CHARACTERISTICS OF FROG CARCINOMA IN TISSUE CULTURE

The adenocarcinoma of leopard frogs may be cultivated with ease in plasma media. In such cultures two types of growth occur with regularity. The first is in the form of tubules which promptly grow out in the solid medium and retain their tubular form as long as they remain completely enveloped by plasma. When, however, they make contact with the surface of the glass, they adhere to it, the part in contact becomes flat, and its cells now grow no longer as tubules but as membranes. The manner of growth in vitro resembles the growth of transplants of the same tumor in the anterior chamber of the living eye, thus suggesting that in each case the habit of growth is determined by the same morphogenetic factors, i.e. those inherent in the cells themselves, and those depending on interfacial forces. The malignant cells of the frog carcinoma have the attributes which in general distinguish malignant cells from normal cells of corresponding type. In comparison with adult kidney cells, their normal homologues, the conspicuous properties of frog carcinoma cells are: larger and more variable size and shape of cell body, of nucleus, and nucleolus; coarser and denser structure of cytoplasm, of nucleoplasm, and of nuclear membrane; increase in number of mitochondria, and more frequent occurrence of mitosis. These cytological characteristics remain unaltered in cultures maintained for as long as six months. Frog carcinoma is a transmissible disease due to an agent which induces inclusion bodies, and which has other attributes indicating that it is a virus. The general correspondence in character between its cells and the malignant cells of mammalian tumors of diverse origin suggests that neoplastic phenomena are essentially alike, no matter in what group of animals they occur or what their causal factors may be

HETEROTRANSPLANTATION OF FROG CARCINOMA; CHARACTER OF GROWTH IN THE EYES OF ALIEN SPECIES

Transplantation into foreign hosts of the kidney carcinoma of leopard frogs has been investigated by grafting the tumor in the anterior chamber of two other species belonging to the same family of frogs (green frog, bullfrog), of a species from a different family (the toad), and of animals of different classes of cold-blooded vertebrates (goldfish, alligator). In the eyes of frogs of alien species, the kidney carcinoma became established as readily as in the eye of the natural host. In toads, the proportion of successful transplantation was slightly less. No progressive growth of transplanted tumor occurred in the fish or the reptile, although in fish characteristic tumor acini persisted and a few mitoses were to be found long after implantation. In the alligator the transplants deteriorated much more rapidly. Thus the success of transplantation into alien species decreased as the relationship to the original species became more distant. An inflammatory reaction to the tumor graft appeared in the eye of reptiles and fish, but no such reaction occurred in amphibians. The character and rate of growth of transplants were followed by continued direct examination with the microscope and were found to be practically the same in the eye of alien species as in the original species. Nor were differences in the finer structure of the tumor observed in the several species. The neoplastic cells retained their acinar arrangement, supported by stroma which developed quite as well from the tissues of alien hosts as from those of the natural host. It was possible to engraft the tumor in the eye of the natural host, then to transplant it into the eye of another species, and finally to retransplant it in the original species. In this way the tumor has been propagated for 443 days. A tissue culture of the tumor has been successfully transplanted into the eye of an alien host. Thus these results demonstrate that, among cold-blooded vertebrates, the humors and tissues of the eye have a high degree of tolerance for foreign tumor grafts

CATALASE ACTIVITY OF LIVER AND KIDNEY IN FROGS WITH SPONTANEOUS RENAL CARCINOMA

The kidney carcinoma of the leopard frog has served for various studies on catalase activity, as a first step in gaining information on enzymatic properties of neoplasms in cold blooded animals. It was found that the activity level of this tumor is reduced to approximately 13 per cent of that of the normal frog's kidney. Systemic effects of the tumor on catalase activity of liver and kidney are evident. Liver catalase in tumor-bearing frogs is diminished to about 50 per cent of the normal, and kidney catalase to an even greater degree; i.e., to about 34 per cent. A positive correlation exists between levels of catalase in tumor and in liver. Frogs kept at 3 different temperatures, 8, 18, and 26.5°C., for upward of 29 days exhibited no significant change in activity levels either of tumor or of livers from normal or from tumor-bearing animals. It is suggested that a seasonal variation may occur in catalase activity of the frog. During the winter months catalase activity of the tumors was found to be higher than during the summer, whereas liver catalase was below the level of normal frogs examined during the summer. Intracoelomic injections of homogenates of tumors promptly lead to diminution of liver catalase lasting for several days. Injection of normal kidney has no such effect. These results with a spontaneous tumor of a cold blooded animal are in essential agreement with the many observations made with transplanted tumors of warm blooded animals. They lend support to the view that neoplasia is a ubiquitous biological process with similar characteristics in all species or classes of vertebrates

THE EFFECT OF TEMPERATURE ON THE GROWTH OF FROG CARCINOMA : I. DIRECT MICROSCOPIC OBSERVATIONS ON LIVING INTRAOCULAR TRANSPLANTS

