Separation of Ascites Tumor Cells Rich in Deoxyribonucleic
Acid by Means of Counter-Streaming Centrifugation*
PER ERIC LINDAHL
(Institute
of Zoophyrzology,
Uppsala, Sweden)
SUMMARY
The possibility of separating ascites tumor cells with different numbers of chromo
somes by means of counter-streaming
centrifugation
has been studied with the hy
perdiploid
Ehnlich
ascites
tumor
(ELD)
and its hypertetraploid
offshoot
(ELT).
From ELD a fraction significantly higher in deoxyribonucleic
acid phosphorus
(DNAP) per cell than the unfractionated
suspension was obtained. Inoculated into
a new mouse this fraction developed into a suspension which was invariably lower in
DNAP per cell than the inoculum. Repeated inoculation of this fraction followed by
further fractionation increased the mean number of chromosomes per cell, giving about
twice the original number.
When another fraction of ELD, which is low in DNAP, was inoculated, the tumor
suspension
producedshowed thesame valueofDNAF as thatoftheinoculum.
Fractionation
oforiginal
ELT gave two fractions
highinDNAP per cell,
one com
posed chiefly of octoploid cells, and another of tetraploid and octoploid cells.
The distribution of the different types of tumor cells upon the three fractions ob
tained with counter-streaming
ce.ntrifugation is not in keeping with that which should
be expectedfrom theirvolumesand contentsof DNA.
It is a well known fact that the chromosome
numbers of tumor cells are subject to great varia
tion (of. [7]). It must be considered of great in
terest to be able to discover and study differences
in biological properties and enzymatic constitution
between tumor cells with high and low chromo
some numbers, but differing as little as possible
in other respects. So far this has been accomplished
only by deriving from the same original neoplasm
two tumor sublines that differ with regard to
chromosome ploidy (2). Such a procedure opens
the possibility that mutations may occur which
would bring about further differences between the
two lines. It would, therefore, be advantageous
to be able to study such differences between cells
‘Thisinvestigation was supported by a grant from the
Swedish Cancer Society, which is gratefully acknowledged.
I
am very much indebted to Professor G. Klein, Karolinska
Institutet,
Stockholm, for material and advice, and to Pro
originating from the same tumor. Since variations
in the chromosome number in ascites tumor cells
are connected with variations in the content of
deoxyribonucleic
acid (DNA) (1), and since this
substance has a rather high specific gravity (about
2; for specific volumes of. [3]) compared with that
of other cellular constituents,
we must expect
a corresponding variation in the specific gravity
or—provided
thatthe cellsizeislinkedwith the
number of chromosomes—in
the sizeof the cells.
In either case the conditions would be such as
to permit the separation of tumor cells containing
high numbers from such with low numbers of
chromosomes by the aid of counter-streaming
cen
trifugation (8). The aim of the present study is
to examine this possibility and to follow the influ
ence of repeated separation and subeultivation
upon the chromosome number.
fessor A. Levan, Lund, for valuable information on the chro
mosome numbers
of some fractionated
material.
I thank
The animals
Mr.
P.-A. Lindstrom and Miss Gertrud Thelin for skilful technical
assistance.
Received
for publication
December
MATERIALS
AND
METHODS
used in these experiments
were
heterozygous
albino mice of both sexes, weighing
25—35 gm. They were kept on a standard
com
pressed diet and tap water ad libitum.
9, 1959.
841
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842
Cancer Research
The experiments
lines of the Ehrlich
ELD
were carried
carcinoma,
out with
two
the hyperdiploid
(2) and its hypertetraploid
offshoot
ELT
(2), obtainedon severaloccasionsfromthe De
partment of Tumour Biology, Karolinska Insti
tutet, Stockholm. The animals were given inocu
lations of about 4 X 1O@cells from ELD or of
4 X 1O@cells from ELT. Ascites was collected
from mice showing marked abdominal distension,
which generally occurs after 7—13days with ELD
and after about 20 days with ELT.
