NEOPLASIA


by Robert C. Mellors, M.D., Ph.D.


Biological Properties of Neoplastic Cells


Introduction

When normal cultured cells are exposed to carcinogens in vitro, they acquire many altered characteristics of morphology, growth, and metabolism, and the cells may exhibit tumor formation in vivo. These phenotypic changes are called transformation. Normal cells when maintained in vitro for a considerable time may sometimes undergo "spontaneous" transformation.

Transformation is usually not a sudden, all at once, change but rather is a stepwise process involving successive generations of cells which become increasingly deviant until final irreversible transformation is achieved. The paramount criterion that cells are fully transformed to the neoplastic state is that they can produce tumors in animals when transplanted to the appropriate host.

Since a combination of many different characteristics are acquired to make a cell neoplastic, the neoplastic phenotype is said to be pleiotropic. There has not yet been found a single crucial metabolic change that defines neoplasia. The various combinations of characteristics combine to overcome barriers, for example, at the basement membrane or the capillary border and so on, to produce a tumor.

There are many possible routes to transformation, and hence the changes in phenotype must be under the control of a variety of genes. The hybridization of tumor cells with normal cells usually results in benign hybrid cells, indicating that the tumor genotype is recessive. In some experiments, the fusion of two different tumor cell types leads (by complementation) to progeny cells with non-transformed phenotype; but, as chromosomes are lost on further division of hybrid cells, the transformed phenotype can reappear. Specific chromosomes must be lost to regain tumorigenic potential, and these chromosomes are thought to carry "tumor suppressing genes". Relevantly, a loss of putative tumor suppressing genes is associated with chromosomal deletions in childhood retinoblastoma (chromosome #13) and Wilms' tumor (#11).

Growth Characteristics of Neoplastic Cells in Experimental Systems


Transplantability. Tumor cells taken from the host of origin will grow to form tumors in syngeneic transplant recipients. Heterografts of tumor cells will grow when graft rejection is inhibited, for example, in the anterior chamber of the guinea pig eye, the cheek pouch of irradiated and cortisone treated hamsters, and immunodeficient "nude" mice. The concept of "tumor cell autonomy" implies that the growth processes in tumor cells proceed independently of host controls. However, many tumors are not entirely autonomous. For example, the sex hormone dependency of human carcinomas of breast and prostate is well known and is the basis of a strategy for palliative therapy of these tumors.

Growth of Neoplastic Cells in Vitro.

    - Establishment.-

    Both tumor cells and normal cells can be established in culture. In general, neoplastic cells become established more readily and have a higher plating efficiency than the corresponding normal cells.

    - Loss of contact inhibition.-

    Normal mesenchymal cells in culture grow in an "ordered" way and cease locomotion and division after a monolayer has been formed ("contact inhibition"). After "wounding" the monolayer, growth resumes to replace only the cells removed. In contrast, transformed mesenchymal cells tend to lose contact inhibition and pile on top of one another in crisscross patterns to create "heaped up" multilayered colonies (transformation foci).

    -Lowered requirement for serum.-

    Transformed cells have a lowered requirement for serum in the growth medium. The serum factors are complex and include insulin, other hormones, and growth factors. Some transformed cells produce their own growth factors and may grow in the absence of serum.

    -Cell immortality.-

    Characteristically, cell cultures obtained from normal tissue can be made to proliferate for a period of months, requiring many subcultures, but thereafter the culture declines ("crisis") and becomes extinct. But after the decline of some cultures, the growth may be resumed and then continue indefinitely ("cell immortalization"). This process of "spontaneous" transformation occurs frequently in mouse fibroblast cultures and is probably due to the activation of endogenous transforming retroviruses known to be present in mouse strains.

    The immortalization of cell cultures is a characteristic of many neoplastic cells and is usually the first phenotypic change in transformation brought about by carcinogenic agents. Cell immortality occurs independently and separately from other properties that comprise cell transformation. In some instances, cells with the property of immortality in vitro fail to produce tumors in animals. Cell fusion studies indicate that immortalization, at least from chemical carcinogenesis, is a recessive trait. Immortalization in many, but not all, transformed cells is associated with the activation of the proto-oncogenes myc and fos and with expression of high levels of the cellular protein p53.

    Cell immortalization occurs in human B lymphocytes infected with Epstein-Barr virus in vitro.

Structural Changes in Cells on Neoplastic Transformation


    Nucleus

    Chromosomal changes are discussed elsewhere (refer to: Chromosomal Abnormalities in Cancer).

    Cell Surface

      - Plasma membrane-

      Tumor cells commonly show a highly developed surface with ruffles and microvilli, which facilitate the uptake of metabolites. The plasma membrane is a major regulatory site for cellular metabolism and proliferation. Agents active on the surface are mitogenic in a number of cell types, particularly lymphocytes.