From the results of these experiments we can now attempt to answer the questions proposed in the introduction. Just as in normal tissues, it has been found that growth and development of a malignant neoplasm are influenced by temperature both quantitatively and qualitatively. The most striking effect was the acceleration in the rate of growth of the tumor at higher temperature, and retardation at lower. The ultimate size attained within periods averaging 3 months was regularly much greater at higher temperature. Also, the character of growth was quite different. At high temperature, there was more efficient vascularization, and the tumors formed long, branching, tubular outgrowths and cysts; at low temperature the outgrowths were short and stubby and cysts were rare. These differences were accentuated by repeatedly passing the tumor from one generation to another. In particular, in such serial passages, there was a remarkable tendency for the tumor to develop greatly dilated tubules and large cysts, which later, however, became solid as the result of ingrowth from the wall. The experiments demonstrate that the carcinoma in the frog can exist over a wide range of temperature. Even a temperature as low as 4°, as in the hibernation experiment, produces no injury to the tumor. This result is consistent with the common occurrence of large, healthy, spontaneous tumors in frogs which have recently hibernated in their natural state

TRANSPLANTABLE EPITHELIOMAS OF THE LIP AND MOUTH OF CATFISH : I. PATHOLOGY. TRANSPLANTATION TO ANTERIOR CHAMBER OF EYE AND INTO CORNEA

The catfish (Ameiurus nebulosus) taken from streams near Philadelphia, is commonly afflicted with an epithelial tumor bearing some resemblance to epithelioma of the lip in man. This neoplasm usually occurs as solitary or multiple, large, red, fleshy masses upon the lips or dental plates, and by reason of its size, may prevent closure of the mouth. The tumor is comprised of epithelial cells, often in papillary arrangement, supported by a delicate vascularized connective tissue stroma. The larger growths frequently invade adjacent normal tissues and force their way into vessels where they are found as emboli. The clinical course of the tumor is one of relatively slow but progressive growth. This neoplasm has been observed from the time of its inception in a number of animals. Thus it has been learned that the proliferative stage of the neoplastic process is preceded and accompanied by a striking vascular reaction. Intense hyperemia invariably occurs in that region of the mucosal surface which later becomes the site of neoplastic proliferation. Furthermore, by direct microscopic observation of the living tumors the atypical structure and arrangement of the blood vessels become apparent. A study of the significance of these vascular phenomena in their relation to the inception and growth of the tumor is now in progress. It has been found possible to transmit the catfish tumor to fish of the same species by implanting fragments of the tumor into the anterior chamber of the eye. Also, by taking advantage of an anatomical peculiarity of the catfish cornea, it has been possible to embed the tumor fragments in normal tissue where it could still be readily observed both in the gross and microscopically. The growth of the transplants in the eye has been followed by periodic examination of the living tumor by means of the slit lamp microscope. In the anterior chamber, the tumor characteristically forms dense membranes which spread over the inner surface of the cornea. In this manner growth continues until the tumor fills the chamber. Between the two layers of the cornea, tumor growth is expansive. Attempts to transplant the tumor to the anterior chamber of two other species of fish and to frogs, were unsuccessful. Implantation of the catfish epithelioma in alien species of fish excited no exudative response, whereas in a less closely related species of animal, the leopard frog, a pronounced exudative reaction resulted

THE EFFECT OF VALENCE OF IONS ON CELLULAR PERMEABILITY TO WATER

1. Permeability to water in unfertilized eggs of the sea urchin, Arbacia punctulata, was studied by measuring the rate of swelling in hypotonic dextrose solution. 2. Permeability is greatly affected by addition of electrolytes in low concentration. 3. A decrease in permeability to water was found with increasing valence of the cation, using a series of cobaltammine chlorides in which the valence of the cation ranged from 1 to 6. 4. Conversely, an increase in permeability to water was found with increasing valence of the anion, using two series of potassium salts in which the valence of the anion ranged from 1 to 4, and 1 to 3, respectively. 5. It is concluded that the effect of electrolytes on permeability to water depends chiefly on the sign and the number of charges on the ion, in the sense that positive ions decrease permeability to water, while negative ions increase permeability to water; and the effectiveness of the ion is greater the higher its valence. 6. Antagonism has been demonstrated between cations and anions in their effect on permeability, and the method employed permits quantitative study of such antagonism

