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 pinocytosis 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 pseudopodia 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 intermittently 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 following 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, especially 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 continuous 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. One can only guess at the forces which move the presumably passive globules centrally. It should be borne in mind that the cells are continuously under tension. This is more pronounced in the body of the cell. This tension thrusts out the pseudopodia by forcing the more fluid cytoplasm into them. It would be expected that this would tend to keep the globules at the periphery unless local tensions or contractions, with perhaps a progressive solation on the central side of the globule, came into play. It 'is especially difficult to picture how a large globule passes centrally along a slender process of less diameter than itself. Such a globule produces a bulge in the process which shifts centrally with the globule. It is possible that here, also, local contraction pressures are responsible for pushing the globules along. It is conceivable that local contraction may be induced by the mere presence of the globule and that the contraction may or may not involve the peripheral cytoplasm. Local solations and gelations are continually going on within cells and are probably responsible for the shifting back and forth, here and there, of mitochondria, neutral red granules, and vacuoles and fat globules. Local contractions are probably tied up in some manner with such local gelations and solations. There are many indications of changes in the condition of the cytoplasm in different parts of the cell from time to time, such as the thrusting out and withdrawal of pseudopodia, the changes in the contraction curves and the motion of mitochondria, granules, and fat globules (2). Sarcoma cells, normal fibroblasts, macrophages and probably all other cells have a definite visible organization more evident in some cells than in others. It is especially evident when cells are spread out on the coverglass. FIGS.3 TO 13. Two MALIGNANTCELLS PROM DIBENZANTIIRACENEbfous~ SARCOMA No. C37. TAKENAT 2-MINUTE INTERVALS.X 1100 FIG.3. At a, pinocytosis just ceased, 5 globules moving centrally; at b, c, and d, ruffle pseudopodia and pinocytosis of small globules. FIG.4. At a, fusion into 3 globules; at b, shift in position of ruffle; at c, fusion of globules, new globule in ruffle; at d, shift in position of globule, fusion and few new small ones. FIG. 5. At a, central globule reduced; at b and c, globules indistinct due to slightly different focus; at d, fusion and shift in position. (See also Figs. 6-13)

670 FIGS.3-5

67 1 FIGS.6-8. LATERVIEWS OF Two MALTCNANTCELLS SHOWNIN FIGS.3-5 FIG.6. At a and b, globules reduced; at b, shift in position of ruffles. FIG. 7. At a, globules nearly gone; at b, globules reduced, new globule in ruffle; at c, globules somewhat reduced; at d, some fusion. FIG. 8. At 4, globules about gone; at b, globules reduced, new globules; at c, globules reduced; at d, globules have remained large because of fusions. (See also Figs. Y-13)

672 FIGS. 6-8

613 FIGS.9-11. LATERVIEWS OF Two MALIGNANTCELLS SHOWN IN FIGS. 3-8 FIG. 9. At a, new ruffle, one small globule left; at b, old globules reduced or gone, large fusion globule moving centrally; at c, 3 new globules have entered, new globules in ruffle, at d, new globules, old globules reduced. FIG. 10. At a, globules gone, small ruffle; at b, most of old globules gone, few new ones; at c, old globules reduced, 1 or 2 new globules, globules in ruffle; at d, fusion of new globules. FIG. 11. At a, no ruffle; at b, fusion and reduction; at c, 3 new globules and reduction of old; at d, 2 new small globules. (See nl~oFigs. 12-23)

674 FIGS.9-11

675 676 WARREN H. LEWIS

The centriole is at the center, the nucleus more or less at one side depending on the size of the central area about the centriole. The mitochondria, the fat, and the granules are arranged about the centriole in sort of radial zones. There is presumably some sort of a gradient in the cytoplasm from center to periphery. All these things and many others will ultimately have to be taken

FIGS.12-13. LATERVIEWS 01 Two MALIGNANTCELLS SHOWNIN FIGS.3-11

FIG. 12. At a, minute ruflle; at 6, reduction of old and some new globules; at c, reduction and fusions; at d, reduction and fusions, large new irregular globule in ruffle. FIG. 13. At 4, small ruffle; at b, reduction of old, some new globules; at c, reduction of old and fusion; at d, recent globule, spherical. into account before the movement centrally of the globules is satisfactorily explained. The rapidity of the movement of the globules centrally and the few minutes involved for the trip from the periphery to the central area varies in part with the distance of the ruffle, which traps the globule, from the center of the cell, and in part with unknown factors. Individual globules can be followed with the eye and in motion pictures. PINOCYTOSIS BY MALIGNANT CELLS 677

Digestion begins soon after the globules enter the cell and continues until all traces of the globules are gone. The assumption that digestion occurs in the globules is based partly on the neutral red reaction. When the culture media contain a suitable amount of neutral red, the globules when first taken in do not show the color. As they move centrally the red tint slowly appears,