The separations, performed with the counter
Vol. 20, July
eter after dilution, first 1 : 20
solution, and then 1 : 20 with
Eighteen A-squares thus give
Since the ascites fluid does not
1960
with the Ringer
Tttrck's solution.
about 1000 cells.
contain any DNA
(5), removal of the liquid and washing of the cells
of sample A was not required. In Tables 1—3
amounts of DNA phosphorus
(DNAF) per cell
are given. The error of these figures may be esti
mated at 15—20per cent (of. [1]).
RESULTS
According to the principle upon which the
streaming
centrifuge
(8), were carried on for 30—40 counter-streaming
centrifuge is constructed the
minutes at 300 r.p.m. and an initial streaming
most easily sedimented
cells, i.e., those having
velocity of 10—17,generally 12 ml. sec.'. The
the highest specific gravity or the greatest volume
decrease in temperature
in the streaming suspen
or these two properties combined, should be cx
sion medium
(8) was 9.0°C./hour.
The tempera
pected to accumulate in fraction M, fractions G
ture at introduction
of the cells was 18°C. in and L being expected to contain cells which are
experiments
with
ELT,
and
in preliminary
cx
progressively
less easily
sedimented.
periments with ELD, whereas in the final experi
ments with ELD the temperature was 230_240 C.
(slightly above that of the laboratory). The sepa
ration chamber used had a greatest diameter of
2.40 cm., with the theoretical point of the cone
at a distance of 18.0 cm. from this diameter. Al
though the efficiency of the separation chamber
A number of preliminary
separation
experi
ments with the original hyperdiploid tumor ELD
were performed in order to find suitable conditions.
When proper streaming velocity and number of
revolutions had been chosen, these experiments
turned out in a most unexpected way, i.e., higher
values of DNAP per cell were obtained in fraction
is generally
G than
of series
somewhat
reduced
in experiments
with model particles (mastic balls) by introducing
a “collecting tube― in series with the chamber
(10), this arrangement
appeared to be advanta
geous here. This tube (of. [10], Fig. 3), with a di
ameter of 2.7 cm., had its inlet and outlet at a
distance of 13.5 cm. from the center of rotation.
Its centripetally directed conical part had a length
of 3.0 cm. In this way three fractions of cells were
obtained,
the first (M) and the second (G) re
maining
in the separation
chamber
and the col
lecting tube, respectively,
and the third (L) leaving
the centrifuge.
The ascites was diluted about 10 times with
Krebs' phosphate Ringer solution, containing 1
per cent glucose and 1 per cent gelatin (cf. [9]).
This
solution
was
also
invariably
used
as the
streaming suspension medium in the centrifuge
(8). The gelatin considerably reduced the damage
caused otherwise by the mechanical stress involved
in the counter-streaming
centrifugation.
The vol
ume of the cell suspension introduced into the cen
trifuge
was 20—30 ml.
After
the
separation
was
finished the volumes of the three fractions were
brought down to about 2 ml. by centrifugation
in an ordinary
centrifuge
at 125 X g. DNA
was
analyzed according to Schneider (11) before frac
tionation (A) and in the three fractions, M, G,
and L in each experiment, and counts of the total
number of cells per ml. performed in a hemocytom
in fraction
M (cf. experiments
No. 1
I—Ill, Table 1): In the series of experi
ments of a first type, each successive mouse was
given an inoculation of fraction G from the pre
ceding mouse for the in vivo production of the
tumor
cells
to
be
centrifugally
separated
in a
following experiment.
The general trend of such a series appears from
Table 1 in which the changes in mean DNAF
per cell during the passage in vivo are given
in a special column. With very few exceptions
this change is positive, i.e., the percentage of cells
with high chromosome numbers in the inoculum
decreased during the multiplication
period. In
the former part of the series this change was small
er than the difference between the mean DNAP
per cell of the sample collected from the mouse
(A), and that of fraction G obtained from this
sample by counter-streaming
centrifugation.
