals for those signals that are normally supplied from other sources. Examples here are the autocrine secretion of modified growth factors and the synthesis of modified growth factors receptors which are permanently activated and ligand independent (Feramisco et al., 1985). Many of these appear to be coded for by oncogenes, and are closely related or analogous to the transforming proteins coded for by the viral oncogenes of oncogenic retroviruses (Bishop, 1985; Duesberg, 1985). There is also evidence that malignant cell acquire inductive properties normally found in heterologous cells (Dawe, 1972). Such properties may render malignant cell populations autonomous from interactions with heterologous tissues on which their normal counterparts depend for the maintenance of normal structure and function. Much current research is aimed at distinguishing those phenotypic characteristics of malignant

cells that are directly related to their transformation to malignancy from others that may be incidental; among the former, it is important to distinguish causes from consequences of the cells abnormal behaviour. Over the years, tissue culture has become accepted as an analytic tool in cancer research and, in particular, in the search for the essential characteristics of malignancy at the cellular level. Such an approach has been justified by the demonstration of the reproducubility in culture of many of the abnormalities in cellular behaviour that seem to be responsible for cancer as a clinicopathologic entity. There cellular abnormalities are expressed in vivo as well as in culture by 1) mitotic activity under conditions inhibitory to normal cells; 2) abnormal migratory activity in relation to like and unlike cells and tissues; 3) the ability of the cells to survive under conditions, or in location, where the homologous normal cells would die due to metabolic or immunologic incompatibility and 4) the expression of struc,tural and functional characteristics that are not part of the phenotype of the normal adult tissue of origin. It is important in the interpretation of such chances in tissue culture, to keep in mind that although cells in culture are removed from: host influence, they do not exist in complete isolation. Rather, they have been transferred to a new artificial environment which, while relatively simple, has in itself very specific and profound effects on the cells. As in any other highly selective situation, cellular characteristics in this environment tend to converge toward a common prototype through selection and adaptation. ,4s a result, properties common to normal and malignant cells that are advantageous in tissue culture become prominent and may obliterate some of the differences that existed between the cells in vivo (Wilson, 1973). Furthermore, explantaltion into the tissue culture environment alters the functional state of the cells: in many normal cell types, such as adult fibroblasts, the change is from a stationary to a proliferative state; thus resembling the response to a regenerative stimulus in vivo. In other cells including some normal

120 embryonic tissue and tumors (Flaxam, 1972; Goldstein et al., 1972) explantation into culture tends to accelerate progress to a terminal, postmitotic state of differentiation. Finally the culture environment accelerates proliferation of many normal and malignant tissues which also grow in viva, but at slower rates. Thus, in most of these instances, the culture environment permits the expression of either proliferative (regenerative) or differentiative potentials that exist in vivo but would never have been realized by all the cells of a tissue during the life of the organism. While the growth characteristics of normal cell populations in culture depend to a considerable degree on the particular makeup of the culture environment, they are predictable under standard conditions and typical for different cell types. This is due to the fact that the range of phenotypic variation possible for a normal cell is programmed in relation to its developmental history and becomes limited with differentiation. Its phenotype is stable as much as it will vary only within these limitations, and always in a predictable fashion, in response to physiologic or pathologic influences. In contrast, the effect of the culture environment on explanted tumors and cells transformed in culture is much more variable and unpredictable. This variability, rather than a difference in any one specific parameter of cell growth or function, represents the most basic deviation of malignant from normal cells in culture, and it is presumably an expression of the autonomy which the cells acquired in the course of carcinogenesis. Tissue culture studies suggest that the autonomy in cancer results from a generalized change which makes the cells refractory not only to normal control mechanisms, but also to influences unrelated to malignancy and even to the unphysiologic, artificial effects of the culture environment: explantation into culture represents a drastic environmental change and one to which most normal cells respond consistently by equally drastic changes in growth and function. In contrast, tumor cells have a much greater tendency to retain sometimes for long periods, their in vivo characteristics upon explantation into culture. It is because of this aspect of tumor autono-

my that malignant cells can retain the capacity to perform complex functions in culture for long periods and under a variety of conditions, while most normal specialized cells tend to lose their characteristics rapidly upon the substitution of the tissue culture environment for the necessary regulatory influences present in the intact organism (Auersperg and Finnegan, 1974). Among striking examples of phenotypic stability in cancer, in terms of differentiation are cell lines of neuroblastoma, which have retained morphological, biochemical and electrophysiologic evidence of differentiation over thousands of cell generation in vitro (Rosenberg, 1982), hepatoma cell lines that continuously synthesize multiple serum proteins (Gaudemack et al., 1973), adrenal cortical tumor cells which produce small amount of steroids in the absence of ACTH (Buonassisi et al., 1962) and lines of malignant pituitary cells which produce hormones in suspension cultures (Bancroft and Tashjian, 1971). No comparable long-term stability of differentiation in established lines of cultured normal cells has been observed. In a few types of cultured normal proliferating cells (e.g. epidermis) the potential to differentiate is maintained for a limited time, but required stringent and complex culture conditions (Yuspa et al., 1981). In the intact organism unrestrained cells proliferation is one of the basic characteristics of cancer. The tissue culture environment also permits indefinite, continued growth for some tumor cell populations, but is restrictive and causes death or differentiation in others. This variation indicates that the capacity of tumor cells for continuing cell proliferation in culture is influenced by the hereditary changes acquired with malignancy and is refractory to the regulatory factors in culture which determine the consistent growth potentials of normal cell populations. For example, under standard culture conditions (e.g. plastic growth surface, 10% fetal bovine serum, DMEM medium), normal human fibroblasts consistently have a proliferative capacity in the order of several dozen cells doublings (Dell’orco et al., 1973), normal squamous epithelial cells replicate a few times only (Wilbanks and Shingleton,

121 1970), while normal adult neurons do not divide at all (Silverberg, 1972). On the other hand, the life span of explanted carcinomas, neuroblastomas and other malignant tumors varies, under identical culture conditions and among neoplasms of one type, from a few days to years of rapid cell multiplication. The hereditary, progressive loss of tissue-specific characteristic which occurs in malignant cell populations in vivo and in culture is commonly interpreted as evidence of irreversible cumulative changes at the gene level. Among the most interesting recent contributions of tissue culture towards understanding the nature of malignancy have been the studies which demonstrate the surprising degree to which some of these changes are reversible, thus supporting other indications that many aspects of transformation to malignancy may be due to epigenetic defect (Pierce, 1983). Such defects may be propagated from one cancer cell generation to the next by mechanisms of genetic regulation similar to those which maintain the differentiated state in replicating populations of normal adult specialized cells, or those active in cells undergoing progressive differentiation during embryonic development. In conclusion, the distinguishing features of tumor cells in culture are based on their autonomy from external influence, whether these be physiologic or artificial in nature. In analogy to the in vivo situation, this autonomy in culture is expressed by unpredictable, abnormal phenotypic variation which is often unrelated, or imperfectly related, to existing environmental conditions. Although when considered collectively, malignant cells in vitro show characteristic abnormalities of movement, replication and differentiation with high frequency, no specific cellular change has been identified that is both consistently present and essential for the malignant state.

Acknowledgements This work has been supported Special Project CNlX ‘Oncology’.

by grant from

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