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PINOCYTOSIS by MALIGNANT CELLS Pinocytosis (Drinking) by Macrophages in Tissue Cultures Is Common (1). Complex Fluids of Culture

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PINOCYTOSIS by MALIGNANT CELLS Pinocytosis (Drinking) by Macrophages in Tissue Cultures Is Common (1). Complex Fluids of Culture PINOCYTOSIS BY MALIGNANT CELLS WARREN €1. LEWIS1 (From the Department of Embryology, Carnegie Institution of Washington, Baltimore) Pinocytosis (drinking) by macrophages in tissue cultures is common (1). Complex fluids of culture media containing proteins and other substances which cannot diffuse into cells are engulfed by the wavy ruffle pseudopodia. The fluid enters the cells as globules which move centrally and are digested. FIG. 1. PINOCYTOSISBY LIVINGMALIGNANT CELL OF CROCKERRAT SARCOMA92 Hanging-drop 2-day culture in chicken plasma plus neutral red, from pure colony of tumor cells cultivated for 485 days in roller tubes. Note ruffle pseudopodia, 2 new pale globules near ruffle, 2 globules near central area, older deeply stained globules in central region among smaller fat globules (white). The second cell has no ruffles, no pinocytosis, but shows accumulated neutral red bodies of unknown origin. X 1000. The fluid then disappears, presumably by diffusion out of the cell, The split products are either utilized by the macrophage or pass out into the fluid. Since macrophages, so abundantly scattered throughout the body in the tissue spaces, always have numerous globules and ruffles similar to those seen in cultures, it seems probable that they also pinocytose in the body. They are only occasionally called upon to clean up dead cells and fibrin produced by injuries, infections, etc., and since they all show globules of fluid and ruffle pseudopodia, it seems probable that, instead of sitting around doing nothing most of the time, they are always actively engaged in drinking tissue juices, digesting them, and passing the fluid and digestion products back into the tissue fluids. It is scarcely conceivable that this does not play an important 1 Aided by a grant from The International Cancer Research Foundation. 666 PINOCYTOSIS BY MALIGNANT CELLS 667 r81e in modifying and keeping the tissue juices in proper condition, and perhaps supplying certain split products to other cells. Malignant cells from both rats and mice also exhibit pinocytosis (1). This was first observed in motion pictures and has been seen in 8 of 17 rat sarcomas examined, namely, Crocker 10, 92, 95, 146, 1548, Walker 315, 319 and 338.' It was common in Crocker 92, Walker 315 and 338, but was seen only rarely in the others (Figs. 1 and 2). It probably would have been observed in more of the tumors had enough series of cultures been made for the purpose. In a recent study of 160 dibenzanthracene mouse tumors pino- cytosis was seen in the malignant cells in cultures from 90 of the tumors and here again it would probably have been seen in many others had more series of cultures been made and examined especially for this purpose. FIG.2. PINOCYTOSIS BY LIVINGMALIGNANT CELL OF WALKER RATSARCOMA 315 Hanging-drop 1-day culture in medium with neutral red, from pure colony of tumor cells cultivated for 568 days in roller tubes. Note ruffle pseudopodia, globules of various sizes moving from ruffle centrally, deeply stained globules in central area. X 1100. Pinocytosis never takes place unless there are ruffle pseudopodia, and not always then. Cells of the same type, in the same culture, not far apart, may or may not have ruffle pseudopodia and may or may not be drinking even though they have ruffles. In one-, two-, three-, and four-day cultures with the same medium there may be few cells, or none, or many showing pinocytosis. As a rule, cultures either have many cells drinking or almost none at all. The long slender spindle cells, which may or may not have small ruffle pseudopodia thrust out at the tips of the processes, rarely show pinocytosis. The large flat cells, which commonly thrust out and withdraw ruffle pseudo- podia from various regions, frequently show pinocytosis in one or more regions at the same time, in varying degrees, intermittently or continuously for a I am greatly indebted to the Institute of Cancer Research, Columbia University, New York, and to Dr. George Walker, for a generous supply of tumor-hearing rats, and to Dr. H. B. An- dervont, U. S. Public Health Service, for many tumor-bearing mice. 668 WARREN H. LEWIS while. No individual cell was followed over a long period, but it seems quite probable that the same cells may keep up the process more or less intermit- tently for several days, until the cultures begin to deteriorate. Most of the observations were made at twenty-four to forty-eight hours, when the cells are in the best of condition. Young daughter cells often show this process a few minutes after division, almost as soon as ruffle pseudopodia