Biological studies of ten human squamous carcinoma cell lines: an overview.

Ten cell lines established from surgical specimens of human squamous carcinomas of the tongue and larynx have been investigated with respect to their motility, ultrastructure, karyotypes, certain biochemical features, interaction with normal epithelial and stromal elements and capacity to infiltrate three-dimensional organoid systems. All the cell lines have maintained several morphological and biochemical characteristics indicating a common origin, although the extent to which each line displays this heritage is variable. The phenotypes of each of the individual cell lines are, however, notably stable. Data are provided for epithelial surface markers (including epidermal growth factor, EGF) and for the synthesis and release of prostaglandins and proteases which may be involved in invasive mechanisms. Encounters between the cell lines and organoid substrata (embryonic chick heart spheroids, human amnion, chick chorioallantoic membrane) are described: the results indicate a scale of invasiveness ranging from lack of penetration to full-thickness infiltration by cells showing various distinctive growth patterns. Correlation between in vitro and in vivo findings is discussed, and it is suggested that the biological heterogeneity of the lines may reflect inherent properties of the original carcinoma cell populations which are more distinctly expressed in vitro.

Ultrastructural evidence for contacts between migrating L5222 rat leukemia cells and extracellular matrix components of the rat mesentery.

The nature of interactions between cells migrating through tissues and their structural surroundings are largely unknown. We have therefore examined the ultrastructural relationship between L5222 rat leukemia cells, moving through the loose connective tissue of the mesentery, and components of the extracellular matrix (ECM). Ultrathin tissue sections, fixed in the presence of ruthenium hexammine trichloride (RHT), revealed the following: Constitutents of fibrillar and nonfibrillar elements of the ECM are in contact with the plasma membrane of L5222 cells. Linear nonfibrillar ECM elements contact the plasma membrane at point-like sites, often associated with root-like structures present within the submembraneous microfilament mesh. Aggregates of ECM material are connected to patch-like cell membrane sites, associated with a condensed, plate-like part of the microfilament mesh. Point-like and patch-like contacts are more numerous at the anterior part of polarized migrating L5222 cells than on the posterior end. In round resting leukemia cells they are evenly distributed around the cell periphery. We suggest that the ECM-cell membrane contacts represent tissue adhesion sites. We therefore hypothesize that in migrating cells a coordinate interaction occurs between the contact sites and the continuous microfilament meshwork which results in a simultaneous backward movement of ECM-membrane contacts on the cell body and in a net forward movement of the whole cell. Since Dembo et al. (1981) present a similar mechanism for in vitro locomotion of granulocytes, we assume that blood cell locomotion in vivo and in vitro depends on similar molecular mechanisms: force generation by the cell, transmembraneous linkage between cytoskeletal and ECM elements, and membrane fluidity. The major difference in blood cell locomotion through a three-dimensional tissue or on a plane substratum would then be given by the distribution of contact sites, occurring around the cell periphery or limited to the ventral cell surface, respectively.

Tumor-associated desmoplasia in the rabbit mesentery characterized by morphological, biochemical and cytophotometric methods.

Intraperitoneal implantation of V2 carcinoma cells in the rabbit leads to invasion of the mesentery and to structural tissue alterations which are concomitantly of a destructive and a desmoplastic type. In this report, we describe the desmoplastic changes which are characterized by the increased formation of collagen and of proteoglycans resulting in an increased thickness of the membrane. Biochemical data indicate that the total amount of collagen increases with time after implantation, whereas the relative amount per unit of dry weight, as well as the contributions of type I (15-25%) and type III (6-8%), stay within the same range. The increased synthesis of extracellular matrix is accompanied by a change in the appearance of the fibroblasts which now show the morphologic features of synthesizing cells. Also, an appreciable number have entered the S-phase. We propose that the desmoplastic changes are tumor-associated, since implantation of epithelial cells from normal rabbit liver does not result in similar alteration. Our findings are discussed in view of the role played by tumor and/or host cells in the increased production of extracellular matrix, of possible factor(s) elaborated by the tumor cells, and of the general significance of desmoplastia for tumor spread.

Lack of mast cell reactivity during leukemic infiltration of the rat mesentery under syngeneic and allogeneic conditions. A study by transmission electron microscopy (TEM).

The ultrastructural morphology of mast cells localized in rat mesenteries was studied after intraperitoneal implantation of L5222 rat leukemia cells in syngeneic and allogenic hosts. It became evident that the mast cells in the syngeneic (BDIX rat) as well as the allogeneic system (BN rat) showed nor morphological alterations. Degranulation was never observed. This is in contrast to the behavior of macrophages which displayed a strong phagocytotic activity in allogeneic hosts. Thus, it seems that mast cells, under the present experimental conditions, remained inactive during a phase of intense tumor rejection.

