Some Aspects of the Ascites Iunior Cell Response
to a Heterologous Antiserum*
K.
A.
O.
ELLEMf
(Department of Bacteriology, University of Sydney, Sydney, N.S.W., Australia)
The potent cytotoxic action of heterologous an
tisera induced in animals against various cell types
is well testified by the many descriptions of the
morphological changes caused by exposing cells to
them (1, 6, 10, 13, 15, 18, 20, 21). Where homol
ogous antisera have been observed to be cytotoxic,
they may also result in considerable structural de
generation (17, 19, 26).
The physiological and biochemical disturbances
of the cell responsible for this apparently charac
teristic (17) set of changes are beginning to receive
some attention (3, 7, 10).
A loss of cytoplasmic ribonucleic acid has been
noted (3, 10, 17). Flax (10) found that antiEhrlich mouse ascites tumor (EAT) -y-globulin, in
the presence of complement, inhibited the respira
tion of the cells with either their endogenous sub
strates or exogenous glucose, while not decreasing
their ability to use Buccinate as substrate. Colter
et al. (3) showed that antisera against ribonucleopro teins of two ascites tumors inhibit the utiliza
tion of glycine-2-C14 by these cells in vitro. In addi
tion to a loss of ribonucleic acid, they also found a
decrease in the total cell protein.
It has been previously shown (7) that rabbit
anti-EAT-serum produced an increase in the cellu
lar volume of about 40 per cent. Accompanying
this swelling was an increase in the permeability of
the cell membrane. Subsequent repeated washing
by centrifugation resulted in cellular disintegra
tion, whereas such treatment caused no com
parable damage to cells suspended in normal rab
bit serum. In the absence of complement, antiserum produced little immediate change in the
cells, but the repeated trauma of centrifugation re
sulted in a small amount of swelling followed by
increasing cellular disintegration. Antiserum alone,
therefore, had increased the cell fragility.
The present report is concerned with defining
* This work was supported by a grant from the New South
Wales State Cancer Council.
t Present address: Wistar Institute, 36th Street at Spruce,
Philadelphia 4, Pa.
Received for publication May 14, 1958.
more precisely the time relationships of the swell
ing and the permeability changes.
MATERIALS
AND METHODS
The tumor cells were maintained by serial transfer of EAT
cells (Lettréhyperdiploid strain) in mature female OS strain
mice. Blood-stained tumors were used neither as antigen nor as
test cells.
Antisera were prepared in rabbits. Cells were harvested and
washed 5 times in Hank's balanced salt solution (28), prepared
without phenol red (HBSS). The cells were diluted to a 10 per
cent suspension. By each of the three portals—intravenous, in
tramuscular, and subcutaneous—1 ml. of the suspension was
delivered on 3 successive days of the week for 3 weeks (30). One
week after the last injection the rabbits were bled, the sera
separated and stored at —¿15°
C. Before use, the sera were
thawed and heated for 30 minutes at 56°-57°
C. Complement
was added in the form of pooled serum from nonimmunized
rabbits and was stored for not more than 6 weeks at —¿15°
C.
The sequence of events induced by antiserum and comple
ment was examined by following the changes in cell volume and
the distribution of inorganic and acid-soluble organic phos
phorus between the suspending medium and the cells, after
different periods of incubation. Variations in the cell reaction,
with changes in the incubation temperature and in the concen
tration of antiserum and complement, were observed.
Cells were harvested after 10 days' growth. They were
washed in 5 changes of HBSS at room temperature, centrifuga
tion being at 980 X g for 10 minutes. In the reaction tubes the
proportions of the volumes of the mixed reagents were
cells: test serum ¡fresh,normal rabbit serum, as 3:5:2.5. The
total volume was adjusted with HBSS to give a final dilution of
the test serum of 1:3.9; and of complement, 1:7.8. During
incubation in 100 X 12 mm. pyrex tubes, the cell suspension
was kept uniform by being shaken manually every few minutes.
After a measured interval, aliquote were placed in chilled
hematocrit tubes of an elongated goblet shape, whose stems
stood in jackets of ice. To insure equivalent centrifugation for
any one experiment, all tubes were centrifuged together at
2,000 X g for 10 minutes. Under these conditions the cells had
sedimented into the stem in less than 2 minutes, at which time
the ice had begun to melt. During the remaining 8 minutes the
temperature gradually rose to 30°-32°
C. It is assumed that
little alteration occurred in cell volume, in the level of cell
metabolites, and in loss of cellular material to the supernatant
during these final 8 minutes. To allow for centrifugation, 2 min
utes were added to the actual incubation times.
