3.1. THE TUMOR CELL

The malignant cell is characterized by: acceleration of the cell cycle; genomic alterations; invasive growth; increased cell mobility; chemotaxis; changes in the cellular surface; secretion of lytic factors, etc.

Morphological and functional characteristics of the malignant cell. Morphologically, the cancerous cell is characterized by a large nucleus, having an irregular size and shape, the nucleoli are prominent, the cytoplasm is scarce and intensely colored or, on the contrary, is pale.

The nucleus of neoplastic cells plays through its changes a main role in the assessment of tumor malignancy. Changes concern its surface, volume, the nucleus/cytoplasm ratio, shape and density, as well as structure and homogeneity. Ultrastructural characteristics are related to nucleus segmentation, invaginations, changes in chromatin, such as heterochromatin reduction, increase of interchromatin and perichromatin granules, increase of nuclear membrane pores, formation of inclusions, etc.

The nucleolus is characterized by hypertrophy, macro- and microsegregation, its movement towards the membrane, numerical increase and formation of intranuclear canalicular systems between the nuclear membrane and the nucleolus.

Mitoses are characteristic of malignant cells. The number of mitoses increases, atypical mitosis forms with defects in the mitotic spindle appear, which results in triple or quadruple asters and dissymmetrical structures and atypical forms of chromosomes.

Nuclear changes explain the presence of different cell clones and genetic anomalies associated with these changes. In intensely anaplastic tumors, the presence of gigantic nuclei and multinucleate cells expresses abnormal divisions.

These morphological characteristics reflect the changes occurring at metabolic level, with the augmentation of structures in relation to cell division and the attenuation of structures associated to other metabolisms.

The cytoplasm also undergoes changes, new structures appear or normal structures disappear. The accumulation of ribosomal and messanger RNA in the cytoplasm makes it basophilic. Malignant cells have a small cytoplasmic amount, frequently with vacuoles.

The granular endoplasmic reticulum has the appearance of a simplified structure. Amorphous, granular of filamentous material can accumulate in the cisternae. Fragmentation and degranulation are frequently found, with the interruption of connections between the granular endoplasmic reticulum and mitochondria. Fingerprint like formations are not uncommon. The decrease of the granular endoplasmic reticulum from tumor cells occurs concomitantly with an increase of free ribosomes and polysomes, which shows an enhanced production of proteins necessary for the cell growth process.

The agranular endoplasmic reticulum is, during the initiation phase, hyperplastic, without being correlated with functional hyperactivity. In other malignancy phases, the endoplasmic reticulum undergoes a reduction.

The Golgi apparatus in malignant cells is generally poorly developed, which involves a positive correlation with the lack of tumor cell differentiation. The cells that have completely lost differentiation sporadically exhibit a Golgi apparatus.

Mitochondria decrease in volume with tumor development. Mitochondria show a high variability of shape and volume, and huge mitochondria can be sometimes observed. Abnormal glycolysis processes occur in mitochondrial membranes, known in the literature as the “Warburg phenomenon”. Changes in mitochondrial crystals occur, inclusions are present in the matrix, and pyknotic images can appear. The longitudinal distribution of mitochondria involves a cytochrome oxidase insufficiency.

Peroxisomes are only present in tumors formed by cells that normally contain these organelles, such as hepatocytes. It has been established that the number of peroxisomes from malignant cells is reversely proportional to growth speed and expresses the degree of differentiation loss [53].

Glycogen in high amounts is a characteristic of malignancy, especially in the liver and kidneys [14], but the already malignant cells generally contain a small amount of glycogen, as it has been found in hepatic and cervical carcinomas. The decrease of glycogen up to its disappearance parallels the increase of lipids.

Lysosomes undergo changes in the process of cell malignization. Thus, secondary lysosomes, myelinic structures and lipofuscin granules appear.

Degenerative cellular changes can be expressed by cytoplasmic inclusions. In some forms of neoplasms, apoptosis occurs, with the presence of apoptotic bodies.

Microfilaments, intermediate filaments and microtubules appear in different proportions, in malignant cells. The capacity of invasion and metastasizing of the cancerous cell depends on its possibility to move, which is ensured by the actin content.

Epithelial carcinomas contain cytokeratins, mesenchymal tumors contain vimentin, and in the central nervous system cells is an acid protein from glial fibers, with a special role in tumor diagnosis [45,136].

Cytostatics act by the depolymerization of tumor cell microtubules, which leads to the inhibition of the metastasizing capacity, as well as mitosis and tumor growth.

The cell membrane plays an extremely important role in the malignization process. Surface molecular changes, associated with malignization, are able to influence the evolution of a tumor, as well as the host reactions to the lesion. Proteins and carbohydrates that act as enzymes and as cell surface receptors can also undergo changes:

• – increase or diminution in the number of surface receptors, changing cell sensitivity to the regulating mechanisms of the host;
• – structural changes of proteins or surface receptors that no longer react with the corresponding ligand;
• – presence of new surface molecules, characteristic of the embryonic tissue, which are hidden at the surface of adult cells.

Abnormal surface molecules are able to act as antigens and are recognized by the mechanisms of humoral and cellular defense. Consequently, tumor cells are covered with immune complexes, which allows the complement to destroy the cells covered by antibodies and allows phagocytes to attack the opsonized cells.

Malignant cells change their enzyme content, such as the reduction of acid or alkaline phosphatase. Changes occur in the relation between sugars and the sialic acid from glycolipids and glycoproteins, and also the negative loading of the cell surface. The plasma membrane of malignant cell favors the accelerated transport of nutritive substances, especially sugars and amino acids.

The surface of malignant cells displays differentiation antigens that express a normal development of the cancerous cell and antigens specific for the tumor, which appear with the oncogenic transformation, by the change of the genetic program of the cell. The distribution of receptors in malignant cells is altered, which modifies the cell agglutination behaviour. On the cell surface there are specific surface proteases that are responsible for the agglutination capacity of cells under the action of plant lectins. By losing contact inhibition, tumor cells also acquire metabolic autonomy, both their proliferation and movement being favored.

On the surface of malignant cells, atypical microvilli, pseudopods and vesicles with extremely active enzymatic equipment appear [117].

Differences between cells from the periphery and the center of tumors have been found. The cell population from the center of the tumor has normal intercellular connections, with the presence of desmosomes and junctional complexes, while these are absent or reduced at the periphery. In areas with a high invasive rhythm, cells are completely detached from the tumor mass, and interconnections disspear altogether [74].

The presence of desmosomes and tight junctions facilitates the establishment of the epithelial origin of the neoplasm, while their absence indicates the mesenchymal origin.

The basal membrane is present in benign tumors, while the invasive growth of malignant cells is characterized by fragmentation, reduplication or disappearance of the basal membrane. During the first phases of malignancy, defects are produced with the interruption of the lamina densa. Malignant cells have lytic factors that destroy the basal membrane [74].

The loss of the basal membrane is considered a fundamental criterion of morphological and biological differentiation between benign and malignant tumors [110]. The basal membrane in malignant cells changes its structure or/and ratios between various components, such as: type IV collagen, laminin, heparan sulfate proteoglycan and fibronectin. Neoplastic cells secrete type IV collagenase that destroys type IV collagen, which facilitates metastasizing through the lysis of basal membranes from blood and lymphatic vessels. Thus, malignant cells are disseminated, but they can also leave the vessels and implant in other tissues and organs, with the formation ofmetastases.

In the process of destruction of the basal membrane, a special role is played by laminin and laminin receptors, receptors that are found in the cell membrane and are reorganized during invasive growth [111].

The functional changes of neoplastic cells cause the formation and elimination of active substances, such as: growth factors, hormones, molecules similar to hormones, lytic enzymes, etc. Lytic enzymes (collagenase, cathepsin and plasmogen activator) favor the increased mobility and dissemination of neoplastic cells.

Major alterations occur in energy metabolism, between normal and malignant cells, especially regarding the use of glucose. The energy production with the highest efficiency in cells is performed by glycolysis in the tricarboxylic acid cycle (TCA cycle of Krebs cycle), where 36 ATP molecules are produced for each glucose molecule. This metabolism is carried out by oxygen use and represents the main energy production pathway, in the majority of cells.

Cancerous cells exhibit anomalies of both glycolysis and the tricarboxylic acid (TCA) cycle. The cancerous cell is particularly characterized by a poor use of oxygen and the massive use of glucose, which is exclusively converted to lactic acid. Consequently, malignant cells take from blood a 5–10 fold glucose amount compared to normal cells and they produce a corresponding lactic acid amount that will be recycled and changed back to glucose in the liver [41].

Tumor cells behave like a metabolic parasite for the organism or they drain its energy.

Proliferation is the main characteristic of benign tumors and especially malignant ones. Cells grow continuously, without being submitted to the local or general control of the organism. Benign growth is maintained within certain limits, while malignant growth is invasive, with quiet phases, followed by intense and uncontrollable growth phases.