Calcium-binding proteins in human carcinoma cell lines.

Elevated levels of Ca2+-binding proteins are reported in transformed cells and thought to be involved in their uncontrolled proliferation and often increased motility. Therefore, three cell lines [LICR(Lond)-HN 1, -HN 2, and -HN 6] derived from human carcinomas displaying various degrees of locomotive activity were investigated for the presence of parvalbumin and related Ca2+-binding proteins. By applying different immunohistochemical methods in conjunction with a monospecific anti-parvalbumin antiserum, an intense staining was seen in cells displaying translocative motility. Often in these cells, an association with filamentous structures located in the nuclear region was observed. Unique Ca2+-binding proteins, absent from comparable normal tissue, were found in the malignant cell lines when analyzed by two-dimensional polyacrylamide gel electrophoresis and high-performance liquid chromatography. From these tumor cells a protein (Mr, 12,000; pI, 4.8) was isolated that crossreacted with antiparvalbumin antiserum. Peptide maps of this protein revealed a further structural homology to parvalbumin (Mr, 12,000; pI, 4.9). In analogy to muscle, where there is evidence for a regulatory role of parvalbumin in the contraction-relaxation cycle, we speculate that this protein is tumor-associated and connected to the motile behavior of carcinoma cells.

Morphology of peritumoral proteoglycan alterations in the rabbit mesentery invaded by V2 carcinoma cells.

After intraperitoneal implantation into Swiss Silver rabbits, V2 rabbit carcinoma cells invade the mesentery where they form nodules of different size and texture: compact (less than 120 microns in diameter), loose (120-250 microns) and mixed (above 200 microns). Together with tumor development, certain changes take place in the loose connective tissue of the mesentery. Application of TEM, together with use of safranin O, has shown that, in areas free of tumor growth, collagen bundles become thick and heavy and proteoglycan density is increased. Concurrently, the number of fibrocytes, now transformed to fibroblasts, increases. Small, compact nodules are surrounded by a concentrically arranged extracellular matrix. Its overall density is similar to that of nodule-free areas. In the immediate vicinity of large, loose nodules, all constituents of the extracellular matrix disappear. Adjacent connective tissue is partly destroyed but still contains collagen fibers and proteoglycans. These findings suggest the following: The presence of V2 carcinoma cells induces marked alterations in the structured and non-structured components of the extracellular matrix. These changes are, at the same time, progressive and regressive and the occurrence of one or the other depends on local tumor progression. Progressive alterations may result from an increased activity of fibroblasts. Since degradative effects, on the other hand, are only seen in the immediate vicinity of larger tumor aggregates, it is assumed that a minimal number of tumor cells is essential for destruction of extracellular matrix.

Comparative morphology of cells located on glass and in the loose connective tissue: a study by scanning electron microscopy.

Most of the studies dealing with cellular shape, surface configuration, and motility are carried out in vitro on plane substrata. During the past years, the direct transfer of results obtained under these conditions to the cellular behavior displayed in the living organism, has been increasingly challenged. For this reason we have investigated the above mentioned functions of different cell classes localized on glass and in the loose connective tissue. The cells utilized were: fibroblasts and macrophages from normal rat and rabbit mesenteries, V2 rabbit carcinoma cells and L5222 rat leukemia cells. The combination of time-lapse cinematography and scanning electron microscopy revealed that motility and surface features are the same, irrespective of the immediate surrounding. Cellular shape and attachment, on the other hand, are dependent on the substrate. Fibroblasts, macrophages and cells of epithelial origin, including carcinoma cells, flatten on glass, but have a rounded configuration in the tissue. The flat leading lamellae displayed during locomotion on glass, are not evident in cells migrating through tissues. What regards attachment devices, extensively studied on glass, their formation and position within a tissue are, at present, a matter of speculation. Although it can be assumed that a similar process is operable in vivo and in vitro, clarification rests upon the use of ultrahistochemical techniques.

The V2 carcinoma of the rabbit as an integrated model of tumor invasion.

The V2 carcinoma, established from skin carcinomas of cottontail rabbits and transplantable in all strains of domestic rabbits, is a paradigm of invasiveness attainable by squamous cell carcinoma. The main mechanisms contributing to this potential are the pressure of incessant cell proliferation, the capacity of the tumor to grow in compact as well as in dissociated formation, the synthesis of proteinases (chiefly cathepsin B and collagenases) by the tumor cells, and the latter's migratory activity. In addition, the V2 carcinoma elicits a large spectrum of host reactions which favor partly the organism, partly the tumor and thus create the complexity of the invasion phenomenon.