After centrifugation, the stems were thrust into crushed ice,
the length of the cell column was measured, and the super
natant was transferred to iced tubes. The cells were homoge
nized in 10 per cent (w/v) trichloroacetic acid (TCA) and, after
standing for 20 minutes in ice, centrifuged. They were then reextracted twice with chilled 5 per cent TCA. The supernatant
1179
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Cancer Research
1180
sera were precipitated with TCA. Total TCA-soluble phos
phorus was measured in the supernatant sera and cell extracts,
following sulfuric acid digestion, by the method of Gomori
(12); inorganic phosphate (IP) on aliquots of the undigested
specimens. In those experiments where P32was used, approxi
mately 10 MC.was injected intraperitoneally into the mice (as a
sterile isotonic solution containing NaH2P32Oi), 24 hours before
the tumor cells were harvested. Subcutaneous streptomycin
was used to prevent infection of the aseites fluid. Inorganic
phosphate (IP) was determined by the method of Ennor and
Stocken (9), the same solution being used for colorimetrie and
radioactive determinations (8). The radioactivity of the total
digested phosphate was also determined on the same solution as
was used in colorimetry. The radiation was counted in a skirted
Vol. 18, November,
1958
liquid counter for a sufficiently long time to reduce counting
errors to less than 2 per cent.
The values of the acid-soluble organic (bound) phosphorus
(ASOP) were calculated by subtracting IP from the total P.
No significant amounts of ASOP were present in the sera before
being mixed with the cells.
The amount of extracellular fluid in the packed cells was
determined by an adaptation of the Evans blue method (27).
The average quantity of extracellular fluid was found to be 20
per cent of the cell mass (variation in several determinations,
17-23 per cent). This value is the same as that found by Crane
et al. (5), using raffinose as the nondiffusible label. By a similar
technic, an average quantity of 0.06 ±0.02 ml. of the super
natant was found as additional contamination of the cells
(packed cell volume, 0.3 ml.), owing to its incomplete removal.
The figures quoted below are corrected on this basis. Of the
chemical values, the maximum correction for cellular ASOP
was —¿18
per cent of the unconnected value, but was less than 5
per cent for all the other fractions. The general trends of the
results were unaffected by the alterations.
To calculate the concentration of a substance in the intracellular water, the volume occupied by the cell solids was as
sumed to be 20 per cent of the original cell volume (2, 5).
RESULTS
Following exposure to antiserum and comple
ment at 20°-21°
C., the cell volume increased rap
idly. Chart 1 represents the changes of a typical
experiment graphically. The rate of increase of the
cell volume was maximal during the first 10 min
utes. After this time the rate of swelling decreased
so that there was relatively little increase in vol
ume after 20 minutes. The maximum increase in
cell volume varied between different samples of
cells—amean increase of 61 per cent after 50 min
20
30
40
50
utes' incubation, with a range of 48-84 per cent in
TIME OF EXPOSURE
IN MINUTES
five experiments. No apparent difference was ob
CHART1.—Interrelationships of volume of the cells (•)
served with guinea pig or rabbit serum in equal
and the total amounts of acid-soluble organic phosphorus
(A) and inorganic phosphate (©)in the suspending medium volumes as the source of complement.
with increasing time of exposure to antiserum and complement.
After the changes in volume were initiated, the
Dotted lines indicate the values of control cells in normal
intracellular
acid-soluble organic phosphates
serum. The phosphorus figures are measured in micrograms
(ASOP)
leaked
out into the suspending fluid
of phosphorus.
TABLE 1
EFFECTSOFANTISERUM
ANDCOMPLEMENT
ONEHRLICHASCITESCELLS
TIME OF
TOTAL
EXPOSURE ACID-SOL.
TO BERUH ORGANIC
ANDCOM- PHOSPHORUS
PLEMENT IN CELLS
(MIN.)
((10. P)
Normal serum
3
53
Antiserum
176
147
XlOO
ICOP]„„„,
1.6
1.6
SPECIFICACTIVITY
OP ACID-SOL.ORG.