THE MANNER OF GROWTH OF FROG CARCINOMA, STUDIED BY DIRECT MICROSCOPIC EXAMINATION OF LIVING INTRAOCULAR TRANSPLANTS

The adenocarcinoma which commonly occurs in the kidney of leopard frogs has been transplanted into the anterior chamber of the eye where its growth characteristics have been studied by direct observation with the slit-lamp microscope. Such observations have been amplified by photographs taken at intervals to furnish permanent and objective records of the mode of development and progress of the growths, from earliest to advanced stages. The fifteen tumors which fumished the transplants were typical large invasive adenocarcinomas having the usual irregular and apparently anarchic arrangement of their component tubules and acini. However, the transplanted tumors developed according to definite and well defined structural patterns, their type depending on the immediate physical environment. Three such morphogenetic patterns were observed. Unimpeded outgrowths into the aqueous tumor characteristically assumed a tubulo-papillary arrangement; the earliest formation consisted of solid, purely epithelial cylinders, many of which at later stages acquired a lumen and thus became tubular; generally only the coarser projections developed vascular stalks. Further growth was made by repeated branching, and lateral outpouching of the tubules. Where the tumor grew in contact with firm, even surfaces, such as lens or cornea, differentiation was lost and broad membranes formed which gradually spread over the surfaces; secondarily, new cylindrical or tubular processes arose from such indifferent cellular carpets. Where the tumor made contact with loose tissue such as iris, it invaded this organ, and, supported by the stroma, assumed an acinar pattern quite like that of the original adenocarcinoma of the kidney. All three types of growth were sometimes found coexisting in different portions of the anterior chamber. The rate of growth of transplanted tumors was followed by photographic records taken periodically. A fairly constant mode of progress was noted: after a variable period of lag and a period of gradual outgrowth, there followed rather abruptly a short period of rapid growth, after which growth increments gradually became smaller. Variations in growth rate due to season and to temperature were evident, but further experiments are required to evaluate the part played by these factors. Attempts to transplant normal kidney in the anterior chamber were unsuccessful. The manner of growth in the vitreus was found to be similar to that in the anterior chamber. The factors that determine the manner of tumor growth are as yet imperfectly understood. Evidence is however accumulating to the effect that neoplastic growth is not as anarchic as is suggested by histologic sections of some tumors (26). The present experiments support the view that cancers are much more responsive to the laws governing growth and organization than is generally supposed

THE EFFECT OF TEMPERATURE ON THE GROWTH OF VIRUS-INDUCED FROG CARCINOMA : II. THE TEMPERATURE COEFFICIENT OF GROWTH IN VITRO

The temperature coefficient of van't Hoff, Q10, for growth of frog carcinoma in vitro over a range of 20–35°C, averages 2.5. This value is closely similar to those obtained for various forms of normal growth. The values of the temperature coefficient slightly but progressively decrease with advancing age of the cancer colonies. A similar relation obtains in many but not in all forms of normal growth. Thus, the law of van't Hoff is found to hold equally for growth of the malignant tissue now under discussion and for normal tissue

THE OSMOTIC PROPERTIES OF LIVING CELLS (EGGS OF ARBACIA PUNCTULATA)

We have attempted to answer the question: How nearly ideal, as an osmometer, is the unfertilized Arbacia egg? The following conclusion have been reached: 1. Volumes can be measured accurately over a wide range of pressures since the cell is in general spherical and does not suffer deformation from its own weight or other factors. 2. The product of volume and pressure is approximately constant, if allowance be made for osmotically inactive cell contents. It is computed that from 7 to 14 per cent of cell volume is occupied by osmotically inactive material. 3. Evidence is presented that no appreciable escape of cell contents occurs while the cell is in hypotonic sea water; that, therefore, the semipermeability of the membrane is approximately perfect, so long as injury to the cell is avoided. 4. In comparison with osmotic pressure the influence of other forces, such as elasticity or surface tension, on cell volume must in these experiments be slight

STUDIES ON OSMOTIC EQUILIBRIUM AND ON THE KINETICS OF OSMOSIS IN LIVING CELLS BY A DIFFRACTION METHOD

1. Osmotic equilibrium and kinetics of osmosis of living cells (unfertilized eggs of Arbacia punctulata) have been studied by a diffraction method. This method consists of illuminating a suspension of cells by parallel monochromatic light and measuring, by means of telescope and scale, the angular dimensions of the resulting diffraction pattern from which the average volume of the cells may be computed. The method is far less laborious and possesses several advantages over direct measurement of individual cells. The average size of a large number of cells is obtained from a single measurement of the diffraction pattern and thus individual variability is averaged out. The observations can be made at intervals of a few seconds, permitting changes in volume to be followed satisfactorily. During the measurements the cells are in suspension and are constantly stirred. 2. Volumes of cells in equilibrium with solutions of different osmotic pressure have been determined. In agreement with our previous experiments, based upon direct microscope measurements, we have confirmed the applicability of the law of Boyle-van't Hoff to these cells; that is to say, the product of volume and pressure has been found to be approximately constant if allowance be made for the volume of osmotically inactive material of the cell contents. The volume of osmotically inactive material was found to be, on the average, 12 per cent of the initial cell volume; in eggs from different animals this value ranged from 6 to 20 per cent. 3. Permeability to water of the Arbacia egg has been found to average, at 22°C., 0.106 cubic micra of water per square micron of cell surface, per minute, per atmosphere of difference in osmotic pressure. 4. Permeability to ethylene glycol has been found to average, at 24°C., 4.0 x 10–15 mols, per square micron of cell surface, per minute, for a concentration difference of 1 mol per liter. This is in agreement with the values reported by Stewart and Jacobs