The cell cycle normally develops along four phases [3]:

• – phase S, the cell synthesizes DNA, in order to prepare mitosis;
• – phase G₂ follows immediately mitosis (phase in which the genome is equally distributed between the two daughter-cells). It occurs between DNA replication and cell division;
• – phase M or mitosis, characterized by the appearance of chromatids migrating separately between the two daughter-cells;
• – phase G₁ is the time interval elapsed between the previous nuclear division and the beginning of DNA synthesis. This phase is very short for bone marrow cells and in enterocytes from intestinal crypts or, in other cases, it can be very long. Cancerous cells have an accelerated cell cycle.

In the case in which the division speed in a tumor is not accelerated, neoplastic proliferation is the result of a disorder in cell maturation, a great number of cells being able to divide within the tissue. In such tumors, a slowing down of the rhythm of cellular apoptosis has also been found, as it happens in lymphoid tumors.

Genomic alterations, a cancer initiating process, persist all through the evolution of a tumor. The combined action of alterations in the mitotic cycle, the deficient synchronization between the nuclear and cytoplasmic divisions and the alterations preceding the existence of the genome induce more and more the instability of cell lines. Aneuploidy, polysemy and chromosomal deficits cause extremely variable morphological and behavioral clones. The depression of some segments of the genome can also enhance the pleiomorphic aspect of neoplastic cells and explains their abnormal secretions.

The proliferation and migration of neoplastic cells from a tumor is unpredictable. The movement of neoplastic cells starts with the formation of irregular cytoplasmic pseudopods, which infiltrate through basal membranes. Between the differentiation grade of a tumor, on the one hand, and its invasive growth, on the other hand, there is a correlation, which means that differentiation processes inhibit the capacity of movement of the cell.

The active locomotion of a malignant cell involves the enzymatic dissolution of the surrounding host tissue, especially of the interstitial matrix. At the beginning of the invasion, a loosening of the interstitial matrix of the host tissue occurs, by the appearance of an edema. The edema is explained by a higher permeability of the capillaries and the lack of lymphatic vessels with a draining role inside and in the proximity of the tumor. The size of the interstitial fluid volume facilitates cell locomotion. With the invasive growth, the destruction of the host tissue, its real lysis occurs, which is partially caused by enzymatic processes, and partially by atrophy through the pressure exerted by the tumor tissue.

The invasion and infiltration of malignant cells is characterized by the fact that they leave the tumor tissue and penetrate the neighboring tissue [116]. But this property is not only specific for malignant tumors, this can also be found in other cells, such as: granulocytes, osteoclasts, endothelial cells and trophoblastic cells. Unlike these cells, the invasive growth of malignant cells is a progressive and continuous growth, ending with the destruction of the host tissue.

The malignant cell that grows invasively has the capacity to move, to produce lytic factors and phagocytose the host tissue. It grows especially in preexisting spaces but it can also create new spaces, by the destruction of the surrounding tissue. Between cytokinases and the invading capacity of cells, there are negative correlations, the cells having in certain phases a proliferative behavior, and in other phases an invasive behavior.

Invading cells have a higher content of actinic filaments and they form plasminogen activator, collagenase, elastase and proteoglycan decomposing enzymes. Proteolytic enzymes are secreted by both malignant cells and certain cells of the host tissue, such as: endothelial cells, fibroblasts, macrophages, mastocytes and lymphocytes.

The hypothesis that lytic factors produced by malignant cells can also initiate the angiogenesis process is advanced.

The loss of differentiation of the malignant cell is an important component. A determining role in this process is played by the reduction of cell organelles, especially the endoplasmic reticulum (which synthesizes proteins) and the Golgi apparatus. The loss of polarity of cell organelles, as well as of some properties of the cell membrane, also occurs. Malignant cells morphologically and functionally become similar to the fetal cells of the host tissue.

In reality, malignant cell complexes are composed of three types of various cells, in which not only the loss of differentiation takes place, but also aberrant processes, along with normal ones, excessive maturation and synthesis of new substances. This explains the histological variety of the cell population of a tumor. Multidirectional differentiation explains the appearance of atypical substances; thus, neoplastic epithelial cells can produce collagen [53].

At ultrastructural level, the main characteristic is not the loss of differentiation and the simplification of malignant cell structures, but structural and functional reorientation [147].

The loss of differentiation can be explained by the reduction of the postmitotic regeneration time, which results in the diminution of the differentiation time. Genetic information defects can also be mentioned. These processes cause changes in the cytoplasmic composition and intermediate metabolism and glycogen anomalies.

The differentiation process differs from one tumor to another, and it can be characterized by:

• – the maintenance of certain structures and functions;
• – the appearance of new cell structures and functions;
• – the appearance of new structures, such as metaplasia and heteroplasia;
• – the appearance of differentiations;
• – the disappearance of functions in malignant cells such as anaplasia and cataplasia. Anaplastic tumor cells lose their specific structural characteristics, having small amounts of granular endoplasmic reticulum and some mitochondria [170].

It can be considered that there is no principle contradiction between malignancy and differentiation, and the loss of differentiation during malignization should be regarded as an epiphenomenon.

In a malignant cell population, subpopulations and subclones develop, which are distinguished in terms of invasiveness, aggressiveness and the capacity of metastasizing. The peculiarities specific for each neoplasm result from heterogeneity, the presence of subclones whose unpredictable appearance and variation, supplemented by the local reaction of the host tissue and of the organism, make difficult tumor therapy. In addition to these peculiarities of tumor cells, the following should be considered: the relation of the tumor to the stroma; the different behavior regarding the invasion and metastasizing of the different subclones; the different structure of cells, in terms of antigenicity and/or membrane glycoproteins and the variable cell sensitivity to cytostatics, radiation, etc.

Cytostatic treatment should aim to eliminate malignant subpopulations, since these have a high proliferation and invasion capacity. It should be mentioned that only a small part of the tumor cells that reach blood circulation have metastatic properties, and only when they find favorable conditions [53].

Depending on the metastasizing subclone, the cellular structure of metastases is similar or different, compared to the primary tumor [158].

The involvement of the host tissue in the development of a tumor is strongly expressed by the growth rhythm and the possibilities of tumor metastasizing. The reactions of the host tissue are initiated by immunological and non-immunological mechanisms [129, 130]. The invasive cell acts on the extracellular matrix of the host tissue, in particular on collagen and elastin. This action develops along three successive stages. In a first stage, the receptors of the tumor cell membrane bind to the glycoproteins of the host tissue, especially laminin and fibronectin. In the second stage, tumor cells secrete hydrolytic enzymes that stimulate the secretory activity of host cells. In the third stage, the dissolution of the components of the host tissue matrix occurs, and desmoplasia of these components is stimulated.

According to CARR and UNDERWOOD, 1974, tumor cells stimulate the following phenomena:

• – the lymphoreticular reaction, with the invasion of lymphocytes, macrophages, lymphoreticular cells, immunologically active cells;
• – the vascular reaction, with the proliferation of endothelial cells and the formation of new capillaries;
• – the fibrous reaction, with fibroblast proliferation and collagen deposition;
• – the inflammatory reaction, with polymorphonuclear infiltration (neutrophils and eosinophils).

During its development, the malignant tumor needs the host tissue to survive and to grow. As part of the non-immunological defense reaction, a special role is played by activated macrophages, which are tightly bound to T lymphocytes. Some biochemical mediators and chemical reagents are able to destroy tumor cells non-immunologically or to inhibit their growth. In the case of the regression of a tumor, the phagocytic and Killer activity of macrophages increases. Spontaneous regressions have been found in some neoplasms: melanomas, choriocarcinomas, neuroblastomas, hypernephromas, etc.

The immunological cellular response controls through its mechanisms the growth of malignant cells. Complex processes with specific and non-specific immunodepressive effects take place. Immunological factors are supposed to eliminate malignant cells rapidly, before the appearance of clinical manifestations [179].

The growth of a tumor depends on its vascularization. It has been found that poorly vascularized or even avascular tumors slow down their development or they even stop growing. In contrast, the appearance of capillaries, the infiltration of the tumor by a great numer of capillaries, stimulates tumor growth and proliferation. Malignant cells secrete some substances that stimulate the formation of new vessels, which are called by BASSERMANN (1984) the “tumor angiogenesis factor” (TAP). Angiogenesis is a normal physiological response that appears in other processes as well, such as cicatrization and inflammation. TAP molecules are probably produced and elaborated by host cells, such as lymphocytes, macrophages, monocytes, etc.

Due to the fact that tumor cell proliferation occurs at a much higher speed compared to the formation of new capillaries, necrobiotic, necrotic and apoptotic processes take place in the tumor.

3.2. APOPTOSIS

Cell death, under the action of external noxious factors, is known as necrosis, and more recently, the term oncosts [115] has been proposed when the cell becomes edematous (oncos = swelling) and represents the first phase of accidental cell death, with morphological changes incorporated in the necrosis process.

