OBSERVATIONS ON THE DIVISION OF SARCOMA
CELLS IE VITRO
ALBERT FISCHER
Prom the Institute of General Pathology of t h University
~
of Copenhagen, Copenhagen, Denmark
It has already been shown (1) t$hattissue cells growing in vitro
are not independent cell-individuals, but form tissues and grow
as a whole, forming a partial undifferentiated organism. It
was concluded that the anastomosis between the individual cells
found in vivo as well as in vitro is significant for the regulation
of their growth. A special method was developed which made it
possible to distribute the cells individually in a suitable medium.
Cell-division was never observed t o occur in isolated cells,
neither was development of a cell colony from a few scattered
cells observed t o take place.
It was furthermore suggested (2) that mitosis of normal cells
may be considered as a phase in a general process of development. I n the periphery of a growing culture of fibroblasts, it
was observed that mitosis set in rhythmically; i.e. periods of
no cell division were succeeded by periods of a relatively high
percentage of mitosis.
I n the present paper, some observations on sarcoma cells in
vitro will be reported, which show that these cells behave quite
differently from normal tissue cells.
As the technique for cultivating chicken tissues has been so
perfected, the experiments were done with the Rous chicken
sarcoma (3). Besides this sarcoma, a few human osteosarcomas
and a gliosarcoma from the human brain were studied.
TECHNIQUE
The tumors were cut into small pieces and placed in the
cultures as soon as possible. The most suitable medium for
71
6
72
ALBERT FISCHER
the Rous sarcoma consists of two volumes of chicken plasma
and a trace of embryonic tissue juice. If too much embryonic
tissue juice be added, extensive liquefaction of the plasma clot
occurs. For the human tissues, various mixtures of human and
chicken plasma, were employed, to which were added extracts
from the respective sarcoma or from human muscle.
It proved to be much less difficult to obtain isolated sarcoma
cells than was the case with fibroblasts. I n all the cultures, it
was easy to find isolated cells a t the periphery of the culture.
These cells are usually very active ameboid and migrate far
out in the culture medium. Observations were made after the
sarcoma cells had been cultivated for about two to four weeks.
The sarcoma cells form but loose connections with other cells
and in addition liquefy the plasma considerably. This property
was utilized to get individual cells by aspirating the liquefied
plasma including the cells and expelling it on a cover glass
containing a drop of fresh plasma and embryonic tissue juice.
By this method, it is possible to get hundreds of isolated cells
on one slide. Ohservations were made under a high power lens,
the microscope being placed in a warm chamber in order to
keep the cultures a t body temperature. The various typical
changes in morphology were immediately drawn on paper.
RESULTS
It can be generally stated that in almost all cultures it could
be observed that the sarcoma cells were able t o divide even
when they were isolated and scattered. The mechanism of the
cell-division of the sarcoma cells was entirely different from that
observed for normal tissue cells in vitro (1). Mitotic celldivision was very seldom observed in the cultures of sarcoma
cells.
The cells which were seen growing out from the explanted
piece of ROUSchicken sarcoma were extremely polymorphous,
and varied considerably in their dimensions. The smallest
were about the size of large bacteria or blood-platelets, the
largest almost 50-100 times the size of the smallest. The
smaller ones moved more rapidly than the larger.
DIVISION O F SARCOMA CELLS IN VITRO
73
I. Description of Cell Types
1. Medium-sized and Small Cells. Figs. I and I V represent
the smallest cells found in the Rous chicken sarcoma in vitro.
Their cytoplasm was very granular and the nucleus could not
be distinguished during life. These cells moved very rapidly,
OF THE SMALLEST
TYPEOF CELLS
FIG.I. SHOWSTHE CELLSHAPE
O~SEIWED
IN THE Rous CHICKEN
SARCOMA
extending and retracting fine or broader pseudopodia. Several
filiform pseudopodia were sometimes extended a t the same time,
and were observed to move like cilia, i.e. the movement de-
THE TYPEOF THE BIGGEST
AMEBOIDCELLS
FI~:.11. REPRESENTS
OBSERVEDIN THE ROUSSARCOMA
The drawings show a pcrfect amalgamation of two cells belonging to the same
type. After separation both cells can be seen to divide, the new cells being
of smaller size than the mother cells. The arrows indicate the travelling direction
of the cells.