This
results in this part of the series in an increase
of the mean DNAP per cell of fraction G from
one experiment to the other. When a value of
about
2.5 in fraction
G was reached,
a turning
point appears, after which the decrease in DNA?
per cell during the period of multiplication
was
greater than the increase produced by the counter
streaming centrifugation
in fraction G. This re
sults in this part in the series in a decrease of the
mean DNA? content per cell of fraction G from
one experiment
to the other. The number of
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TABLE 1
SERIAL SEPARATION
EXPERIMENTS
WITH ELD
First mouse inoculated with original tumor. Ascites tumor suspension obtained (A of first experi
ment) was separated into fractions M, G, and L. Each successive mouse was given an inoculation of
fraction G from the preceding mouse. DNAP per cell in @g
X 10-6.
CELLOF
No. or
EXPERI
PER
MULTI
PLWA
RENTPERIODDNAP
TION
A—'G at
(days)AMGLChange
fractionat.Change
SERIES
1
9
2
11
3
in vivo
(G—+A)
I
1.90
1.02
2.12
0.92
+0.22
8
2.03
1.26
2.25
0.99
+0.22
4
12
2.14
1.67
2.31
1.24
+0.17
5
12
2.21
0.65
2.52
1.32
+0.31
6
11
1.52
0.97
2.04
1.2.5
+0.52
7
12
1.36
0.85
1.74
1.14
+0.38
—0.09
—0.11
—0.10
-1.00
—0.68
8
91.15
1.281.04 0.791.89
—0.46SERIES
1.460.81 0.79+0.74
+0.18+0.01
II15
1
2
13
1.61
1.07
1.86
0.91
+0.25
2.28
1.15
+0.63
—0.21
3
9
1.65
1.02
4
13
1.81
1.55
2.37
1.29
+0.56
5
12
2.06
1.22
2.50
1.22
+0.44
6
18
1.36
1.01
1.62
7
111.27
1.190.80
0.741.86
1.410.99
—0.47
—0.31
—1.14
0.96
—0.43SERIES
+0.26
1.10+0.59
+0.22—0.25
Ill
1
9
1.19
0.96
1.97
0.96
+0.78
2
13
1.56
0.94
2.11
1.13
+0.55
S
10
1.89
1.12
2.29
0.91
+0.40
4
12
2.00
1.72
2.30
1 .25
+0.30
‘5
12
1.97
0.97
2.07
1.05
+0.10
6
10
1.55
1.13
1.70
1.06
+0.15
7
13
1.16
—0.41
—0.22
—0.29
—0.33
—0.52
—0.54
1.50
+0.34
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
Cancer Research
844
experiments
preceding
this turning-point
in each
stant.
series varied. In series I—Il of Table 1 the turning
point was associated
with a very great decrease
in the DNA? content per cell during the multipli
to the
highest
value
obtained
of the cell population
a slight decrease
(A) and in fraction
1960
in the collected
M should
be noted.
On application of the same conditions of sepa
ration to the hypertetraploid
tumor ELT the
highest
DNA?
content
per
cell
was
found
fraction L, and the next highest in fraction
experiment
No.
1 in Table
8). When
in
M (of.
the original
ELT material was separated, the DNA? content
per cell of fraction L attained a value which was
about twice (or somewiiat less) that of the col
lected suspension. This result has been obtained
in three out of four experiments.
In the fourth
in fraction
G was 2—2.5times, this ratio depending on the
composition
Perhaps
suspensions
cation period. In some preliminary series all fur
ther growth of the inoculated cells ceased after
this high mean value of DNA? per cell had been
reached. The total rise in the mean DNA content
per cell from that of the original inoculum of the
series
Vol. 20, July
of the starting
material.
In a second type of experimentalserieswith
ELD, each successive mouse was inoculated with
fraction L from the preceding mouse (Table 2).
Here alsothe separations
gave thehighestDNA?
values per cell in fraction G. All through the series
the DNA? content per cell remained fairly con
experiment
the separation
was not successful.
In
the single series of separation experiments per
formed with ELT each successive mouse was given
an inoculation of fraction M from the preceding
mouse. In this series the DNA? content per cell
in all fractions except M dropped between the
first and the second
TABLE
experiment,
whereas
no such
2
SERIAL SEPARATION EXPERIMENT
WITH ELD
First mouse given inoculation of original tumor. Ascites tumor suspension obtained (A of first experiment) was
separatedintofractions
M, G, and L. Each successive
mouse was givenan inoculation
offraction
L from the pre.
ceding mouse. DNAP per cell in @g
X 106.