Apoptosis is the term proposed by KERR et al. (1972), being composed of apo, which means from and ptosis, which means fall. Apoptosis in Greek expresses the fall of leaves or petals. In cell biology, the term suggests the arrest of vital functions and consequently death occurring without the intervention of external factors. The cell has its own biological and chemical mechanisms that are capable of determining cell death. Apoptosis is a physiological process that is triggered by the activation of genetic self-destruction programs existing in the genome of all cells. In this way, the multicellular organism destroys the undesired cells from a tissue [54].

Apoptosis is an active form of cell death characterized by biochemical and morphological processes, especially by chromatin condensation, poly-nucleosomal DNA fragmentation and the fragmentation of the cell into apoptotic bodies. The apoptotic process plays a central role in the development and functioning of the immune system. Apoptosis can be partly genetically regulated and it can also be related to physiological and nonphysiological signals. Apoptosis can represent a defense mechanism at cellular level against cancer, by the participation of protooncogenes and tumor suppressor genes in the regulation of apoptosis [118]. Each cell receives multiple signals, which by means of specific receptors can induce the cell to enter the cell cycle or apoptosis. The alteration of a specific receptor can lead to the appearance of a malignant clone, due to the imbalanced relation between apoptosis inducing or repressing signals and proliferation [27, 43, 162]. The antiapoptotic Bcl-2 protein may inhibit apoptosis induced by the absence of growth factos, neurotrophic factors and cytokines [5, 96].

For the correct use of terminology, it should be emphasized that a differentiation is proposed between apoptosis, which strictly refers to morphological cell changes, and programmed death, which occurs in the course of biochemical (physiological) processes specific for cell death.

The term apoptosis is currently used either as a synonym for programmed cell death, or to indicate morphological cell changes that are associated with programmed cell death. According to some authors [54] the correct expression would be “programmed cell death by apoptosis”; at present, the concepts of apoptosis and programmed cell death are used with the same meaning, being considered synonymous. The authors consider that apoptosis can be defined as a strictly physiologically regulated process, which is partly characterized by nuclear condensation and cell volume decrease, with the maintenance of an intact plasma membrane, and which reaches its highest point with the irreversible destruction of nuclear chromatin and genomic DNA digestion.

In apoptosis, by the mechanisms in function, the cell actively participates in its own death. Morphologically, it is characterized by compact cytoplasm, vacuoles in the cytoplasmic membrane, nuclear chromatin condensation, DNA fragmentation and the formation of apoptotic bodies. Unlike the images observed in necrosis, in the case of apoptosis chromatin does not flocculate, mitochondria are not swollen and the cell membrane is not permeable to staining agents. Apoptosis does not trigger inflammatory reactions, since lysosomal enzymes are not released [42].

For the understanding of the phenomenon of cell death through apoptosis or necrosis, the clarifications made by ZEISS (2003) are edifying and essential. The author defines apoptosis as a highly regulated process, characterized by specific morphological and biochemical properties. The apoptotic process is initiated by both physical-biological and pathological stimuli, and the full expression of apoptosis requires a signal cascade in which caspase activation plays a central part. By the elimination of the genes that control caspase-dependent apoptosis, the apoptotic phenotype is transformed into a necrotic phenotype both in vitro and in vivo. This suggests the fact that necrosis and apoptosis represent the morphological expression of a similar biochemical mechanism by both a caspase-dependent mechanism and a non-caspase-dependent mechanism with effectors such as catepsin B and apoptosis inducing factor. The program of cell death, either by apoptosis or by necrosis, is mediated by an integrated cascade, which can be accessed from multiple sites and propagated by many ramification points. A cell can die either by apoptosis or by necrosis, depending on the physiological environment, developmental stage, type of tissue and nature of the cell death signal.

Developing tissues follow a fine line between proliferation and death; in order for a tissue to develop and grow, this should resist to apoptosis. Cell subpopulations at a given time and location must submit to apoptosis so that the tissue maintains its normal shape and function. This proves the close relationship between the mechanism of neoplastic development and proliferation and reflects the intersection between apoptotic pathogenic pathways and cell cycle mechanisms. The coexistence of apoptosis and necrosis characterizes both developmental processes and acquired morbid processes; the morphology of apoptosis is caspase-dependent, and any deviation from this pathogenic pathway results in death through necrotic morphology [185].

In the mechanisms of apoptosis, endonuclease activity plays a major role, being responsible for chromatin condensation, nuclear segregation and dispersion. As a result of chromatin cleavage and chromatin condensation, a dramatic reduction in nuclear volume occurs. Small masses of nuclear chromatin are formed, apoptotic bodies, which contain morphologically intact cell organelles. Apoptotic bodies are phagocytosed by viable neighboring cells or macrophages. DNA cleavage in low molecular weight material is extremely important and represents a protective function, limiting the probability of transfer of potentially active genes from dead cells to the nucleus of viable neighboring cells [4, 9, 10].

Scheme of programmed cell death (according to BERGES and ISAACS, 1993)

Apoptosis is involved in physiological processes and especially pathological ones, the latter aspect being extremely important for oncogenesis, evolution and even treatment. The understanding of the mechanisms of apoptosis and the clarification of the biochemical pathways in the activation of the apoptosis inducing genetic program will change the actual concept of treating diseases, in general, and cancer, in particular [4, 11].

Some bacteria are pathogenic through the activation of the genetic program that induces the “suicide” of the affected cell. Some substances elaborated by microbes can enter the genetic programs of cells, causing apoptosis. This could explain the affinity of some pathogenic bacteria for certain cells or tissues [184].

Role of oncogenes in the regulation of programmed cell death (according to LANE, 1992)

Viral and chemical oncogenesis offers attractive research directions regarding the balance/imbalance between apoptosis and proliferation. Spontaneous or induced tumor cell apoptosis regulates tumor growth. Tumor growth represents an imbalance between proliferation and apoptosis, starting with the first stages of carcinogenesis. Experiments have demonstrated that in a preneoplastic hepatic focus, initiated by a chemical carcinoma, the number of apoptotic cells is much higher than that of the unaffected adjacent tissues. Contrary to the generally accepted idea, the tumor initiation stage is not irreversibile, because 80–90% of the initiated cells are eliminated by apoptosis. The promotion stage is also subject to apoptosis [35,162].

Cytotoxic cells induce in target cells morphological changes that trigger apoptosis. Apoptotic cells are in higher numbers in tumors that are strongly infiltrated with cytotoxic T lymphocytes or NK cells.

New hypotheses and researches have appeared considering oncogenesis as a possible deregulation of apoptosis. Thus, it has been assumed that an oncogene, such as bcl-2, may not stimulate cell proliferation, but inhibit cell apoptosis. It has been shown that under the action of growth stimuli, the c-myc oncogene is involved in cell proliferation but, in the absence of mitogenic signals, this oncogene can induce apoptosis. Finally, p53, whose disappearance or inactivation is frequently observed in malignant tumors, can orient the cell towards proliferation or apoptosis, depending on internal or external signals and the physiological cell status [69,73,148].

In veterinary oncology, there are few studies on the apoptotic index in various types of neoplasms. In mammary carcinoma in dogs, the apoptotic index is very low, between 0 and 1 % [55].

The inhibition of the apoptosis of cells from germinative centers plays an important role in the development of malignant lymphomas. The mitotic index in the germinative centers from malignant non-Hodgkin lymphoma is significantly lower compared to unchanged lymph nodes. So, apoptotic inhibition in malignant lymphomas results in the increase of the number of cells per volume unit, not by an increased mitotic index, but by a low apoptotic index [87,112,181].

In pluricellular organisms, survival and functioning according to physiological parameters is mediated by a balance between cell proliferation and destruction, an optimal number of various cellular categories being ensured. The elimination of undesired (old, abnormal, tumor) cells from the organism occurs by a genetically controlled process, which has structures for the transmission of messages to effector elements (caspases), a process termed programmed cell death or apoptosis. This process is initiated by a multitude of external and internal cellular factors that, in a certain context, trigger apoptosis. The cells that are about to enter apoptosis show a series of changes, some of which are evident (nuclear material alterations), others inapparent or undetectable by actual investigation means [54]. The authors reproduce the data published by THOMPSON (1995) regarding diseases in which apoptosis is inhibited and diseases in which it is stimulated.

The diminution in the capacity of cells to enter the apoptotic process when they receive internal or external signals to enter this process, and to be eliminated, has two major causes: either the cell has no functional apoptotic equipment, which is destroyed, or the cellular pathways for apoptosis are blocked.

Diseases associated with apoptotic inhibition [172]:

1. Cancer:
   • –follicular lymphomas;
   • –cancers withp53 mutations;
   • –hormone dependent tumors: mammary neoplasms, prostatic neoplasms and ovarian neoplasms.
2. Autoimmune diseases:
   • –systemic lupus erythematosus;
   • –immune glomerulonephritis.
3. Viral infections:
   • –herpetic viruses;
   • –poxviruses;
   • –adenoviruses.

In the appearance and development of the neoplastic process, the role of apoptosis consists of an insufficient development of programmed cell death, accompanied or not by excessive proliferation. Apoptosis counteracts a tumor proliferation process, but at the same time the majority of substances used in anticancer therapy induce apoptosis by triggering cell lesions that cannot be repaired by cellular mechanisms [54].