      The membrane is a "fluid mosaic" of proteins (especially glycoproteins) suspended in a lipid bilayer, with the sugars of glycolipids (among other groups) on the external surface.

      Cell division is controlled by the action of hormones and other polypeptide growth factors present extracellularly. Many of the factors are present in serum, particularly epidermal growth factor (EGF) and platelet derived growth factor (PDGF). Growth factors bind to specific surface receptors which have three domains: external, intramembranous, and internal. The action of these factors is part of normal growth, repair, and healing processes but is under very tight cellular and extracellular regulation.

      - Loss of glycoprotein complexity-

      In general, the surface of tumors shows the simplification of certain complex groups, as compared with normal cells. In human tumors, A and B blood group glycolipids may be absent, and simpler molecules (e.g. Le or the Forssman antigen) appear. Surface molecules which specify attachment between cells of the same organ may be absent. Furthermore, there is an inability to lengthen polysaccharide chains on cell contact, and this may correlate with lower adhesiveness of neoplastic cells.

      - Lectin probes-

      The structure and dynamics of cell membranes have been examined extensively using lectins as probes. These substances (plant and animal proteins which bind with specific sugars and which are equivalent to polyvalent antibody) attach to exposed groups on the cell and in some circumstances causes agglutination. Transformed (neoplastic) cells are more readily agglutinated by lectins than non-transformed. The reasons for this in relation to cell surface physiology are not understood, despite considerable study.

      - Fibronectin (LETS)-

      The external surface of non-transformed fibroblasts in culture usually displays a fibrillary covering of fibronectin, a glycoprotein of 240,000 molecular weight. After transformation fibronectin decreases greatly in amount, and the cells become more rounded, hence one of its names: LETS (Large External Transformation- Sensitive). The protein is also present in normal plasma as a globulin which is insoluble in the cold. Addition of fibronectin to the medium in which transformed cells are grown restores their morphology towards their normal flattened and adherent state.

    Cytoskeleton

    The presence of fibronectin on the cell surface appears to be associated with the development of thick bundles of 60 Angstrom microfilaments ("actin cables") extending in the cell cytoplasm to the surface at which cells are attached to the culture flask. The cytoskeleton tends to be sparse and simplified in malignant tumors. This alteration is associated with a change in cell shape, to a round morphology. The surface area also becomes enlarged through the development of microvilli and ruffles.

    Although very frequent among tumors, none of the changes which have been described above have proved to be an invariable accompaniment of the tumorigenic state.

    Proteases


      The invasion of normal tissue is a fundamental characteristic of cancer cells. In this process, as with epithelial cancers, the tumor cells must degrade and traverse the barriers of the basement membrane and the stromal extracellular matrix. Cancer cells characteristically display higher external proteolytic (protease) activity than most normal cells. Much of this protease activity, involving the plasminogen and matrix metalloproteins (MMPs) systems, is contributed by stromal cells (fibroblasts, inflammatory cells, and blood-vessel cells) that become activated as they interact with tumor cells (Edwards, D.R., and Murphy, G., Nature 394: 527-528, 1998).

    Angiogensis

      Tumor cells like fetal and some other normal cells produce a number of biological products that stimulate the proliferation of capillary endothelial cells and encourage the development of new blood vessels (angiogenesis) for the growing tumor. The balance between the promoters and inhibitors of angiogenesis, as determined by genetic (oncogenes, tumor suppressor genes, etc.) and other controls is critical to tumor growth and metastasis (Folkman, J., Nature Med., 1:27-31, 1995).

      Promoters of angiogenesis include vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).

      Inhibitors of angiogenesis in animal tumors include angiostatin, a peptide fragment of plasminogen, and endostatin, a peptide fragment of a collagen protein ( O’Reilly, M. S., et al., Cell 71: 315-328, 1994; ibid., 88:277-285, 1997). Synthetic small molecule inhibitors of matrix metalloproteinases ( see Proteases, above) that inhibit animal tumor angiogenesis are reported to be in Phase I clinical trials (Edwards, D. R., and Murphy, G., Nature 394: 527-528, 1998).

Inappropriate Expression of Genetic Information

    Oncofetal Proteins (Antigens)

      - Alpha-fetoprotein (AFP) -

      This protein is found plentifully in liver cells and plasma during fetal life, but afterwards only in a minute amount. In the blood it is found in much larger amount in:

      • 60-90% of patients with hepatocellular carcinoma:
      • 60% of those with teratoid gonadal tumors;
      • 13% of those with metastatic liver tumors; and rarely in infectious hepatitis and other inflammatory conditions of the liver.