PHOSPHORUS
(COUNTS/MIN/VOP)
In
In
cells
medium
39.8
30 5
41.1
38.7
SNOPt+COP(„G.
CELLS(ilO.
P)81
P)lili178183177191221MÃ-23«TOTALINORGANICPHOSPHATEIN
977.891.991.989.586.586
TOTALNO.OF
CODNTS/MIN
FROMINORO.
PHOSPHATE
SXIP5
CIP
IN MEDIUM
IIH
667
H5
14:i
4SS
41.0
SO.9
1.4
164
644
40.4
5.6
34.1
136
146
821
39.2
37 4
11.8
11«
108
«02
35.4
43
2
35
0
71
104
486
481.3
39.0
34 6
48.8
71
108
692
34.0
35.9
47.8
76
n
* ISNOPkon«.,[COPUo.«.= concentrations of acid-soluble organic phosphorus in medium and cells, respectively.
t SNOP + COP = sum of acid-soluble organic phosphorus ¡ncells and medium.
ÃŽ
ISNIPJeone., ICIPJcono. = concentrations of inorganic phosphate in medium and cells, respectively.
§SNIP + CIP = sum of inorganic phosphate in cells and medium. Antiserum provided a barely significant 2 ¡tg.P more in inorganic phosphate to the
medium than did normal serum, owing to slight differences in the level of inorganic phosphate in the sera.
The concentrations of acid-soluble organic phosphorus and inorganic phosphate in the medium and cells were calculated as >igP/ml of medium and ¡igP/ml
of cell water, respectively.
S
8
11
U
93
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ELLEM—-Ascites
Cell Response to Antiserum
(Chart 1). The point at which the rate of this loss
was greatest was at least 3 minutes later than the
point of maximum increase in cell volume. The
leakage tapered off after 20 minutes' exposure and,
like the volume, increased little after this. There
was thus a rise in the total ASOP of the super
natant and a fall in that of the cells. The sum of
these shows that, in the system as a whole, there
was an increase in ASOP (Table 1, SNOP +
COP). The degree of loss of ASOP from the cells to
the supernatant may be expressed as the ratio of
the concentration of ASOP in the supernatant to
that in the cells. After 50 minutes' incubation at
20°C., the concentration of ASOP in the medium
was 48 per cent that of the cells. Presumably, com
plete cell lysis would result in a value of 100 per
cent.
Since the ASOP are a mixed group of com
pounds of varying rates of metabolic turnover,
there is little meaning attached to the specific ac
tivity of this fraction. However, although its spe
cific activity was consistently lower than the
ASOP of the cells (Table 1), it was so much higher
than the specific activity of the IP of the super
natant (Chart 2) that it rules out the latter as its
source. It must, therefore, have come from the cell
interior.
The inorganic phosphate also undergoes consid
erable changes (Chart 1 and Table 1). After the
cell swelling was under way, the IP of the medium
surrounding the cells began to disappear. At ap
proximately the same time as the cells were losing
ASOP at a maximum rate, IP of the medium was
falling at a maximum rate. After 20 minutes' ex
posure this ceases, and there was little change be
tween 30 and 50 minutes, the final amount being
less than half of the original value. By contrast,
the total amount of cellular IP altered little; in a
series of experiments there appeared to be a small
increase. The actual concentration of cellular IP
fell as the cell volume increased.
Experiments with cells in which the intracellular
phosphorus compounds had been labeled with P32
showed that during the first 10 minutes of antiserum action, the rate of loss of inorganic phos
phate (IP32) from the cell was greater than the
control cells in normal serum (Table 1). Between
10 and 20 minutes' incubation, when the rate of
increase in cell volume was falling, when the rate
of loss of ASOP from the cells was maximal, and
when the rate of fall of IP in the medium was
greatest, the IP32 level of the medium also fell.
There appeared to be free interchange across the
cell boundaries between the IP of the medium and
that of the cells after 25-30 minutes' exposure,
1181
since their specific activities became and remained
equal after this time (Chart 2).