In neurodegenerative and heart diseases, apoptosis is increased, these diseases being according to THOMSON [54]:

1. AIDS;
2. neurodegenerative diseases: Alzheimer's disease; Parkinson's disease; lateral amyotrophic sclerosis and cerebellar degeneration;
3. myelodysplastic syndrome: anaplastic anemia;
4. ischemic lesions: myocardial infarction and changes related to reperfusion;
5. toxic hepatic diseases: alcohol.

Apoptosis occurs as a physiological mechanism for the protection of the organism that allows the elimination of undesired cells, in particular those containing pathological mutations. Recent studies have shown that a deficiency in the apoptotic process can play a fundamental role in the genesis or development of cancer. Numerous cellular genes involved in the regulation of apoptosis have already been identified, such as protooncogenes (c-myc, bcl-2) and tumor suppressor genes (p53). It seems that apoptosis is a complex network of pathways that interact, causing the intervention of proapoptotic and antiapoptotic antagonist factors [118].

In the neoplastic tissue, even in the case of a high growth rate, many cells die at a rate similar to that of cell formation and multiplication. In the proliferation or the death of neoplastic tissue cells, a major role is played by vascularization. Endothelial cells from tumor capillaries are directly involved in the proliferation process. The study of vascular endothelium has revealed the fact that the proportion of cells in the S phase is approximately 10 times higher than in endothelial cells from normal blood vessels. Blood vessels within tumors are formed by endothelium arranged on the basal membrane, having a continuous structure or partially lacking vascular walls. The tumor vascular network is poorly oriented, which allows the blood flow to change its direction or have stasis periods. The result of this irregular flow is that tumors larger than several millimiters show deep hypoxic areas, with low pH, due to the accumulation of acid metabolites of anaerobic glycolysis. The cell groups from the proximity of blood vessels, which are in increased supply, continue proliferation, and old cells will be progressively driven towards hypoxic areas, where they will gradually die. Necrotic areas will be surrounded by tumor cell cords that benefit from oxygen supply and substances necessary for proliferation.

Tumor cells usually produce angiogenic factors (TAF), which stimulate endothelial growth. However, by the alert rate of development of the neoplastic tissue, their vascularization capacity is exceeded. Hypoxic tumor necrosis is a sign that rapid tumor cell proliferation continues. In numerous neoplastic types (gliomas, sarcomas, soft tissues, etc.) the presence of necrosis is correlated with an unfavorable prognosis. The death of endothelial cells through apoptosis can be induced by the fluctuation of angiogenic factors; tumor necrosis factors, tumor macrophage and lymphocyte products may have a similar effect.

Apoptotic cells within tumors may appear among neoplastic cells during proliferation, but they appear in great numbers around and at the periphery of necrotic foci. The incidence of apoptotic cells varies widely from one tumor type to another or even from one patient to another, always depending on the phase and developmental stage of the tumor. In some cases, the presence of apoptotic cells represents a diagnostic and/or prognostic factor.

The factors responsible for tumor cell apoptosis are not well understood; internal factors, the action of cytotoxic cells, NK cells and macrophages are suspected. The intimate phenomena by which cytostatics act are not completely elucidated. The fact that tumor apoptosis can be instrinsically programmed also has interesting implications for therapy and has led to attempts to discover the regulation mechanisms of apoptosis in tumor cells. Thus, human c-myc and H-ras oncogenes were inserted in a rodent fibroblast cell line. The cells obtained this way were tumorigenic, but their capacity of local invasion and metastasising varied. All c-myc tumors had high mitotic and apoptotic rates, with a slow general expansion rate. In contrast, ras tumors had low apoptotic rates and were more aggressive. In ras tumors, necrosis was much more extensive, as a result of the non-proliferation of vascular endothelium, which favors hypoxia and necrosis.

In therapy, it has been demonstrated that some cytotoxic drugs act on cancerous cells, inducing apoptosis. Experimental studies aim to discover cytostatics with a maximum efficiency on tumor cells, by either preventing apoptotic block or decreasing the apoptosis induction threshold, while increasing the difference between the therapeutic dose and the toxic threshold in the action of these drugs.

By synthesizing the results of the researches in this field, KAHN (1994) noted the existence of cell apoptosis in the absence of growth factors, neurotrophic factors or cytokinins. Apoptosis can be avoided by an increased synthesis of Bcl-2, which suggests that this antiapoptotic protein can alter at least certain effects of the above mentioned factors. Between Bcl-2 (apoptotic inhibitors) and Fas (apoptotic inducers) proteins there are no known direct interactions with the control systems of the cell cycle, and proliferation and apoptosis may both be supposed to act on tumor growth, but their mechanisms are different. Each cell receives multiple signals (hormones, cytokines, etc.) that, by means of specific receptors, can induce the cell to enter the normal cycle or apoptosis. The alteration of a specific receptor may result in the appearance of a malignant clone, due to the imbalanced relation between the inducing or repressing signals of apoptosis and proliferation. The stimulation of proliferation and apoptosis occurs by the activation of common genes, such as c-fas and c-myc genes [162].

Viral oncogenesis offers interesting research alternatives regarding the interaction between apoptosis and proliferation, providing at the same time, like in the case of c-myc and bcl-2 genes, an exciting hypothesis on the mechanisms of cooperation of oncogenes.

The spontaneous or induced apoptosis of tumor cells regulates tumor growth. Tissue homeostasis requires a constant balance between cell death and proliferation. Apoptosis mediates the selective elimination of undesired cells (damaged, old, preneoplastic cells). Tumor growth represents an imbalance between proliferation and apoptosis, starting with the first stages of carcinogenesis.

SOLARY et al. (1993) have experimentally demonstrated that in a preneoplastic hepatic focus initiated by a chemical carcinoma, the number of apoptotic cells is much higher compared to the surrounding tissue. So, contrary to the generally accepted idea, the tumor initiation phase is not irreversible, because 80–90% of the initiated cells are eliminated by apoptosis. The promotion stage is also subject to apoptosis. In a tumor, cells disappear periodically by differentiation, desquamation, migration or death.

Cytotoxic cells induce in target cells morphological changes that evoke apoptosis. The number of apoptotic cells has been found to be much higher in tumors that are markedly infiltrated with cytotoxic T lymphocytes or NK cells.

The consequence of oncogenesis being regarded as a possible deregulation of apoptosis has been an enlargement of perspective in the formulation of new work hypotheses in this field in which scientific research has an unlimited scope. Thus, it has been supposed that an oncogene such as bcl-2 can play a role not in the stimulation of cell proliferation, but in the inhibition of cell apoptosis. Recently, it has been demonstrated that under the action of various growth factors, the c-myc oncogene is involved in cell proliferation but, in the absence of mitogenic signals, this oncogene is capable of inducing apoptosis. Finally, p53, whose disappearance or inactivation is frequently found in malignant tumors, can orient the cell towards proliferation or apoptosis, depending on various internal or external signals and the time of the cell cycle.

The mechanisms responsible for apoptotic cell death in untreated malignant non-Hodgkin lymphoma, as well as in other tissue neoplasms, are not yet completely understood.

The results of the study performed by LEONCINI et al. (1993) suggest that an increase in the apoptotic index and the lack of the cell protein Bcl-2 could be an unfavorable prognostic factor, regardless of the histologically established malignancy grade, for malignant non-Hodgkin lymphoma. However, it can be estimated that a relative increase in cell apoptosis and a better understanding of its mechanisms could have major implications in the establishment of new principles in the treatment of malignant diseases. A deeper knowledge of apoptosis and its induction by various means could assist in the more effective destruction of neoplastic cells.

In 1989, LIU et al., and in 1990, WILLIAMS et al. suggested that the apoptotic inhibition of cells from germinative centers could play an important role in the development of malignant lymphomas. Starting from this hypothesis, HOLLO WOOD and MACARTNEY (1991) have studied the apoptotic index and mitotic index in the germinative centers from malignant non-Hodgkin lymphoma. Results have demonstrated an extremely high apoptotic index in the germinative follicles of the control group compared to the neoplastic group. This favors the hypothesis that, by the inhibition of apoptosis in malignant lymphomas, apoptosis is directly involved in the pathogenesis of these tumors.

Results suggest that an increase in the apoptotic index and the absence of the Bcl-2 cell protein could be more reliable predictive elements in establishing a prognosis for malignant lymphomas compared to histological prognosis. The precise evaluation of the relation between apoptotic diminution and cell proliferation from malignant follicles requires further investigations, as well as the development of adequate supporting techniques.

Regarding the perspectives and implications of the apoptotic process in cancer therapy, we note the observation of some researchers, according to which the sensitivity of cancer cells to chemotherapy, ionizing radiation, etc., seems to be related to the induction of an apoptotic program.