Cathepsin B containing cells in the rabbit mesentery during invasion of V2 carcinoma cells.

Localization of cathepsin B was studied in the rabbit mesentery during invasion of V2 carcinoma cells. Cathepsin B was visualized immunohistochemically by using monospecific sheep antibodies and the avidin-biotin-peroxidase complex (ABC) method. Horizontal and vertical semithin Epon embedded sections of stained mesenteries showed that histiocytes always displayed the strongest staining reaction independently of the presence of V2 carcinoma cells. Fibroblasts, mesothelial cells and the invaded V2 cells were less stained. Strongly stained peritoneal monocytes were frequently found on the surface of the mesentery in association with tumor foxi. The role of these various cathepsin B containing cells with respect to extracellular matrix degradation during tumor invasion in the mesentery is not clear; some aspects of this problem are presented in the discussion.

Morphologic aspects of the leading lamella in locomotive normal and neoplastic blood cells. A study by scanning electron microscopy (SEM).

A leading lamella is part of every translocative cell, irrespective of nature and origin. The morphological aspect of this cytoplasmic extension, however, varies and, among the different cell types, locomotive blood cells show the greatest diversity with regard to position, size and shape. In this communication, SE micrographs of benign and malignant blood cells of different classes are presented. The dynamic interpretation attempted for the description of the static photographs is based on analyses of time-lapse films which show the activities of the live cells.

Morphological studies on V2 carcinoma invasion and tumor-associated connective tissue changes in the rabbit mesentery.

The mesentery is a duplicature of the peritoneum consisting of loose connective tissue and covered on both sides by mesothelium. Rabbit V2 carcinoma cells implanted i.p. adhere to the mesenteric surface between contracted mesothelial cells. While invasion from these sites sets in, progressive changes of the connective tissue, reflecting fibroblast stimulation, become apparent and comprise multiplication of connective tissue cells, transformation of fibrocytes into fibroblasts, and enhanced production of fibrillar and nonfibrillar constituents of the extracellular matrix. Tumor invasion into this increasingly dense tissue proceeds in 2 ways. (a) Single cells penetrate into and locomote within the interior, where they divide and give rise to nodules which become surrounded by zones of tissue destruction. (b) Proliferation of surface-attached tumor cells results in the formation of nodules which, preceded by zones of tissue damage, extend into the interior. While evidence for lytic effects in the microenvironment of single tumor cells is lacking, degradation of the fibrillar extracellular matrix is regularly found around tumor nodules and indicates a collective lytic action achieved by tumor cells and, possibly, host cells. These morphological findings are discussed in relation to published bio- and histochemical data on spread of the V2 carcinoma.

Cellular motility on glass and in tissues: similarities and dissimilarities.

Four neoplastic cell populations, two leukaemias and two carcinomas, were compared with regard to their motile behaviour on glass, on the surface of, and within the mesentery. This natural membrane was chosen because cells with invasive capacities can penetrate into its loose connective tissue where their movements were recorded by time lapse cinematography. Scanning electron microscopy was utilized to better visualize shape and surface specializations of cells moving in the three localizations. Analysis of time lapse films have shown that two populations, the L5222 rat leukaemia and the V2 carcinoma of the rabbit, contain cells with translocative motility. The non-locomotive cells of these populations and all cells of the other two populations (the BNML rat leukaemia and the human tongue carcinoma cell line LICR-HN1) exhibited stationary (non-translocative) motility. These basic patterns of cell motility remained the same irrespective of the environment. Regarding the influence of the structured elements of the loose connective tissue on overall shape and surface features, it was found that cells from the two leukaemias were the least affected. While BNML cells displayed their non-locomotive activity in a spherical configuration, the translocative L5222 cells migrated in the same shape as on glass. The anterior cytoplasmic extensions, however, were smaller and less variable. The two carcinoma cell populations were more influenced in both shape and cytoplasmic extensions. The well-spread configuration assumed by the stationary LICR-HN1 cells on glass was not maintained in the tissue. There the cells were more spherical. V2 carcinoma cells locomoting within the mesentery showed an increased ability to adapt their shape to the structured environment. The large leading lamellae, characteristic for V2 cells moving on glass, were not evident in the mesentery. They were replaced by smaller and longer cytoplasmic extensions. These observations indicate that the display of cellular locomotion as such does not depend on the environment. The latter, however, can induce adaptations of the cells' locomotive machinery which are expressed as modifications of overall shape and surface features.