74
ALBERT FISCIIER
scribed by their tips was circular and concentric with the axis
of the cell (Fig. IV, 4,5, 6).
I n fixed and stained preparations Fig. VII the cytoplasm
took up the stain (Azur I1 or methylen blue) very intensively.
The nucleus or nuclei were either rather distinct or (Fig. VII,)
represented by fragments scattered about the cytoplasm.
s
6
r
7
FIG. 111. SHOWS
THE AGGRESSIVE
ACTIVITY
OF A MEDIUM-SIZED
CELLTOWARDS
A FIBROBLASTIC
TYPEOF CELLOF THE Rous SARCOMA
Note the plasticity of the fibroblastic cell.
These cells were rather numerous in cultures of the chicken
sarcoma. Besides this type, there occurred a medium-sized
cell with a rather clear, very finely granular cytoplasm containing a few vacuoles (Fig. VI, the cells marked z). The
movement of these cells was also very lively. The changes in
their outline can be followed in Fig. VI; the time taken by the
cell in adopting the various shapes illustrated in Fig. VI was
about 10 minutes.
DIViSION O F SARCOMA CELLS IN VITRO
'
75
2. Large Ameboid Cells. Characteristic of these cells is the
slow movement of their cytoplasm. Their shape is approximately spherical, their cytoplasm highly granular, and their
FIG.IV. SHOWSTHE ACTIVITY
OF
THE
SMALLEST
TYPEOF CELLS
OBSERVED I N THE R O U S SARCOMA
1, 2, 3, 4, 5, 6 and 7 show the changes of outline of a cell during 15 minutcs.
The pseudopods of the 4 and 5 are very characteristic. 8 and 9 show the amalgamation between the cell marked 5 and the cell marked 21.
nuclei rarely visible during life (Fig. 11). When stained, the
nucleus is rather distinct and usually contains one large nu-
6
CELLDIVISION
OF ONE SINGLE
FIG.V. SHOWSTHE CHARACTERISTIC
ISOLATED
CELLFROM THE Rous CHICKEN
SARCOMA
In 2 is shown the separation of the two daughter cells and in 4 and 5 the mother
cell is dividing into seven small cells, which all exhibited lively amcboid movement.
cleolus and two smaller ones (Fig. IX,N). Very characteristic
is the peculiar way in which the granules or vacuoles are ar-
76
ALBERT FISCHER
ranged. Often the big vacuoles are arranged as a single layer
in the periphery of the cell, or as a circular layer around a fine
granular area in the central portion (Fig. IX,). At other times
a distinct colony of uniform sized vacuoles can be seen, sur-
X
FIG.VI.
ILLUSTRATES
THE MOVEMENT
A N D CHANGES
IN SHAPE
OF A
MEDIUM-SIZED
CELLTYPEOF THE ROIJSSARCOMA
The cell has a very clear cytoplasm containing a single vacuole. The cell can
be seen wandering from cell to cell and during 14 hours it has moved a distance of
about two times the microscopic field.
I
FIG.
a
VII. SHOWS T H E C E L L S O F T H E SMALLEST TYPEO F T H E ROUS
IN A FIXED
AND STAINED
PREPAI~ATION
(METHYLENE
BLUE)
Note the big chromatic substances in the cytoplasm
(a).
SARCOM.4
DIVISION O F SARCOMA CELLS I N VITRO
77
rounded by a thin membrane (Fig. IX2A). Sometimes structures can be seen in the cytoplasm resembling crystals of
fatty acids.
FIG.VIII.
TYPEO F T H E ROUS
FIXED
AND S T A I N E D P H E P A R A T I O N
Note the chrorriatic substance scattered in the cytoplasm outside the nucleus
(1 and 2).
S H O W S T H E C E L L S O F T H E FIBROBLASTIC
C H I C K E N SARCOMA IN A
3. Spindle-shaped Cells. These look like rather large fibroblasts, or, sometimes, like the transitional forms between large
mononuclear lymphocytes and fibroblasts described by Carrel
(4). That is to say, one end of the cell is pointed, while t h e
other end may show filiform or tongue-like pseudopodia. T h e
cytoplasm is usually filled with granules. The nucleus may
contain one to four nucleoli (Fig. IX,). Other cells of this
type may show peculiar chromatic fragments distributed in the
cytoplasm (Figs. VIII, and VII1,).