Based on the results obtained in vitro, FUKUDA, KOJIRO and CHIU (1993) suggest that tumor apoptosis can represent a residual autoregulation attempt, aimed at the expanding tumor population and/or could be the result of mild cell injuries, such as hypoxia, nutritional deficiencies or other unknown noxious factors. The authors have demonstrated that apoptosis can be induced in vitro, in hepatoma cells, by the successive action of low intensity injuries or stimuli. The clarification of the biochemical pathway in the activation of apoptosis should lead to the finding of new possibilities in cancer treatment.

Spontaneous apoptosis in tumors can be amplified by therapy, e.g. by hormonal deprivation of hormone dependent tumors. It should be considered that the induction of apoptosis depends on the cellular terrain, the inducing signal and its intensity [17,162]. Apoptosis can be activated by an increase in speed as well as by the activation of cell cycle regulating genes. The increase of the apoptotic speed starts with the activation of non-specific oncogenes (c-fas; c-jun; c-myc), in response to extracellular signals, which modulate their activity (inhibitory signals or their absence). The cell is at the same time unable to complete its cell cycle, probably due to the absence of the mitogenic factor, and dies through apoptosis.

Some chemical substances used in the treatment of tumors result in the arrest of the cell cycle in phase G₁ or especially in phase G₂. This arrest of the cell cycle evolution is interpreted as a possibility for the treated cell to gain time for lesion repair, in order to resume its cell cycle. If during malignant transformation the control points, such as p53, have disappeared, the cell does not interrupt its cycle for lesion repair and rapidly accumulates lethal lesions. If the processes involved in DNA repair are deficient, cells cannot eliminate DNA lesions, such as those produced by ultraviolet rays, when the cell accumulates genetic abnormalities. Over the past years, the first human genes involved in DNA repair have been successfully isolated.

The apoptosis of a great number of cells, shortly after anticancer treatment, suggests that apoptosis induction does not depend on the cell cycle [162]. All happens as if the cell was programmed for the activation of the final phase of apoptosis, probably by the activation of an endonuclease. The alteration of the genetic programme of the cell undergoing differentiation or the change of signals sent to the cell can modify cellular sensitivity to apoptosis induction. The capacity of tumor cells to enter apoptosis becomes the key of the response to the tumor disease treatment.

SOLARY et al. (1994) estimate that apoptosis modulation can have various implications, such as:

• – potential antitumor activity of cytotoxic agents;
• – reduction of undesired (toxic) effects, inhibiting apoptosis in non-tumor cells;
• – orientation of research towards the obtaining of new products, which selectively induce apoptosis in malignant cells.

The occurence of apoptosis during the interphase can only be delayed by molecules that alter the structure of chromatin (polyamines) or inhibit nuclease activity (zinc). The study of cell death, apoptosis, seems to develop a new branch of biology, cell thanatology, which will certainly serve life.

It may be concluded that a knowledge of the mechanisms and development of the apoptotic process phenomena and the clarification of the biochemical pathway in the activation of genetically programmed death will change the current concept regarding the treatment of diseases, in general, and of neoplasms, in particular. Researches in this direction bring hope that more efficient means to fight tumor disease and, why not, to prevent the multiplication of neoplastic cells, may be found. The possibility to influence the apoptotic process, sometimes by accelerating it, at other times by slowing it down, gives hope for the efficient treatment of some diseases, but also for the prolongation of life.

3.3. TUMOR DEVELOPMENT AND GENERALIZATION

Cell homeostasis consists in the maintenance of a balance between the loss and repair of cells. In the case of tumors, an imbalance occurs between reduced cell loss and excessive cell growth. The time of duplication of a tumor volume is clinically estimated. The great majority of tumors develop from the oncogenic transformation of a number of simultaneously transformed cells, but only one of these will survive and grow excessively. Although neoplasms have a clonal nature, it is clear that any tumor will evolve, becoming composed of a highly genotypically and phenotypically heterogeneous neoplastic cell population. This heterogeneity is dynamic, changing continuously in time, and can be accompanied by an irreversible gradual increase in the biological aggressiveness of the tumor, in terms of invasiveness and metastatic characteristics [134].

Cell heterogeneity is a manifestation of tumor progression, by the gradual appearance of new clones, or more properly “subclones” of the original neoplastic clone [98].

The local infiltration of cells is favored by local tissue factors in which neoplasms develop, as well as by some peculiarities of tumor cells. Thus, it has been confirmed that a glycoprotein, laminin, present in basal cells, favors the attachment of neoplastic cells to the tissue matrix. Fibronectin, a glycoprotein present in fibroblasts, endothelial cells, collagen fibers and fundamental interstitial substance, has similar properties. Collagenase is present in tumor cells, in a higher amount compared to normal cells, having properties that favor tissue invasion.

The loss of contact inhibition of neoplastic cells seems to be an intercellular communication anomaly specific for these cells. It has been found that orifice junctions between tumor cells disappear, so that communication between adjacent cells is interrupted.

Metastasis or secondary tumor is defined as a colony of cells developing at a distance from the primary tumor from neoplastic cells that have migrated and fixed to tissues completely different from the original tissue. Metastases can appear at the same time as the primitive tumor or later, as late metastases. There are also metastases that precede the primitive tumor; such metastases have been identified in the case of melanomas and malpighian carcinomas.

Metastases are favored by rich capillary vascularization, without this being a compulsory condition. All cancer types produce metastases with the same frequency, and some organs or tissues are more or less the site of secondary tumors.

Some neoplasms produce early metastases almost constantly, such as: osteosarcomas, tonsillar carcinomas, oropharyngeal melanomas, splenic sarcomas, etc. In other cancers, metastases are exceptional and occur at a late stage. This is the case of the basocellular epithelioma, Sticker’s sarcoma, seminoma, etc.

All vascularized tissues can be locations for neoplasm metastasis. There are also surprising locations: maternal cancer metastasized in the fetus, the metastasizing of a neoplasm into a benign tumor, such as the metastasizing of a mammary carcinoma into a uterine leiomyoma. A higher frequency of metastases occurs in filter-organs: lymph nodes, capillary-rich organs, such as the lungs, liver, kidneys, bone marrow [114].

Metastasized cells, as well as secondary tumors, show some morphological peculiarities compared to the primary tumor. It should be emphasized that not all the cells of the primary tumor have the same capacity to develop metastases, which proves the fact that the heterogeneity of tumor cells is not only morphological. Some cells have a higher capacity to enter vessels and metastasize, but not all cells that reach filter-organs (lymph nodes, liver, lungs, etc.) will develop metastases. Cell clones with different capacities of producing metastases have been isolated from the same primitive tumor.

The cells of a primary tumor are not identical, they differ through: their capacity to react to immune defense; their growth speed; their metabolic characteristics; their sensitivity to one or more antimitotic substances, etc.

In producing metastases, the cells of the primary tumor must overcome some obstacles, passing through several stages:

• – the cell or the cell group must detach from the primary tumor;
• – enter a blood or lymphatic vessel;
• – migrate into the blood or lymph flow;
• – through lymphatic circulation, neoplastic cells can stop in a lymph node, multiply and disseminate;
• – through blood circulation, cells can stop in the lung or the liver, they form growth centers and subsequently disseminate;
• – some migrating cells reach and stop in other organs (brain, bone, kidney, etc.) [154].

The detachment of cells from the primitive tumor is favored by collagenases and other histolytic enzymes. The poor adhesion of cells to one another may be the result of alterations at the level of the junctional complexes that link cell membranes. For example, the oncogenic Rous sarcoma virus produces a phosphokinase that turns vinculin, a junctional complex protein, to phosphovinculin, which causes a weakening of structural adhesions.

The penetration of neoplastic cells among the cells of the capillary endothelium is a complex enzymatic process. The evolution of metastasis depends on: the tumor type, organ vascularization and enzymatic content of tumor cells.

The margination of neoplastic cells at the site of metastasis is the result of a mechanical blockage in the capillaries and of a selective identification between vascular endothelium and tumor cells. It is known that neoplastic cells can migrate selectively in a certain organ.

Experimental observations and researches demonstrate the existence of an affinity of cancer cells for a certain organ or tissue. Thus, a certain type of melanoma metastasizes particularly in the lung. KINSEY [114] experimentally grafted pulmonary tissue in the thigh, after cicatrization he intravenously injected melanoma cells, which developed in both the rat lungs and the pulmonary tissue grafted in the thigh.

The proteins or carbohydrates present at the surface of tumor cells are supposed to be responsible for the specific migration. On the cell surface there are enzymes or carbohydrates that allow the cells to adhere to and remain in an organ or not. Thus, lymphocytes are the only cells programmed to stop in a lymph node, carrying a surface protein corresponding to the endothelial membrane receptors of lymph node veins. Similar mechanisms of recognition also function for tumors. On tumor cells, there are specific receptors, formed by carbohydrates.

In vivo experimental studies have demonstrated the adhesion capacity of tumor cells to undamaged vascular endothelium, as well as in the case of mechanical endothelial lesions. In both cases, the interaction between the endothelium-platelet complex and tumor cells has been noted. In patients with cancer, platelet anomalies appear, as functional quantitative and/or qualitative alterations, which predisposes to venous thromboembolism. In advanced stages of cancer disease in the lung, colon, stomach, ovary and mammary, reactive thrombocytosis is found [142]. These changes, induced directly or indirectly by cells or the whole neoplasm, favor the dissemination and development of cell clones in tissues, at a distance from the primary tumor, i.e. they metastasize.