      - Carcinoembryonic antigen(s) (CEA)-

      These are related glycoproteins (at least six) found in fetal intestinal epithelium but only in trace amounts in adults. CEA is found in the blood in many cases of gastrointestinal carcinoma, particularly of the colon. It has also been found in the blood in cases of bronchogenic carcinoma and mammary carcinoma and in the urine in papillary carcinoma of the bladder.

    Enzymes

      - Intracellular-

      Numerous enzymes of tumor cells show the fetal type of isoenzyme rather than the adult. This aspect has been studied particularly in enzymes of glucose metabolism, such as hexokinase, aldolase, and pyruvic kinase.

      - In the blood-

      An isoenzyme of alkaline phosphatase (Regan enzyme) has antigenic and other properties which are identical to those of normal human placenta. This enzyme is detectable in very low amounts in normal serum, but in about 12% of patients with various forms of cancer is present in much increased (3-300 fold) amounts.

    Hormones

    Tumor cells may produce hormones which are appropriate, or inappropriate, for the tumor tissue of origin (Table; also refer to: Hormone Production and Paraneoplastic Syndromes).

    Hypercalcemia accompanying some malignant tumors is produced by the secretion of a (17 kD) polypeptide whose aminoterminal sequences are homologous to parathormone. This ectopic hormone is expressed at a low level in normal keratinocytes but at high level in squamous carcinomas of the lung associated with hypercalcemia, as well as in some other tumors.

    Table: Hormone Production by Human Tumors

    TissueTumorHormoneClinical Features

    Hormones appropriate for the tumor tissue of origin (endocrine tumors):

    Adrenal CortexAdenoma,
    carcinoma
    Cortisol
    Aldosterone
    Androgen
    Cushing's syndrome
    Conn's syndrome
    Adrenogenital syndrome
    Adrenal MedullaPheochromo-cytomaNorepinephrine,
    epinephrine
    Hypertension
    Pancreas IsletAdenomaInsulinHypoglycemia, to 1492
    hyperinsulinism
    EnterochromaffinCarcinoidSerotoninCarcinoid syndrome
    PlacentaChoriocarcinoma,
    hydatidiform mole
    Gonadatropin

    Ectopic hormones, inappropriate for tumor tissue of origin (non-endocrine tumors):

    LungOat cell carcinoma

    Squamous cell
    ACTH
    ADH
    PTH-like
    Cushing's syndrome
    Inappropriate diuresis
    Hypercalcemia
    KidneyAdenocarcinomaPTH-likeHypercalcemia
    LiverHepatomaGonadatropinPrecocious puberty,
    gynecomastia
    LungOat cell carcinomaGonadotropinGynecomastia

    Symbols: ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone; PTH-like, parathyroid hormone-like.

    1492 Islet cell adenoma of the pancreas. Note clusters of uniform, monotonous, small cells, the characteristic appearance of this tumor.

Intracellular Metabolism


An enormous number of metabolic abnormalities have been found in tumor cells. Many of these findings have been in rapidly dividing, undifferentiated cells of experimental tumors, but studies of more differentiated types, particularly the "minimal deviation hepatomas", have failed to reveal any pattern of metabolism unique to the neoplastic state. Nevertheless, many observations have theoretical and therapeutic significance.

    Convergence

    Poorly differentiated tumors show many similarities ("convergence") in metabolic patterns, possibly linked with specialization for growth. Degradative enzymes, such as esterases and aminoacid deaminases, are greatly decreased in activity. Enzymes involved in specialized function are frequently lost. Tumors may be dependent on external sources of substances which are synthesized by the normal cell, for example, L-asparagine, L-glutamine, L-serine. By contrast, enzymes involved in DNA synthesis, membrane synthesis, and so on are greatly increased in activity.

    Methylation of certain CpG islands in the cell DNA forms a consistent pattern in each cell type and is related to inactivation of specific genes. The methylation pattern differs in tumor cells from that in the cell of origin.

    Respiration

    Under anaerobic conditions both tumor cells and normal cells utilize glucose and produce lactic acid (glycolysis). In the presence of oxygen, glycolysis decreases greatly in normal cells (Pasteur effect) while in tumor cells the glycolytic rate may remain high. This respiratory pattern was long considered to be characteristic of the neoplastic state. Nevertheless, it is now known that some normal tissues, such as embryonic tissues and the retina, may exhibit equally high, or even higher, rates of glycolysis than the majority of tumors and that well differentiated "minimal deviation tumors" show normal glycolytic activity.

Growth Control Mechanisms and their Relation to Neoplasia

    Growth Factors

    In addition to salts, aminoacids, and other defined constituents of culture media, serum is necessary for the growth of many cell types in vitro. The effect of serum is now known to be largely due to its content of growth factors. Many growth factors have now been identified (Table) and some are produced as secretions by cells in culture. Inhibitory factors are also produced, some of which can induce differentiation.