11. The Activity of the Various Cells in V-itro
Characteristic for all cell-types of sarcoma tissue is their
great activity. Even the slowly moving ones exhibit greater
activity than normal fibroblasts. Secondly, i t is characteristic
of sarcoma cells to unite with other similar cells. The union
seems to be an actual fusion, not, a mere contact or cohesion,
such as can be observed so often to take place between normal
FIa. Ix. SHOWS
‘rM.0 CELLS OF THW FIBltoRLASTlC T Y P E O F T H E I t O I T b
SAHCOMA IN A METHYLENII:
B L U E STAINED PREPARATION
Thesc cells arc olmrved through the 1/12 oil immersion and comp. ocular 18.
1 shows a distinct nwleus with onc big mass of chroniatin and two small nuclcoli
with :t peculiar arrangement of the chromatic suhst:mcc. In the cytoplasni, which is
filled with big vacuoles, can be seen peculiar formations which look like crystals of
fatty acids.
2 Yhows also a distinct nricleus ( N ) with thrce ni~cleoli. Note the characteristic
arrangement of the vacuoles. A colony of even-sized vacuoles can be seen, stirrountlcd with a rather distinrt mcmt~r:~nc.
79
DIVISION O F SARCOMA CELLS I N VITRO
k'IG.
s. REPHESENTINC
CELLS
FROM A
HUMANOSTEO-SARCOMA
IN VITRO
The various figures show the activity of the cells and the changes of the outlines.
Thc cell which is marked 5 is the main cell for the observation. The convex !ine
80
ALBERT FISCHER
13
IS
on the northeast side of the cell, indicates the border line of the still solid plasma
clot. The arrows indicate the direction in which the cell moves.
1, 2, 3, 4, 5, 6, 7 and 8 show the active motion of the cell, drawn with intervals
DIVISION O F SARCOMA CELLS IN VITRO
81
of half an hour. In 9 the cell is shown to wander away from the plasma clot towards
another cell zl.
I n 10, it looks as if the two cells are going to unite and it seems that a part of
z1is separating, namely z2.
I n 11, granules can be seen to flow from zzinto the protoplasm of z.
I n 12, it can be seen that z is wandering away with a part of 21 namely 22. The
united z and x2 is wandering now towards the bordcr of the solid plasma clot and
x1 changes into a spindle-shaped ccll.
In 13, the z and xz are perfectly united. Broad pseudopods are spread out.
21 is suddenly seen t o move out of the stagc and changc into a spherical type.
Another cell za arrives. In 14. all three cells unite into one cell. In 15, thcy separate
again into thrce individuals.
I n 16, a new cell 5 4 arrives arid x2 is seen to wander towards this newcomer.
I n 17, z3has adopted the spindle shape and x2 and 5 4 are nearing towards each other.
The observations are made continuously in about 13 hours and drawings made
with an interval of about .5 to 10 minutes with the exception of the first 9 pictures.
fibroblasts. Shortly after amalgamation of two or more individuals has taken place, they generally separate into as many
individuals as united. This is illustrated in Fig. X, which
represents cells from a human osteosarcoma, and can also be
seen inFigs. I1 and IV.
This phenomenon of amalgamation was observed in all
cultures of malignant cells, but never in the case of normal
fibroblasts. I n a few cases i t was observed that two cells of
the large ameboid type amalgamated perfectly; shortly after
their separation (about one to two minutes) each divided in
two a t the same moment, the new ones being of smaller size
than the mother cells. This was a rather surprising fact,
because it has never been observed for normal tissue cells.
The observations were carefully made in order t o see what
really happened, and it was easy t o see that the two cells
actually divided and that all four moved away in different
directions with lively protoplasmic motion. I n one case it was
possible t o fix and stain the culture immediately afterward, and
thereby control observations on the living cells with the stained
preparation. This conjugation, or whatever it may be called,
followed by cell-division, is illustrated in Fig. 11.
It was often observed that isolated individuals divided and
that the new cells were of smaller size than the mother cell.
Karyokinesis has not been observed. Fig. V shows an extreme
82
ALBERT FISCHER
case in which one single cell separated into seven cells, many
times smaller than the mother cell. It is noteworthy that all
the small new cells were motile, and it is no wonder that sarcomata should appear so polyniorphous when their cells are
able to divide and separate into cells of uneven size.