CELLAMGLChange
.
PER
OF
.
NO.OF
MVLTIPLI
EXPERI
RENTPERIOD
CATION
A—ILat
(days)DNAP
fractionation1
13
2
in vivo
A—'G at
fractionationChange (L—A)Change
1.01
1.22
1.34
—0.11
0.90
+0.33
—0.05
10
3
0.85
1.08
1.25
0.79
—0.06
+0.40
+0.09+0.36
151.14
411
0.881.26
1.141.50
1.400.95
TABLE
SERIAL SEPARATION
@
0.94—0.19 +0.06+0.06
+0.52
3
EXPERIMENT
WITH ELT
First mouse inoculated with original tumor. Ascites tumor suspension obtained (A of first experiments) was sepa
rated into fractions M, G. and L. Each successive mouse was inoculated with fraction M from the preceding mouse.
DNAP per cell in
X l0@.
PER CELL
OF
.
No. OF
-
MULTIPLI
EXPERI
RENTPERIOD
CATION
(days)DNAP
A—@M
at
fractionationChange
AMGLChange
fractionation1
in vivo
A—+L
at
(M—A)Change
.48
2
17
1.65
3.37
0.854.65
4.02
+0.72
+2.37
-1.77
3
10
160
415
272.25
1.783.13
2.97w1.76
3.08+0.88
+1.37—1
+1.48
—1.19+2.40
* Also
inoculated
for
preliminary
chromosome
studies
by
Dr.
A.
Levan.
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LINDAHL—Counter-Streaming
Cenlnfnçjation
of Ascites
Cells
845
(or octoploid) cells during the fractionations,
these
cells then multiplying in about the same propor
tion as the diploid ones. After a few such frac
DISCUSSION
The number of chromosomes of ELD is only tionations and inoculations the DNA content per
cell in the unfractionated
collected suspension has
slightly higher than that normal for the mouse
(2). The value 1.11 X 10@ jzg. DNA? per cell in increased at most about 100 per cent. That means
ELD (2) comparedwiththat of inflammatorya doubling of the mean chromosome number.
On reaching this stage, however, the decrease
cells, 0.77 X 10—ijig. (1) is, therefore, remarkable.
in DNA? per cell which occurs during the period
The difference depends partly on the spontaneous
of multiplication in vivo becomes greater than
ly appearing
minority
of polyploid
(tetraploid)
earlier.
This must be caused by a reduced rate
elements (about 5 per cent [2]) which always
of multiplication
of the true tetraploid cells com
occurs in the hyperdiploid
tumors, and partly
pared with that of the diploid ones. At the next
upon the fact that a fraction of the hyperdiploid
separation the increase in DNAP per cell in frac
cells which are in a preparatory
phase related
tion G over that of the unfractionated
suspension
to the following mitosis have doubled their DNA
thus
becomes
smaller,
and
this
is
repeated
in the
content and their number of chromosomes. The
following experiments of the series until the DNA?
size of this fraction of incidentally tetraploid tumor
per cell of the un.fractionated
ascites tumor (A)
cells depends upon the mitotic index and must
has
assumed
about
the
same
value
as that of the
be expected to be greater during the fast phase
original
tumor.
In
other
series
the
appearance
of
of growth, i.e., the first 6 days after the inocula
this turning-point involved the complete cessation
tion, than later. A variation in the size of these
of growth of the inoculated material.
two fractions of tumor cells, the true tetraploid
Some comparatively high values of DNA? per
and the incidentally tetraploid cells, probably causes
cell occur also in the M and L-fractions. At least
the variation in DNA? per cell of the original
for one of these two fractions this is always the
populations which have been in no way experimen
case, when the DNA? per cell was high in the
tally influenced, and which is presented at the
top of column A of the three series in Table 1. suspension to be separated. This must most prob
Further, these cell populations include about 10 ably be looked upon as a weakness of the method
of separation. The introduction into the centrifuge
per cent leukocytes (inflammatory cells).
of too great an amount of cell material may pos
To simplify the following discussion, the mean
sibly also contribute
to this. It appears from
DNA? content per cell is approximated as follows:
Table 1 that these comparatively high values gen
for hyperdiploid cells, 1.0 X 10'; for hypertetra
ploid cells, 2.0 X 10'; and for hyperoctoploid cells, erally appear in fraction M or in fraction L.
