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. PINOCYTOSIS
BY LIVINGMALIGNANT
CELL OF CROCKERRAT SARCOMA
92
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 1 7 rat
sarcomas examined, namely, Crocker 10, 92, 95, 146, 1548, Walker 315, 319
and 338.' I t was common in Crocker 92, Walker 315 and 338, but was seen
only rarely in the others (Figs. 1 and 2). I t 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 RAT SARCOMA 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 a t 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 t o Dr. H. B. Andervont, 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.
I t can thus scarcely be considered as a degeneration phenomenon.
A mouse sarcoma cell with five globules followed for five hours took in
2 7 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 2 7 globules were taken in and, although the cell started
the latter period with 1 2 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 disappearance 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 decrease 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. I t '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. I t 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. I t is especially evident when cells are spread out on the coverglass.
FIGS.3
TO 13.
T w o MALIGNANT
CELLS PROM DIBENZANTIIRACENE
b f o u s ~SARCOMA
No. C37.
TAKENAT 2-MINUTE INTERVALS.
X 1100
FIG.3. At a, pinocytosis just ceased, 5 globules moving centrally; a t b , c, and d, ruffle pseudopodia and pinocytosis of small globules.
FIG.4. At a, fusion into 3 globules; a t 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; a t b and c, globules indistinct due t o slightly different
focus; a t d, fusion and shift in position.
(See also Figs. 6-13)
670
FIGS.3-5
67 1
FIGS.6-8. LATERVIEWS OF Two MALTCNANT
CELLS SHOWNIN FIGS.3-5
FIG.6. At a and b, globules reduced; a t b, shift in position of ruffles.
FIG.7. At a, globules nearly gone; a t b, globules reduced, new globule in ruffle; a t c, globules
somewhat reduced; a t d, some fusion.
FIG. 8. At 4, globules about gone; at b, globules reduced, new globules; a t 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 MALIGNANT
CELLSSHOWNIN FIGS. 3-8
FIG. 9. At a, new ruffle, one small globule left; a t b, old globules reduced or gone, large
fusion globule moving centrally; a t 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; a t b, most of old globules gone, few new ones; a t
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 n l ~ 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.
LATER
VIEWS
01
Two MALIGNANT
CELLS SHOWNIN FIGS.3-11
FIG. 12. At a, minute ruflle; a t 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; a t 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. PINOCYTOSIS
AND ACCUMULATION
OF GLOBULES:LARGE
BINUCLEATE
MALIGNANT
CELL
FROM DIBENZANTHRACENE
MOUSETUMOR
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 diffused 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 pinocytosed, may be only faintly or not at all colored.
I t 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 disappearance 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 temporary 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 accident. 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 concerned 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., A N D 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.