are thrust out. It can thus scarcely be considered as a degeneration phenomenon. A mouse sarcoma cell with five globules followed for five hours took in 27 globules during the first forty-five minutes, and at the end of the period had thirteen. There were a number of fusions of globule with globule and some of the ones taken in earlier vanished. Occasional observations during the next four hours showed pinocytosis. During the last twenty-three minutes of the five-hour period 27 globules were taken in and, although the cell started the latter period with 12 globules, there were only 11 at the end. There were some fusions and disappearances, and the total amount of fluid at the last observation was about the same as at the beginning. Pinocytosis is easily seen in motion pictures. This type of observation is in some respects better than following the process directly with the eye in living cells. The next best method is by a series of photographs, such as shown in Figs. 3 to 13. These figures show, at two-minute intervals and 1100 diameters, a living sarcoma cell and part af a second one in a simple hanging drop four-day culture from a dibenzanthracene mouse tumor (C37, 17th generation, mouse-to-mouse transplantation) in 2 parts of chicken plasma plus 2 parts of Locke solution plus 1 part of beef embryo juice. Most of the mitochondria are concentrated about the nuclei; a few are scattered in the thin ectoplasm. Each nucleus contains two medium-sized nucleoli. By fol- lowing the nucleoli one can see that the nucleus of the left cell has rotated counter clockwise about 230°, and the one on the right a little more than 90°, in the course of twenty minutes. Rotation of the nucleus is common, espe- cially in prophase. I shall have more to say about this in a later article. Both cells show long curved contraction borders except where pseudopodia are thrust out by the contraction of the cell. By following events at a, b, c, and d, where ruffle pseudopodia are active, one can see that globules taken in there move centrally, often fuse, and finally shrink and disappear. Sometimes cells show more or less lively pinocytosis with relatively few globules at any one time followed by cessation of the process and disappear- ance of all the globules. At other times cells may show a continuous rapid pinocytosis with a steady increase in the number of globules retained. The fate of such cells has not been followed (Figs. 1, 2, and 14). It is probable that both the condition of the cell and the condition of the medium play a part in this process. Both are subject to more or less con- tinuous change in the cultures. Since the process takes place only when ruffle pseudopodia are present, and the latter are present only when the cells are in good 'condition, it is not a degeneration phenomenon. When globules are first taken in, they are entirely enclosed by surface membrane. How long this persists unaltered is problematical. The surface membranes of these cells are of invisible thickness and are probably interface membranes. The latter are constantly subject to rapid changes in extent, as PINOCYTOSIS BY MALIGNANT CELLS 669 when pseudopodia are thrust out and withdrawn, and even when they persist for some length of time they undergo ceaseless changes in form and size. It is quite evident, then, that the surface membrane is a very plastic affair and is automatically formed and dissolved as the pseudopodia increase and de- crease in surface area. If the surface membrane persists for a while on the globules, it must ultimately disappear when the globules disappear. Globules when first taken in vary greatly in size. Often several fuse quickly to form larger ones. Fusion also occurs as globules move centrally and after they have arrived in the central region of the cell. When a part of a ruffle fuses around and encloses a bit of the surrounding fluid to form a globule, the latter is frequently at first somewhat irregular in outline. On entering the cell it soon becomes spherical, the result presumably of surface tension forces. The primary distortion may be due to contractions of the ruffle. Not infrequently fluid apparently enclosed escapes, and no globule results, due to incomplete fusion of the ruffle about the fluid. Almost immediately after globules enter the cell they begin to move more or less centrally; they may shift about somewhat. During this movement they pass through the cytoplasm, often close to mitochondria or small fat globules without disturbing them very much except sometimes to push them slightly to one side. They finally reach the central part of the cell in the neighborhood of or close to the nucleus, but rarely touch it. There they may shift about a little among the mitochondria, the fat globules-when the latter are present-and the neutral red granules. They remain in this region until the fluid diffuses out and only a small granule is left.
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