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.

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.

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.

Use of reflection contrast microscopy in the analysis of cellular motility.

Recognition and determination of the following activities of living cancer cells on glass substrates can be greatly facilitated by the use of reflection contrast microscopy: 1. stationary versus translocative motility, 2. migration over/under other cells, 3. actual locomotory activity of cells with a polarized shape usually associated with this type of motility. In addition, reflection contrast is useful for recognizing the presence of fibroblasts in cancer cell populations.

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.

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.

In vitro motility of cells from human epidermoid carcinomas. A study by phase-contrast and reflection-contrast cinematography.

The motile behavior of six cell lines derived from human squamous carcinomas (two from the larynx, four from the tongue) was studied by cinematography under phase- and reflection-contrast illumination. The recorded cell activities consist in spreading, stationary and translocation motility, and aggregate formation. Within this common pattern, quantitative modifications ("sub-pattern") are stable properties of the individual cells lines. Such modifications are particularly evident with regard to the dynamic texture of the aggregates which ranges from loose, netlike structures to compact islands with smooth borders. Accordingly, the intensity of cell traffic within and around the aggregates varies considerably. It is discussed to what extent the in vitro motility of the carcinoma cell populations reflects their behavior in the organism and thus the significance of cell movements for invasion.

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.

Motility of L 5222 rat leukemia cells in the flattened state. Evidence against emperipolesis.

Emperipolesis is the term for the assumed penetration of living cells into other living cells. As reported earlier, L 5222 rat leukemia cells, migrating in vitro, change from a spherical to a spread configuration when they meet flat cells, and continue to move in this shape within the contours of the target cells. Whether or not this close cellular association corresponded to emperipolesis could not be determined with phase and interference contrast cinemicrography alone. In combination with transmission electron microscopy, it could be demonstrated that the compartment, in which the spread leukemia cells move, is not the cytoplasm of the target cells, but the narrow space created by the target cells and the underlying glass surface. Thus, emperipolesis could be ruled out for L 5222 leukemia cells. On this basis the reported observations on emperipolesis are reviewed, and a critical attitude regarding the occurrence of emperiopolesis in general is advocated.

Motility of l5222 rat leukemia cells.

The locomotive behavior of cells of the transplantable rat leukemia L5222 was studied by means of microcinematography. It was found that these cells exhibit a homogeneous pattern of movement resembling that of normal lymphoblasts and stimulated lymphocytes. This is in contrast to cytochemical and ultrastructural evidence according to which the cells are completely undifferentiated. Another phenomenon, recorded by timelapse, is the ability of the cells to move in a spherical and in a flattened state. Treatment with cytochalasin B in a concentration of 30mug/ml leads to loss of locomotion. Incubation with colchicine, 40mug/ml, results in a greatly reduced locomotion, while the on-spot motility is not impaired. The suitability of this model for investigations on the role of locomotion in penetration and tumor cell dissemination is emphasized.

Reappearance in vivo of neuraminidase-sensitive sialic acid in L 5222 rat leukemia cells.

Cell electrophoretic data and quantitative sialic acid determination show that, 16 to 20 h after i.p. implantation of neuraminidase-treated L 5222 rat leukemia cells, the original sialic acid content at the cell periphery is reconstituted.

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.

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.

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.

Dynamic morphology applied to human and animal leukemia cells.

Dynamic morphology, which describes the shape and surface architecture of fixed cells in terms related to their behavior in the living state, is based on the concurrent use of two methods: scanning electron microscopy and microcinematography. This combination has both advantages and disadvantages. In this study on leukemic cells, we were able to draw the following conclusions about the usefulness of dynamic morphology. It confirms that white blood cells do not flatten on a glass substrate; they stay spherical and are either round or polarized. Round cells of similar size, whatever their origin, cannot be classified by dynamic morphology. Polarized cells can be classified as blasts, promyelocytes, myelocytes, granulocytes and lymphocytes, although polarized blast cells of different origins cannot be differentiated. Dynamic morphology cannot classify the same cell type as benign or malignant.