FIG.14. PINOCYTOSISAND ACCUMULATIONOF GLOBULES:LARGE BINUCLEATE MALIGNANT CELL FROM DIBENZANTHRACENEMOUSE TUMOR C37 Seventeenth generation mouse-to-mouse transfer. Three-day culture in 2 parts chicken plasma, plus 2 parts Locke solution, plus 1 part beef embryo extract, plus 1 part 0.5 per cent neutral red solution in water. Numerous globules between small ruffles and center. Accumulated globules in central region among small fat globules have considerable neutral red. X 1100. and by the time they reach the center of the cell they may be deeply stained (Figs. 1, 2 and 14). They retain the neutral red as long as they persist, and the granule that remains after the red fluid contents of the globule have dif- fused out is deeply stained. If neutral red is added to a culture containing cells drinking, the globules at the center of the cell almost instantly become deeply stained, while those located more peripherally, and recently pinocy- tosed, may be only faintly or not at all colored. It has been known for a long time that when macrophages in culture media containing a little neutral red ingest dead cells, the latter, when first taken in, are colorless, and later take up neutral red, their color gradually increasing in intensity as digestion progresses. If neutral red is added to a culture medium where macrophages have been ingesting and digesting dead cells for some time, the partly digested cells take up neutral red almost instantly but the recently ingested ones do not. The accumulation of neutral red in phagocytosed cells seems to parallel digestion and presumably indicates the process of digestion or the presence of digestive enzymes. Koehring (3), in a series of beautiful experiments with neutral red on lower organisms, presents much evidence that neutral red is an indicator for digestive enzymes, both intracellular and extracellular. She refers to theswork of Robertson (1907), Halz- 678 WARREN H. LEWIS bert (1913), Marston (1923), and Epstein and Rosenthal (1924), who found that azine dyes, which include neutral red, precipitate the enzymes, pepsin, trypsin, and erepsin. The theory that the neutral red reaction is an indication of the presence of digestive enzymes is a valuable working hypothesis and fits well with my observations on phagocytosis, pinocytosis, and the accumulation of what I have termed degeneration granules and vacuoles (4) which may be due to autodigestion. Whether or not all neutral red staining granules and vacuoles are to be included is uncertain. Presumably complex substances such as proteins, which are present in the culture fluid and in the globules, are split by the digestive enzymes into simpler products which can be utilized or can diffuse out of the cell. The disappear- ance of the globules is probably linked in some way with the completion of the digestion of their contents. They persist for varying lengths of time, five minutes to an hour or more, after which they slowly shrink in size and disappear, leaving a small granule deeply stained with neutral red when the latter is present, The fate of individual granules has not been followed but presumably they too are ultimately digested and disappear in healthy cells, since the latter do not become filled with granules even after prolonged pinocytosis. I have assumed that the fluid diffuses out of the cells when the globules disappear, because cells do not increase in size in spite of active periods of drinking when they may take in several times their volume of fluid in the course of a few hours. The factors involved in the diffusion of the fluid out of the cell are as mysterious as most of the other processes which take place. There has been nothing to suggest that globules open to the outside as presumably in some protozoa. They shrink slowly in size, and partly shrunken ones may suddenly enlarge by fusion with another and then slowly shrink again. Concerning the significance of the process for malignant cells there is little to say. Meltzer (5) in 1904 suggested the hypothesis that ‘( all cells might be endowed with the submicroscopic act of ( sipping ’ the adjacent fluid, which might indeed be a subsidiary or even an essential factor in the process of nutrition of all cells.” He also suggested that (‘ we might in imitation of this term ( phagocytosis ’ designate the ability of cells to drink solutions by the term potocytosis.’ ” Submicroscopic drinking would, of course, be invisible, but since ruffle pseudopodia take in fluid globules down to the limits of visibility, they may also take in submicroscopic ones. There is now no way of demonstrating submicroscopic drinking (potocytosis) by sarcoma or other cells. Pinocytosis, visible drinking, is evidently not necessary for either tem- porary survival or the multiplication of many malignant and normal cells in cultures. Is drinking by malignant cells sort of an accidental affair of the ruffles? The movement centrally and digestion of the globules, like that of globules and phagocytosed dead cells in macrophages, indicate that malignant cells can produce abundant intracellular digestive enzymes. This is no acci- dent. Malignant cells have been occasionally seen to phagocytose and digest dead cells. Koehring’s observations with neutral red indicate that intracel- PINOCYTOSIS BY MALIGNANT CELLS 679 lular digestion occurs within the endodermal cells of Planaria, Hydra, Rotifers and Chaetogaster (lining cells of the gizzard). The endodermal cells con- cerned in this process have mechanisms for engulfing undigested food. Ruffles and the ability of cells to pinocytose and phagocytose are probably related in some way to special intracellular digestive ability.

SUMMARY Malignant sarcoma cells from rat and mouse tumors often show in tissue cultures active, wavy, ruffle pseudopodia which engulf complex fluid media containing proteins and other substances that cannot diffuse into them. The fluid enters the cells as globules which move centrally. The contents are digested and the fluid then diffuses out of the cell. Thus many sarcoma cells may at times exhibit considerable intracellular digestion. Pinocytosis by malignant cells is similar to pinocytosis by normal macrophages.

REFERENCES 1. LEWIS,W. H.: Pinocytosis, Bull. Johns Hopkins Hosp. 49: 17-27, 1931. 2. LEWIS,M. R., AND LEWIS,W. H.: Mitochondria (and other cytoplasmic structures) in tissue cultures, Am. J. Anat. 17: 359-401, 1915. 3. KOEHRING,VERA: The neutral-red reaction, J. Morphol. & Physiol. 49: 45-130, 1930. 4. LEWIS,W. H.: Degeneration granules and vacuoles in the fibroblasts of chick embryos cultivated in vitro, Bull. Johns Hopkins Hosp. 30: 81-91, 1919. 5. MELTZER,S. J.: Edema, American Med. 8: 19-23; 191-199, 1904.