    Thus, the control of cell growth is thought to involve an interplay between growth factors (and also inhibitors) and the cell. Different growth factors act on different phases of the cell cycle. Furthermore, during cell differentiation a number of distinct "differentiation windows" are recognizable, each having a specific combination of factors necessary for growth stimulus, until eventually with full differentiation many cells become refractory.

    Growth factors have a number of characteristics in common:

    • they are all polypeptides which bind to specific high affinity membrane receptors;

    • they are produced and act on cells at "short range", unlike endocrine action;

    • each cell has receptors for multiple factors;

    • some factors, such as interleukin 2 (IL2) act on very few cell types, while others, for example, epidermal growth factor (EGF), act on many;

    • factors vary in the point of the cell cycle in which they act;

    • factors may, according to cell type, inhibit cell division and cause differentiation.

      -EGF-

      This factor exists in many cells in the form of an inactive high molecular weight precursor. In the cells' microenvironment, the active form binds to a transmembrane receptor which then becomes interiorized and activated as a tyrosine kinase. This enzyme phosphorylates proteins on their tyrosine residues, and the proteins themselves become "activated". Later stages in the sequence leading to cell division are not yet known, but one effect is the cellular influx of Ca++ from the exterior. Squamous carcinoma cells have a greatly increased number of EGF receptors on their surfaces as compared with normal keratinocytes. A truncated form of the EGF receptor is encoded by the v-erb B oncogene.

      -TGF-Alpha-

      This factor was first discovered in the supernatant fluid of tumor cells in culture. For some tumor cells, the factor acts in an autocrine fashion, maintaining growth. When appropriate cells are treated with the factor, they not only divide but temporarily acquire properties of transformed cells (hence the factor's name). Actually, TGF is probably a fetal form of EGF, and it binds to the EGF receptor.

      -PDGF-

      This factor is of great importance in wound healing. When the PDGF receptor is stimulated, a phosphodiesterase in the cell membrane is activated. This enzyme hydrolyzes phosphoinositol diphosphate in the membrane to form inositol triphosphate (IP) and diacylglycerol (DG). IP is a potent second messenger which causes the release of intracellularly bound Ca++. Numerous proteins are activated by the raised Ca++ level, leading to a cellular response. DG activates phosphokinase C (PKC) bound to the internal surface of the plasma membrane. This enzyme causes the phosphorylation (activation) of, amongst others, proteins controlling ion movement through the membrane. The result is that H+ ions are extruded and Na+ ions enter, resulting in a rise of intracellular pH thought to be significant in inciting cell division. It can be noted that certain tumor promotors, particularly phorbol esters, act directly on and activate PKC.

      The association of receptors for PDGF, EGF, and phorbol esters with protein kinase activity emphasizes the importance of this mechanism in neoplastic development.

      -TGF-Beta-

      This is a multifunctional peptide which in different circumstances can stimulate proliferation, inhibit proliferation, induce differentiation, or affect other cell functions.

      -Bombesin-

      This small peptide (2kD), normally present in the central nervous system, is secreted as an autocrine factor by small cell carcinoma of lung, a tumor thought to be of Kultchitsky cell origin.

    Table: Cellular Growth Factors

    FactorFull NameSize (kD)SourceTarget Cell

    EGFEpidermal6Salivary gland
    GI cells
    Many epithelial and mesenchymal cells
    TGF-ATransforming5.6Tumor cells
    Placenta & embryos
    Same
    PDGFPlatelet derived32Platelets
    Endothelium
    Placenta
    Mesenchymal cells
    Smooth muscle
    Trophoblast
    TGF-BTransforming25Platelets
    Tumor cells
    Kidney
    Fibroblasts
    Keratinocytes
    Mammary cells
    Carcinoma cells
    IGF1Insulin-like (Somatomedin C)7Liver
    Smooth muscle
    Epithelial and mesenchymal cells
    IGF2Insulin-like (Somatomedin A)7Fetal liver
    Placenta
    Same
    IL2Interleukin15T-helper cellsT-lymphocytes
    FGFFibroblast16Brain, Tumors,
    Salivary gland
    Endothelial cells
    Fibroblasts
    CSF1Colony Stimulating (macrophage)70L-cellsMacrophage precursors
    CSF2(macrophage-granulocytic)15-28Lung
    Placenta
    Macrophage & granulocyte precursors
    CSF3MultiCSF (IL3)28T-lymphocytesMany types of leukocytes
    Bombesin2Brain
    GI cells
    CNS, GI tract
    Oat cell carcinoma