Only in one experiment in each series do such high
4.0 X 10@ pg. These values are too high rather than
values appear simultaneously
in both these frac
too low.
The fractionation of ELD most probably brings tions, and it is remarkable that this very experi
ment is always closely associated with the maxi
about an accumulation
of the two kinds of tetra
mum of DNA? per cell of fraction G.
ploid cells in fraction G. After this fraction has
According to the discussion above fraction L
been inoculated the incidentally tetraploid cells
of an experiment performed with original ELD
probably divide synchronously
for some time.
material must be expected to contain hyperdiploid
It can be expected that later the divisions of these
tumor cells and inflammatory
cells, whereas
the
cells will become evenly distributed in time. This
two
kinds
Of
hypertetraploid
cells
are
probably
implies a smaller fraction of incidentally tetraploid
rare. The results of the experimental series (Table
cells than in the inoculum and thus a decrease
2) in which only fraction L was inoculated are
in the mean DNA? content per cell as observed
in keeping with this view. A significant decrease
in the experiments.
This decrease
is, however,
in the DNA content per cell does not occur during
generally smaller than the increase in DNA?
the periods of multiplication (of. Table 2). Accord
content per cell attained in fraction G by means
ing to the reasoning advanced above, this means
of the preceding fractionation
as compared with
that no or very few incidentally hypertetraploid
that of the original suspension (A of experiment
No. 1). Thus, the unfractionated
suspension (A cells are present in these inoculates. Although
rather low in DNA per cell, the suspensions ob
of experiment No. 2), obtained from the mouse
tained from mice given an inoculation of fraction
given an inoculation of fraction G, has a higher
L furnish G-fractions with a significantly higher
DNA? value than the original suspension. This
DNA? content per cell than that of the unfrac.
applies to all the early links of the series and must
b@ ‘@aused
by the accumulationof truetetraploid tionated material (of. Table 2). This increase
decrease
third.
occurred
between
the second
and the
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
Cancer Research
846
Vol. 20, July
1960
is, however, small compared
with the correspond
ing one at the fractionation
of original suspensions
after the phase of fast
is being investigated.
of ELD (Table 1).
ELT lines contain a residue of 5—15per cent
hyperdiploid
cells and > 86 per cent polyploid
(2), i.e., tetraploid and possibly octoploid cells.
ing to (2) a volume about twice that of the hy
perdiploid
cells. Provided with the double amount
Since
the
tumors
are always
mixed
with
about
10 per cent inflammatory cells these figures, when
applied to the suspensions collected from the mice,
must
be reduced
to 4.5—13 and
The very high DNAP
>78
per
cent.
values per cell found
in
fractions L and M (Table 3) must mean that these
are to a great extent composed
of incidentally
or true octoploid
cells. The DNA?
content
per
cell of fraction
in this
series
M which was used as inoculum
has not
changed
appreciably
after
two fractionations
and passages. A continuous
decrease was, however, observed in fraction L,
and both in fraction G and in the series of A-values
there was a sudden decrease between the first
and
the
tendency
second
experiment.
Thus,
in this series is a decreasing
is a consequence
of the
the
general
one. If this
accumulation
of cells
with very high ploidy (of. the results with ELD)
or a manifestation
of the
tendency
some number of 2 s or still higher, often 4 8,
the normal chromosome number of the hyperdip
bid Ehrlich tumor being 8.
The counter-streaming
centrifugation unfortu
nately does not admit in the present experiments
to distinguish between true tetraploid and inci
dentally tetraploid
tumor cells of ELD origin.