The understanding of the mechanisms of onset and development of metastases has led to the initiation of experimental studies and the formulation of hypotheses and theories. According to GIAVAZZI et al. (1980), metastases would be a random manifestation in cell populations present in primary tumors.

In an extensive study, KERBEL et al. (1988) maintain and experimentally demonstrate the fact that metastasizing is a selective process, in which only cells equipped with special metastasizing capacities can overcome the various elimination stages and form colonies at distance.

The great majority of neoplasms develop by the oncogenic transformation of a single normal cell. Theoretically, it is possible that a tumor may develop from a great number of simultaneously transformed cells, but only one of these will survive or grow excessively, to finally manifest clinically as a neoplasm [134]. The nature of neoplasms is clonal, and the cell population is composed of genotypically and phenotypically heterogeneous cells.

The selective theory of metastasis development has been demonstrated based on observations on spontaneous tumors, by the analysis of clonal dynamics of tumor growth and of the linear ratio of primary tumor development to the non-randomized appearance of metastases. The heterogeneity of neoplastic cells is dynamic, changing continuously in time, and it can be accompanied by a gradual irreversible increase of aggressiveness, invasiveness and metastasizing properties [133]. The development of cell heterogeneity is a first manifestation of tumor progression, cell clones or subclones gradually appearing in the primary neoplastic population. These have other characteristics, a high growth rate, exceeding the number of primary cells that they can even replace. In primary tumors, cell lines with a high metastatic capacity develop. These cells may occur during the early stages of the development of a neoplasm or, on the contrary, the primary tumor may develop late metastasizing cell lines. This explains the possibility of appearance of early metastases or even metastases that are concomitant with the development of the primary tumor, and implicitly, the importance of excision during the first developmental stage of a neoplasm.

Metastases are selectively derived from a genotypically distinct cell population compared to the primary tumor. On average, metastatic cells are much more metastasizing than their non-metastatic equivalents from the primary tumor. So, not all the cells of a primary tumor can metastasize, which is a selective property that only a minority of mutant cells or cell variants possess [70,144]. The same authors prove the existence of a correlation between the phenotypic characteristics and the presence of certain genes in the malformed cell with metastatic properties, such as the detachment and mobilization from the primary tumor, the invasion in and from blood or lymphatic vessels, survival in the circulatory system, growth in an ectopic microenvironment, angiogenesis, etc.

The metastatic process is selective, and metastatic potential proves to intensify based on a successive selection of neoplastic cells.

Metastatic cells are much more independent from the growth factor compared to their non-metastatic or less metastatic partners. Experience suggests a progressive selection of tumor cell progenitors in the course of development from primary tumors into metastases. Metastases appear from the same clonal progenitor, and primary tumor growth is exceptionally clone selective.

The clonal dominance of metastatic cells in primary tumors is proved by the fact that during the late stages of human colon carcinoma, metastatic cells are predominant, compared to early stages [98]. The experiments of the cited authors have demonstrated beyond any doubt that the preference of a cell clone for selection, in primary tumors, is correlated with its metastatic potential. This is explained by the fact that metastatic cells have a shorter duration of duplication or increased tumorigenicity. Metastasizing cell clones manifest an increased growth autonomy. The same authors conclude that there is a cell – cell interaction; thus, the presence of a metastatic cell subpopulation can suppress the development of non-metastatic cells; alternatively, non-metastatic cells can stimulate the autonomy of the growth factor of the metastatic subpopulation. Experiments have also proved that in time, in tumors obtained by experimental inoculations using different cell clones, metastatic cells will be selected and become dominant, sometimes almost exclusive. So, metastases are formed in a certain selective way from a distinct genotypic cell population.

In the case of some primary solid tumors, in which no aggressive malignant and metastatic cells appear, the following explanations could exist:

• – metastatic cells do not have in the primary tumor a growth advantage, compared to non-metastatic cells;
• – the primary tumor has not been metastatic and no further selection of metastatic cells has been possible;
• – the process of serial propagation has not developed over a long enough period to allow the appearance of metastatic cells that have been previously “silenced” [98].

In conclusion, the cited authors emphasize the fact that the establishment of the mechanisms that mediate clonal dominance will allow to understand the way in which the progress and metastasizing of a tumor take place, as well as the importance of cell-cell or tumor-host interactions in influencing the evolution of tumor disease.

Some aspects regarding the two parameters, the invaded tissue and the cancerous cell have been clarified. Thus, the invaded tissue can by a parenchyma rich in cells, poor in collagen fibers and fundamental substance, structure that favors the penetration of neoplastic cells and metastasis. In contrast, there are tissues whose structure opposes cell migration and metastasizing, such as: cartilages, tendons, aponeuroses, etc. Other tissues have a medium resistance, such as: myelinic nerve sheath, elastic artery media, etc.

The factors related to cancerous cells would be: external plasticity, increased mobility due to diminished cell cohesion, by the appearance of an electron negative charge and the intervention of enzymatic systems. The instability of the plasma membrane is added, allowing continuous sprouting and the exit of “aggressive” proteolytic lesional vesicles, favoring the detachment of the cells and their fixation to the tissues [100].

The primitive tumor can perforate the organ capsule and graft in cavities, ulcerate and fix downstream, along a natural duct, or penetrate a vessel and form secondary tumors after embolization.

Mechanical dispersion and grafting may involve the serous membranes, different mucosae, leptomeningeal spaces and skin. Grafting on serous membranes occurs after the capsule rupture, in the case of pulmonary or mediastinal tumors in the pleural cavity, and for splenic, ovarian, mesenteric tumors, in the peritoneal cavity [121].

Grafting on mucosae can occur as autografts, such as ulcerous mucosal tumors (bronchi, intestines, etc.) and holografts, such as canine venereal sarcoma, which is transplanted from one mucosa to another.

The tumors of the nervous system do not produce metastases outside the central nervous system. The arterial wall is resistant, while lymphatic vessels and veins are susceptible of being traversed under the action of neoplastic cells.

The great majority of sarcomas diffuse and metastasize early, through blood, compared to the majority of carcinomas that progress by lymphatic route, from one lymph node to another.

3.4. TUMOR ANGIOGENESIS

Vascularization in normal tissue is essential for its growth and evolution, and in tumor tissue it represents a determining factor for the alert rhythm of development, as well as for the dissemination, the metastasizing of neoplastic cells. Some laboratories have specialized in the study and the understanding of neoangiogenesis from tumor tissues. The incidence of metastases directly correlates with the vascular density of tumor tissue. The angiogenic potential of neoplasms represents a predictive factor and an essential element in prognosis.

Microvessel density by surface unit is higher in malignant than benign neoplasias, and in highly malignant neoplastic forms, that are intensely anaplastic, the capillary network is denser, having the basal membranes fragmented. These data show the importance of vascularization, neoangiogenesis, in tumor growth and neoplastic cell metastasizing. Tumor neoangiogenesis generates numerous vascular malformations.

The mutations that occur in tumor cells can induce the appearance of cell clones that possess angiogenetic capacities. This process can involve either an increase of angiogenetic stimuli, or a decrease in angiogenetic infiltration, or the two factors can act concomitantly. More precisely, malignant transformation and/or the malignancy grade can relate to the disturbance of suppressor genes, determining the non-coding (non-synthesis) of angiogenetic inhibitors, such as angiostatin or thrombo-spondin-1, which results in the activation of oncogenic genes that induce angiogenesis, by an increase in the factor that causes the development of vascular endothelium, in the fibroblast growth factor or the increase of both factors. In the mass of anaplastic neoplasms, with a high malignancy and metastasizing grade, a decrease in the diameter of large blood vessels, and an increase in the number of small vessels and numerous abnormal vessels are noted. Very many isolated endothelial cells appear concomitantly, which proves a high angiogenic but also metastatic potential. There is evidence that endothelial cells can favor the development of metastases not only by the induction of neoangiogenesis, but also by the elaboration of collagenases and other enzymes that facilitate their mobility and penetration in the new capillaries. Endothelial cells may stimulate the development and proliferation of tumor cells. In the development and metastasizing of tumors, a central point is represented by the angiogenic balance that involves, on the one hand, the capacity of endothelial cells to form new capillaries, and on the other hand, the capacity of neoplastic cells to favor neoangiogensis [170].

The vascularization of tumor cells has been the object of studies, experiments and formulation of new theories. The main theories, with morphofunctional support, are:

• – the theory of multistage angiogenesis;
• – the theory of vasculogenic mimicry;
• – the theory of cooption of preexisting vessels by the tumor.