The sum of the ASOP in the cells and medium
represents the total ASOP of the system. Compari
son of this sum (Table 1, SNOP + COP) and the
sum total IP of the system (Table 1, SNIP +
CIP) indicated that, during the first 10 minutes,
there was barely any shift between inorganic phos
phate and bound phosphorus. By 20 minutes there
had been an increase in the ASOP, quantitatively
accounted for by the decrease in the IP of the
whole system. After 30 minutes the total ASOP
had increased by 23 per cent over the control value,
owing to the binding of IP. After 50 minutes there
O
¡o
20
30
TIME OF EXPOSURE
40
50
IN MINUTES
CHART2.—Specificactivities (counts/min//jg P) of inor
ganic phosphate in cells (•) and in suspending medium
(©) after exposure to antiserum and complement. Dotted
lines indicate the values of control cells in normal serum.
was a further small increase in ASOP, with no
further fall in IP. If this excess of ASOP (2-3 per
cent of the total in a number of experiments) was
larger than experimental error, it may be ac
counted for by the breakdown of polynucleotide,
which has been reported (3, 10, 17) and confirmed
in this laboratory.
When the criteria for equilibration of concen
tration of IP between cells and supernatant were
applied, a pattern, at variance with that of the
ASOP fraction, was consistently observed. Instead
of the rise in concentration in the supernatant and
fall in the cells found with ASOP, the opposite ob
tained. Thus, after 50 minutes of antiserum action,
the concentration of IP in the medium was only 7
per cent that in the cells—lessthan the value of 10
per cent found with normal serum.
The control cells suspended in normal serum
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1182
Cancer Research
remained essentially unchanged. In a number of
experiments a decrease in cell volume was ob
served, in some cases amounting to 15 per cent;
but this was variable, and in the experiment re
ported there was no significant change. There was
slight increase in the leakout of ASOP, but the
final concentration
of ASOP in the medium was
less than 2 per cent of its concentration
in the cell
water. There was no significant change in the IP
of either compartment.
There was little inter
change of IP between cells and medium, the spe
cific activity in the latter rising relatively little
(Chart 2).
TABLE 2
EFFECTSOFANTISERUM
DILUTIONONEHRLICH
ASCITESTUMORCELL
Time of incubation, 42 minutes. Temp., 20°-21°
C.
Complement constant (dilution 1:7.8)
Vol. 18, November,
1958
in volume and twice as much ASOP in supernatant
as control) (Table 2).
The highest effective dilution of unheated nor
mal rabbit serum as a source of heat-labile comple
ment was 1:62 (volume increase, 17 per cent; gain
in ASOP of medium, 1.7 times control) (Table 3).
The action of antiserum in inducing the observed
changes was completely dependent on the presence
of heat-labile complement,
since with high dilu
tions of complement no difference was observed
between cells suspended in normal serum and
those in antiserum.
Temperature
variations.—The
reaction
at
37.5°C. was too fast to allow analysis of the early
changes in the cells when a relatively low-speed
centrifuge was used. However, after 30-40 min
utes' incubation, there were certain points of dif
ference from the cells incubated at 20°C. The
final volume of the cells was 5-15 percent less than
that of the cells incubated at 20°C. Instead of a
Volumeof
aspercentageof
cella
TABLE 3
Dilutions
of
antiserum
1:4
1:8
1:16
1:31
1:02
1:125
1:250
Normal
cells
EFFECTSOFCOMPLEMENT
DILUTIONON
in normalserum1421511411S8lil115110Totalacid-solubleorganicphosphorus
inmediumG«.
xioo
P>143137997(1r,5M38[SNOPlcono*[COPlcono.48.442.022.011.28.56.08.5
EHRLICHASCITESTUMORCELL
Time of incubation, 42 minutes. Temp., 20°-21°
C.
Antiserum constant (dilution 1:3.9)
serum
1.4
1:4
100
19
* [SNOPJoonc.,[COPlconc.= concentrations of acid-soluble
organic phosphorus in the medium and cell water, respectively.
Dilution of antiserum and complement.—In gen
eral, dilution of either component led to a decrease
inf the volume and permeability
changes in the
cell. Curves relating either of these characteristics
to the amount of the antiserum or complement
were concave to the axis that expressed the quan
tity of active serum in arithmetic (volume) rather
than logarithmic (dilution) units. Sufficient experi
mental results are not available to evaluate the
shape of the curves at high dilutions of antiserum.