This impairs the separation of clean tetraploid
cells which might possibly have been achieved
otherwise. Further, the diploid elements, being
thus included in the otherwise tetrapboid fraction,
will tend to multiply synchronously when trans
ferred into a new mouse. Depending on the stage
of mitosis of these synchronized cells, determina
tions of the content of DNA per cell from samples
secured
in tnvo shortly
after
the inoculation
of the
mouse would show an increased variability. Most
probably the synchronization
would then gradual
ly weaken and might possibly have disappeared
problem
of DNA they should have about the same specific
gravity. Because of their greater volume the tetra
ploid cells should sediment faster than the diploid
ones
tube
and should thus be retained
(fraction
M). Instead
they
in the conical
appear
in the
collecting tube.
It might be expected that in ELT the hyper
diploid and hypertetraploid
cells should have the
same properties
determining
mentation
as in ELD. From
their rate of sedi
the figures in Table
3 it appears that, in experiments with ELT, frac
tion L is dominated by octoploid and tetrapboid
cells. The comparatively
low DNA? values of
fraction G seem to indicate that this fraction,
at least in two experiments,
was made up chiefly
by diploid cells. This means that the tetrapboid
cells of ELT did not accumulate in fraction G as
did those of ELD. Further, the octopboid cells of
ELTwerepartlyretainedintheseparation
cham
“to ber (fraction M) and partly
of the hy
tion leaving the centrifuge
size. All mitoses which could
the first week had a chromo
This
cells of ELD have accord
of ELT
revert gradually toward predominance
perdiploid cell minority― (2) cannot be decided
from the present experiments.
Five mice inoculated with fraction M of the
third experiment were sent to Dr. A. Levan, who
had kindly offered to make preliminary studies
of the ploidy of this material. Of the five mice
only one contained tumor cells. The ascites cells
from this animal were striking from the beginning
for their enormous
be counted
during
The hypertetraploid
growth.
released into the frac
(L), thereby
the collecting tube (fraction
that two kinds of octoploid
in ELT
with
rather
escaping
G). This suggests
tumor cells occur
different
rates
of sedimenta
tion. Possibly these two kinds are represented by
incidentally and true octoploid cells.
From the above discussion it is obvious that
the rate of sedimentation of the different categories
of cells is also influenced by other factors than
those—size and DNA content—mentioned
in the
introduction.
When observed under the micro
scope the tumor cells of both ELD and ELT
exhibit
a varying
number
of bulging
with clear fluid and situated
surface.
These
structures
blisters,
just beneath
are
of quite
filled
the cell
another
kind than the bulges described by Shear (12)
as resulting from the treatment
of tumor cells
with salt solutions. They occur already in tumor
cells freshly secured from the mice and are of lim
ited size, 1—2.5p diameter. Such blisters have
been photographed
by Klein et al. ([4], cf. Figs.
2, 3, and 6) in ascites
mice given inoculations
lich carcinoma,
The number
in the
leukocytes
tumor cells obtained
from
intraperitoneally
of Ehr
and are spoken of as “vacuoles.―
of the vacuoles which are absent
increases
with
increasing
size
of the tumor cells; number of vacuoles per cell
plotted against diameter of cell raised to the
2nd power gives a straight line. With increasing
numbers
of vacuoles
the
specific
gravity
most
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
LINDAHL—Counter-Streaming
Centrifugation
probably will decrease, and the frictional drag
on the cell surface increase. Both these factors
will cooperate in a decrease in the sedimentation
rate which will reduce or dominate over the in
crease in sedimentation
rate due to the rise in
ploidy and therewith in the size of the cells. Most
probably this is the cause of the unexpected dis
tribution
of the different types of tumor cells
upon the three fractions
obtained
with counter
streaming
centrifugation.
Further studies on these
relationships
are in progress.
Further
differences in the chemical composition
may contribute
to differences
in specific gravity.
Of interest
in this connection
is the observation
of Ledoux and Revell [6] that the content of ribo
nucleic
acid
in ELD
may
decrease
as much
as
50 per cent during the aging of the tumor.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1960 American Association for Cancer Research.
Separation of Ascites Tumor Cells Rich in Deoxyribonucleic
Acid by Means of Counter-Streaming Centrifugation
Per Eric Lindahl
Cancer Res 1960;20:841-847.
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