FIG. XI. REPRESENTS
A FEW CELL TYPESFROM A HUMANGLIO-SARCOMA,
IT HAS BEENPOSSIBLE
TO KEEPGROWING
FOR MORETHAN Two MONTHS
VITlEO.
The drawings were made from a preparation, stained with Azur 11.
WHICH
IN
I n some preparations there was observed a struggle between
two cells of different type. Thus Fig. I11 illustrates the attempt
of a cell belonging to the medium sized type to enter a much
larger cell belonging to the fibroblastic type. It can be seen
that the small active cell is intruding upon the protoplasm of
the larger one. The only change which could be observed in
the larger cell was the appearance of two vacuoles moving
toward the place where the small cell attacked.
DIVISION OF SARCOMA CELLS IN VITRO
83
SUMMARY
.
The behavior of scattered and isolated tumor cells from the
Rous chicken sarcoma and from a few human sarcomas was
studied in vitro. Malignant cells have already been cultivated
in vitro by Carrel and Burrows ( 5 ) (chicken sarcoma, rat
sarcoma, and the Flexner-Jobling rat carcinoma), Lambert and
Hanes (6) (mouse carcinoma), and Losee and Ebeling ( 7 )
(a human sarcoma). All agree that it is rather difficult to
keep tumor cells growing outside the body. Extensive liquefaction generally occurred, so that good cultures could not
be obtained.
I n the experiments reported here, it was not so important to
keep the tumor cells growing over long periods of time, for
microscopic observations could be made after the tissue had
undergone a few passages.
The individual cells in the cultures were followed under the
microscope for several hours a t 39" C., and observations on
living cells were always controlled by stained preparations.
The cells which migrated from the explant were very polymorphous and the motility of all the cells seemed t o be much
greater than that of fibroblasts.
The main conclusion is, t h a t sarcoma cells are able to divide
when isolated and scattered. This was not found to be the
case for isolated and scattered normal fibroblasts (1). Still
more interesting is the fact that many of the new cells were
much smaller than the mother cells. I n all the cultures examined it was observed that certain cells wandered about among
other cells, establishing perfect union with them, after which
they separated again and wandered to other cells, uniting and
separating, and so on. I n a few cases perfect amalgamation of
two cells took place; they subsequently separated, each cell
divided, and the four individuals moved about with a lively
ameboid motion. Cell-divisions were often seen without preceding amalgamation, so that i t is difficult to draw any conclusion respecting the significance of amalgamation for celldivision.
84
ALBERT FISCHER
Compared with the normal fibroblast (l),where no division
of isolated individuals was seen, it must be said to be a general
occurrence for sarcoma cells. I n almost all the cultures of
scattered sarcoma cells examined, cell division could be observed.
CONCLUSIONS
1. Various types of sarcoma cell are able to divide in vitro
when isolated and scattered. This was not observed to be
true of isolated and scattered normal fibroblasts.
2. Sarcoma cells were seen to amalgamate in such a way
that it was impossible to discover more than one cell after
fusion had taken place. Generally they separated again shortly
atterward.
3. I n a few cases it was observed that cells which had amalgamated and separated underwent division immediately afterward.
4. Mitotic cell division was never observed among the cells
examined. The new cells were always found to be much smaller
than the mother cell.
I wish to acknowledge my indebtedness to Professor Rovsing
for permission to use the material from the surgical clinic, and
to Professor Ellermann for presenting me with the Rous chicken
sarcoma.
11EFERENCES
(1)
(2)
(3)
(4)
(5)
(6)
(7)
FIYCHEIL,
A.: J. I+:xpcr. M., 1'323, xxxviii, 667.
FISCHER,
A.: J. Cancer ltcs., 1925, ix, 50.
Rous, P.: J. Exper. M., 1010, xii, 696.
CARREL,
A., AND EBELING,
A. H.: J. Exper. M., lY22, xxxvi, 365.
CAI~REL,
A, AND UURI~OWS,
M. T.: J. Exper. &I., 1911, xiii, 571.
LAMBERT
AND HANES:Proc. Soc. Uiol. and Med., 1911, viii, 59.
LOSEE,J. It., A N D EBELIN(:,
A. €I.: ,J. Exper. M., 1!314, xx, 140.