The theory of multistage angiogenesis admits and demonstrates that angiogenesis is a complex process that includes the mutual influence between endothelial and neoplastic cells and the factors of the extracellular matrix components. The formation of tumor tissue vessels is a complex process, with the contribution of several factors: release of protein kinases from activated endothelial cells; degradation of the basal membranes of vessels; migration of endothelial cells in the interstitial space; proliferation of endothelial cells; elaboration of active enzymes by neoplastic cells; formation of the lumen; generation of new basal membranes, by the contribution of pericytes; fusion of newly formed vessels; maintenance of blood flow.

The endothelial cells of the existing blood vessels must degrade the basal membrane and invade the adjacent stroma, in order to initiate neoangiogenesis. The process of invasion of endothelial and migrating cells requires the cooperation with the urokinase plasminogen activator and the metalloprotein kinase matrix system [134].

Following the process of proteolytic degradation of the extracellular matrix, the endothelial cell “leader” starts to migrate through the degraded matrix. Then, the proliferation of endothelial cells occurs, which are stimulated by a variety of growth factors, of which some are released by the degraded extracellular matrix. Other products of the extracellular matrix, such as fibrin or hyaluronic acid peptide fragments, also stimulate the angiogenesis process [181].

The multistage theory of tumor neoangiogenesis considers the intervention of three categories of factors that act successively. The first category contains the vascular endothelial growth factor and angiopoietin related growth factor, which act on vascular endothelia. The second group of factors consists of active molecules with direct action, including cytokines, chemokines and angiogenic enzymes, which activate a wide range of target cells, situated around endothelial cells. The major factor of this group is the fibroblast growth factor [37, 38, 137]. The third group of angiogenic molecules includes factors that act directly, and their effect is mediated by the release of direct action factors from macrophages, endothelial cells and tumor cells. The tumor necrosis factor and the growth transformation factor, which inhibit endothelial cell proliferation in vitro, belong to this group.

The growth transformation factor induces, in vivo, angiogenesis and stimulates the expression of the tumor necrosis factor, platelet derived growth factor and vascular endothelial growth factor, by the attracted inflammatory cells [75, 162]. The tumor necrosis factor increases the expression of the vascular endothelial growth factor and its receptors, as well as of interleukin and fibroblast growth factor, by endothelial cells, this explaining its own angiogenic proliferation in vivo [92, 183].

Cell invasion, migration and proliferation do not exclusively depend on angiogenic enzymes, the growth factors and their receptors being also mediated by cell adhesion molecules. In order to initiate the angiogenic process, endothelial cells should dissociate from the surrounding cells before they can invade adjacent tissues. Over the duration of invasion and migration, the interaction of endothelial cells with the extracellular matrix is mediated by interin. The final stage of the angiogenic process, including the construction of capillaries and the determination of the polarity of endothelial cells that are necessary for the formation of the lumen, involves intercellular contact and interactions between cells and the extracellular matrix [26]. In neovascularization, cell adhesion molecules are involved, such as: selectins, immunoglobulins, superantigens, cadherins and integrins [142].

The proteolytic degradation of the basal membrane and the migration of endothelial cells is followed by the synthesis of new basal membranes at the periphery of newly formed capillaries. Over the duration of this extracellular process, local proteolysis should be inhibited, in order to allow the deposition and assembling of extracellular matrix components. After the formation of capillary buds, the newly formed extracellular matrices are degraded again at the tip of the capillary bud, which subsequently allows invasion. Thus, the formation of capillaries results from an alternation of activation and inhibition cycles of extracellular proteolase. Endothelial cells form ramifications that will connect with other ramifications, forming capillary connections. Cell adhesion molecules establish the polarity of endothelial cells, as well as the luminal space that they delimit from the adjacent abluminal area. The subsequent stabilization of the new capillaries requires the recruiting of pericytes and smooth muscle cells, which are regulated by the platelet growth factor.

After neovascularization is completed, angiogenic factors are downregulated or the local concentration of inhibitors increases, and as a result endothelial cells become passive and the vessels stagnate or regress.

The theory of vasculogenic mimicry is based on the fact that tumor cells can form vascular channels without the presence of vascular endothelia, being considered a new concept in the biology of tumor vascularization [65].

Older observations have noted the presence of tumor cells that line “vascular channels and blood pools”, which in their turn communicate with microvascular structures. Ultrastructural studies have demonstrated that cancerous cells contribute to the formation of vascular walls in tumors. Structural and ultrastructural evidence has determined the use of the expression “endothelial mimicry” for tumor cells that form vascular channels [95, 101, 177]. The fact that cancerous cells can become cells that line and participate in the formation of intratumoral vessels has a particular pathophysiological importance, with major implications on both tumor tissue and treatment, causing the arrest or even the regression of a neoplasm.

For the final clarification of the contribution and involvement of tumor cells in tumor neoangiogenesis, there are extremely accurate methodologies (identification of genes, markers, histochemistry, histoenzymology, electron microscopy, etc.). Studies of tumor vascularization have been performed on spontaneous, experimental or in vitro neoplasms. Thus, the retinoblastoma, an intraocular tumor in children, offers good study opportunities due to the fact that it is highly angiogenic, having a great number of vessels lined by endothelia. In this neoplasm, necrotic areas are present distally to tumor cells, surrounding intratumoral blood vessels. Angiogenesis in retinoblastoma is regulated by the growth factor [71].

In aggressive primary and metastatic melanomas, tumor cells generate acellular microcirculation channels, composed of extracellular matrix and lined on the exterior by tumor cells. These vascular channels formed in the area of aggressive and metastatic tumor cells are not, in a strict sense, a vasculogenic phenomenon, because real vasculogenesis is produced by the “de novo” formation of endothelial cells that line the newly formed vessels.

The process by which channels bordered by extracellular matrix, into which blood flows, arise in aggressive tumors has been termed “vasculogenic mimicry”. The study of vasculogenic mimicry in uveal melanomas has shown the existence of a PAS positive extracellular matrix, which forms a tube outside which tumor cells are found. These channels delimited by PAS positive extracellular matrix form a specific network, with erythrocytes, especially in high malignancy areas. The aggressive cells of uveal melanomas, but not the non-aggressive cells, produce type VI collagen, which is at the origin of vascular channel histogenesis [71]. In experiments in vitro, cells derived from aggressive melanomas are able to form channels similar to those of melanomas in patients with high lethal risk. These channels are found in high malignancy cell cultures, without the presence of endothelial cells, fibroblasts and without the addition of the growth factor. These experiments demonstrate that the PAS positive extracellular material that forms the extracellular channels is not a response of connective tissue to the presence of the tumor.

Similar images, the presence of endothelium free vascular channels, have also been found in cutaneous melanomas [171]. Consequently, the vasculogenic mimicry phenomenon does not only define uveal melanomas, which are rare, but also cutaneous melanomas, where these channels have been present both in vitro and in vivo [71].

Investigations performed in other tumor types have reported the presence of tumor cells that line sinusoids in sarcomas. Electron microscopy has revealed the presence of endothelium free vascular channels in sarcomas [114, 172]. The observations according to which tumor necrosis does not usually appear in vasculogenic mimicry areas are noteworthy, as well as those indicating extremely rare microthrombi in canalicular vasculogenic mimicry models.

Mammary adenocarcinomas in the mouse show an intense vascularizaton of perivascular connective tissue, while the tumor proper is much more poorly vascularized. We have made similar observations in mammary carcinomas in female dogs. Mammary carcinoma in women presents an abundantly vascularized stroma, which develops and precedes local dissemination and distant metastasis. The more aggressive the carcinoma, the more marked the induction of vasculoconnective development [37].

According to the theory of cooption of preexisting vessels by the tumor, tumor tissues coopt preexisting vessels, especially from peripheral vasculoconnective tissue. This tissue develops under the influence of the tumor, and active angiogenesis is stimulated by the factors released by tumor cells. Through neoangiogenesis, the vascular network develops from the periphery to the center of the tumor.

It may be concluded that each tumor type can develop a vascular network by one of the three modalities or all three modalities mentioned. Peculiarities consist in the possibility that one of the three modalities may be obvious or present, with differences from one tumor type to another.

The understanding of tumor angiogenesis allows to find ways for the slowing down or even the regression of neoplasms, by acting on the formation and development of the capillary network from a tumor tissue.

3.5. TUMOR IMMUNOLOGY

Organisms have the natural capacity to prevent the onset and progress of tumors but, when the mechanisms of control are diminished or absent, tumor disease appears. These control mechanisms can be exceeded by the incapacity of formation of immune effectors, the deficient recognition and processing of antigens, their masking or the reduced life duration of immunity effects.

Antitumor defense involves the elimination of cells infected with exogenous oncogenic viruses or cells transformed by endogenous oncogenic viruses, and the nonself recognition of neoplastic cells. The neoplastic process develops in the presence of immunological depression.

Experimental oncology has demonstrated that tumors induced by chemical carcinogens, such as immunizations with tumor cells exposed to radiation or treated with mitotic inhibitors can protect the organism from the subsequent treatment with the same viable malignant cells. The development of a malignant tumor requires the action of an oncogenic agent and a deficiency in the immune host response.