Cytoskeletal organization in locomoting cells of the V2 rabbit carcinoma.

The relationship between the organization of cytoskeletal elements and locomotory activity was studied in single cells of the V2 rabbit carcinoma. Like migratory fibroblasts, and unlike colony-forming epithelial cells, these cells show a pronounced horizontal polarization, and develop a large lamella at their leading front. With affinity-purified antibodies and a combination of light and electron microscopic techniques, actin and alpha-actinin (but not myosin and tropomyosin) were found highly concentrated within the marginal region of the leading lamella, both in ruffles and in the underlying zone of contacts with the substratum. Close contacts prevailed in the locomotory cells and small focal contacts developed only in cells detaching from others. Focal contacts always contained small microfilament bundles. Reorganization of actin filaments is suggested as the fundamental event for the dynamic contact formation of the leading lamella. Large microfilament bundles (stress fibers) were absent in all stages of locomotion. Since locomotory behavior and shape changes of V2 cells are the same on glass as on the surface of a natural membrane, the rabbit mesentery, organization and distribution of contractile elements of cultured V2 cells probably reflect the in vivo situation.

Cell adhesion: a general appraisal.

Most data on cell adhesion relate to in vitro conditions; for this reason the subject of this review is adhesion of cells to plane inorganic substrata. Adhesion is conceived of as a process requiring energy and comprising distinct steps, most notably the secretion of attachment proteins, the build-up of attachment sites, and the attachment site--induced organization of the cytoskeleton. The grip and stick concept (Rees et al. 1977) is a very adequate interpretation of this course of events. Agents and functions involved in the stages of adhesions are briefly outlined and a few possible extrapolations to cell adhesion in vivo are indicated.

Interactions between normal epithelial and squamous carcinoma cells in monolayer culture.

Confrontations of rings of adult human oral mucosa epithelial cells enclosing islands of similar normal epithelium, fibroblasts, and cells of three established lines of human squamous carcinoma in monolayer culture were investigated by phase and reflection microscopy and by time lapse cinematography. Measurements of the dimensions of the rings and islands of cells revealed that, while normal epithelial rings confronted with normal epithelium or fibroblasts migrated continuously inwards, similar rings confronting islands of the carcinomas retreated progressively outwards from the tumor islands. The persistence of substantial cell-free space between the epithelium and tumor cells indicated that the outwards migration of the epithelial rings was not solely due to proliferation of the tumor cells. The tumor-induced migration of normal epithelium in monolayer culture may reflect the response of normal epithelium to carcinoma cells in certain in vivo situations.

Cell locomotion, a contributing factor in spread of the V2 rabbit carcinoma.

In histological sections of s.c. transplanted V2 rabbit carcinoma, single tumor cells and small tumor cell groups were found at some distance from the main tumor mass. This led to the question of whether locomotion could represent a contributing factor in the invasiveness of the V2 carcinoma. The behavior of V2 cells was therefore recorded under experimental conditions of increasing complexity: on glass, on the surface of a normal explanted rabbit mesentery, and on and within mesenteries of rabbit which had received intraperitoneal implants of V2 carcinoma. Time lapse cinematography showed the locomotory activity of V2 cells to be unaffected by the different substrates. In all instances the carcinoma cells migrated singly, on the two plane substrates also in small groups, under production of large leading lamellae. Intraperitoneally implanted V2 cells, in addition to their migration on the surface of the mesentery, penetrated into the interior with continuation of their characteristic translocative motility. Although cell locomotion could be established as a mechanism in the invasiveness of the V2 carcinoma, we do not consider it to be the only relevant factor. Tumor cell proliferation and destructive effects of proteinases appear to be other mechanisms contributing to the functional complex of local spread.

Shape and motility, two interdependent features.

By the concurrent use of scanning electron microscopy (SEM) and time lapse cinematography the interdependence of cellular shape and motility was visualized for leukemia cells and for normal and malignant epithelial cells. During the different modes of motility (stationary without net translocation of the whole cell, and locomotion), the cells display a distinct configuration. This allows the interpretation of static images in terms usually reserved for the description of activities of living cells. The ability of cells to change shape is of special importance when they migrate in the body. Irrespective of nature and origin, these cells must continuously adapt their configuration to the structural elements which they encounter in their pathways. The existence of such an adaptability could be shown by time lapse films for leukemia cells and for cells of the V2 rabbit carcinoma moving in mesenteries of rat and rabbit, respectively.