The term immunogenicity is not synonymous with the term antigenicity, because a tumor can be antigenic, which means that it can have antigens different from those of normal tissues but, for various reasons, it may not generate an immune response from the host, so that the malignant process is considered non-immunogenic [21].

Experiments have demonstrated that cell malignant transformation includes a series of antigenic changes that consist of: loss of antigenic specificity of normal tissues; acquisition of new tumor-specific antigenic properties; the appearance of oncoembryonic or oncofetal antigens that are normally found only in embryonic or fetal tissue, which disappear after birth and may reappear in the case of the development of malignant tumors in the organism.

The most important tumor antigens that could be used in the understanding of oncogenesis, as well as in specific antitumor immunotherapy, are of several types:

• – antigens associated with tumors induced by chemical or physical agents;
• – antigens associated with virally induced tumors;
• – antigens associated with spontaneous tumors;
• – oncoembryonic or oncofetal antigens [21].

Tumor antigens induced chemically, by carcinogenic substances, or physically, by radiation, are unique antigens for each tumor. Thus, a tumor induced by methylcholanthrene presents tumor antigens different from the antigens of any other tumor induced by the same substance. If cutaneous tumors are induced in the same animal in two different locations, the two tumors are antigenically different [51]. Aspects are similar in hepatocarcinomas induced by N-acetylaminofluorene, in sarcomas induced by aromatic hydrocarbons, as well as in tumors induced by physical agents, such as those produced by cellophane implantation or radiation [6]. There is evidence that chemical agents activate a virus that becomes carcinogenic [125]. The detection of antigens by monoclonal antibodies is used in the diagnosis of a wide variety of human tumors, and in veterinary medicine in feline lymphomas.

Monoclonal antibodies can be used in both diagnosis and cancer therapy, either unchanged, using for example antiidiotype antibodies, or after coupling with cytotoxic agents or radioactive isotopes [135].

Virally induced tumor antigens are antigens common to all tumors induced by the same virus, regardless of their morphological picture.

Oncogenic DNA viruses cause in particular the appearance of induced antigens (Papova and Adeno viruses), more rarely structural antigens (SV 40 virus) or oncofetal antigens (herpesviruses).

Common antigens induced by viruses appear through oncogenic DNA viruses, in which case antigens are synthesized based on the genetic information contained in the virus genome, which will be integrated in the cell genome as a provirus. In the nucleus of the transformed cells, T antigens (Thomsen-Friedenreich antigen, shown in human carcinomas) are found, and on membranes, the specific transformation tumor antigen. Virally induced antigens are not components of the virus structure, but may be the product of viral genes required only for the assembling of the virus body. Immune reactions directed against these antigens do not confer viral immunity and, vice versa, the presence of (neutralizing) antiviral antibodies does not protect the animal from the tumor induced by the same virus (in the transformation of a tumor from a syngenic animal). In Marek's disease, herpesvirus causes lymphoproliferations with malignant aspect, and the positive response after vaccination emphasizes the role of immune control in the prevention of the lymphomatous transformation of the disease [21].

The papillomavirus may induce tumors of the digestive tract (BPV-4) or urinary bladder (BPV-2). Based on the DNA recombination technique, papillomaviruses will be used for antiviral vaccines [169].

Structural viral antigens are inconsistently found, as a rare event in cell malignant transformation through DNA viruses.

Oncofetal antigens are determined by herpesviruses, of which the best known is the Epstein-Barr virus, incriminated in the etiology of human malignant tumors.

Oncogenic RNA viruses, oncornaviruses or retroviruses, cause tumors containing in particular structural viral antigens, components of viriona, and more rarely, induced antigens and oncofetal antigens.

Structural tumor antigens appear on the membrane of malignant cells and can function as both TSTA (tumor-specific transplantation antigens) in the rejection of tumor transplants, and TSSA (tumor-specific surface antigens), in the induction of antibody formation, reported in murine leukemia.

Virally induced antigens, less common in tumors induced by retroviruses, function as TSTA and have been described in murine, feline and avian leukemia. During their replication, retroviruses form a DNA copy or a provirus of the RNA genome. This retrovirus migrates in the nucleus and becomes integrated in chromosomal DNA, where it will be transcribed and translated in proteins. Endogenous proviruses are found in many animals, but remain latent; in some species, for example in cats, endogenous elements recombine with horizontally transmitted exogenous viruses [135].

Spontaneous tumor antigens are considered non-specific antigens or even if they exist, they are very weak, non-immunogenic, the previous inoculation of malignant cells in syngenic animals does not result in the rejection of transplants derived from the same spontaneous tumor. The rejection reactions obtained so far in spontaneous tumors are nothing else but experimental artifacts [39].

Spontaneous tumors of animals with oncofetal antigens prove the fact that malignant cells are destroyed by the lymphocytes of normal gestating animals, sensitized to embryonic antigens. Spontaneous tumor antigens, although weak, can induce an immune response, which has been demonstrated using tumor cells radiated or treated with glutaraldehyde, exclusively from primiparae [52].

Oncofetal (oncoembryonic) antigens are normally present in the ontogenetic development of the organism, during the embryonic and fetal stages, being absent during adulthood. These antigens are coded by information existing in the cell genome prior to malignization and not by external information (like in the case of viral oncogenesis) or information resulting from genomic alterations, through mutations determined by chemical carcinogens.

The appearance of these antigens is the result of the activation of genes that encode them, these genes only being active during the embryofetal developmental stages of the organism. During the adult stage of the organism, these genes are repressed, becoming inactive. In the case of cell malignization (spontaneous, by radiation, carcinogens or oncogenic viruses) these genes are activated and they start to function and code for the formation of oncofetal antigens [21].

Oncofetal antigens have different locations and generate different immune responses:

• –cell membrane antigens, if recognized as non-self antigens, can induce a reaction of tumor rejection;
• –intracellular antigens, which cannot be detected, cause destruction reactions only under special conditions;
• –circulating soluble antigens, resulting from the release of intracellular antigens or the exfoliation of membrane antigens, do not influence tumor evolution, as they do not remain at the site of their production. However, they are particularly useful in the diagnosing of malignancy, and their presence in blood can be evidenced using specific antibodies.

Oncofetal antigens differ depending on the differentiation of embryonic tissue or malignant adult tissue, but also depending on the endo-, ecto- or mesodermal origin of the tissue. These antigens can be evidenced in serum and change with the disease evolution, which is why they are considered as markers.

The oncofetal antigens that are best known in humans are: carcino-embryonic antigen (CEA); alpha fetoprotein (AFP); gamma fetoprotein (gamma FP); alpha-2-H-globulin or alpha-2- hepatic ferroglobulin (α₂-H-Fe); gamma fetal antigen (γ-FA); common fetal antigen (CoFA); human embryonic prealbumin-1 (EPA-1); T globulin (TAL); pancreatic oncofetal antigen (POA); tissue polypeptide antigen (TPA); Tennessee antigen (TAG).

Immunological tests have evidenced other markers, such as: human chorionic gonadotropin (HCG); galactosyl transferase isoenzyme II (GT-II); placental alkaline phosphatase (Regan isoenzyme); prostatic acid phosphatase; serum creatin kinase isoenzyme (CK-BB); fetal aldolase; terminal deoxynucleotide transferase (T_dT); F hemoglobin.

By using monoclonal antibodies, other antigens useful for clinical and histopathological diagnosis have been evidenced, which are tumor associated and differentiation antigens [21].

Tumor defense mechanisms are complex, involving a certain sequence of events, such as the activation of factors, of which some possess recognition capacities, others destruction capacities, and at the same time the mobilization of relations of intercooperation and regulation of all phenomena involved. The recognition of foreign antigens is performed by immune-dependent cells with antigen receptors, such as T and B lymphocytes, in the presence and with the help of macrophages. Lymphocytes sensitized to antigens reach peripheral lymphatic organs: lymph nodes, spleen, tonsils, Peyer platelets, etc., where they undergo blastic changes and proliferate, each giving rise to a clone of activated lymphocytes. By lymphokine release, these recruit and activate other cells in order to destroy the antigen against which they have been directed [21].

Effector elements include T effector cells, cytotoxics (cytotoxic T lymphocytes), K cells, NK cells and activated and armed phagocytic cells –macrophages, to a smaller extent polymorphonuclear cells. B lymphocytes act through their products: antibodies or immunoglobulins, which have the capacity to destroy malignant elements – in collaboration with the complement or, more efficiently, with the cells involved in antibody dependent cellular cytotoxicity (ADCC). In parallel to the evolution of the immune response, cooperation relations take place between effector and recognition elements, as well as regulating interactions for the adequate functioning of immunological mechanisms, by their intensification or, on the contrary, their limitation. The regulation of these complex mechanisms can be:

• –direct, performed by regulatory T lymphocytes: helper (Th) and suppressor (T_S3) lymphocytes and by macrophages;
• –indirect, through chemical mediators synthesized by immune cells: lymphokines, synthesized by lymphocytes; monokines, elaborated by monocytes; antiidiotype antibodies, synthesized by lymphocytes against antigen receptors [21].

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