No comparison is therefore available with the sigmoid curves obtainable for the distribution of redcell resistance to various lysins, such as are dis
cussed by Ponder (23).
Since the volume of the cells in some cell
samples decreased on exposure to normal serum,
dilutions of both antiserum and complement were
made in heat-inactivated
normal serum, thus
keeping the total amount of serum proteins con
stant. Significant effects were still found at an
antiserum dilution of 1:250 (10 per cent increase
Dilution
of fresh nor
mal rabbit
serum
+ antiserum
1:8
1:16
1:31
1::62
1::125
1:250
+Normal
Volumeof
aspercentageof
cells
cells¡n
normalserum163142141117110100Totalacid-solubleorganicphosphorus
mediumO.g.
xioo
D1331338043U25[SNOPlconc.*[COPlcon
serum
100
1.8
* [SNOPlconc, [COPlconc.= concentrations of acid-soluble
organic phosphorus in the medium and cell water, respectively.
1:8
fall in IP of the medium, there was a rise, there
being no over-all synthesis of ASOP from IP but,
in fact, the reverse—a breakdown of ASOP in the
system as a whole to increase the IP. The final dis
tribution of ASOP concentration between cells and
supernatant was the same as at the lower tempera
ture. Instead of being maintained against an unal
tered concentration
gradient, the intracellular IP
fell, and the ratio of its concentration to that of the
medium rose, as was the case with the cell ASOP
fraction.
DISCUSSION
It has been shown that antiserum developed
against intact Ehrlich mouse ascites tumor cells, in
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ELLEM—-Ascites
Cell Response to Antiserum
the presence of complement, induces a characteris
tic series of changes in the cells. Soon after being
mixed, the cells begin to swell, at first rapidly; but,
after about 15 minutes at 20°C., the rate becomes
progressively slower. Almost concomitant with the
swelling is an increased diffusion of inorganic phos
phate outward across the cell membrane. Further
changes in the permeability of the cell membrane
are represented by an increasing rate of leak out of
organically bound phosphorus, which follows the
other changes. Synchronous with the maximal loss
of ASOP from the cell occur a number of events :
the rate of swelling of the cells decreases, the in
organic phosphate no longer leaks out of the cell,
and inorganic phosphate disappears rapidly from
the medium.
The control cells in normal serum showed small
interchange between the cell IP and the super
natant IP. The rate of movement of phosphate
across the cell membrane has been shown to be
slow by comparison with the cations of sodium and
potassium (4, 25). The cellular barrier to inorganic
phosphate appears to be destroyed very early,
while the permeability to the larger molecules of
the ASOP fraction is altered somewhat later. By
producing increased permeability of the cell mem
brane to anions and cations (while preserving that
for sucrose) in leukocytes with n-butyl alcohol,
Wilson (29) produced cell swelling in an environ
ment of isotonic salt. The explanation for this
swelling is attributed to the osmotic pressure of the
intracellular colloids, which is no longer balanced
by the active transport of ions and the passive
movement of water out of the cell. The probable
influence of the colloid osmotic pressure on the
movement of ions and water in erythrocytes is rec
ognized in that form of hemolysis known as "col
loid osmotic," which has been discussed by Jacobs
and Stewart (16) and Ponder (23). If it can be
shown that there is an early loss of permeability to
other ions and cations before that of the intracellu
lar colloids, as would seem probable from the pres
ent results, then the swelling of the cells resulting
from antiserum and complement damage might be
explained on the basis of colloid osmosis.
While many of the earlier descriptions of the
morphological changes brought about by heterologous antisera differ widely, recent studies with the
use of interference (6) and phase (11, 18, 20) mi
croscopy agree on many points. All four authors
reported marked "blebbing" of the cytoplasm, an
affected cell showing one or more clear vesicular
protrusions from its surface. Occasional blebs con
tained one or two organelles moving about inside
them under Brownian movement. Few cells devel
oped blebs in normal serum. With time such
1183
blisters enlarge both outward and also by extend
ing around to involve more and more of the cell
surface until, as is well seen in the photomicro
graphs of Easty and Ambrose (6), the central nu
cleus and clumped mitochondria are surrounded
by a halo of clear cytoplasm with a distinct bound
ary. This series of changes has also been observed
in this laboratory. It is of interest to note that, in
the two cases where blistering was specifically ab
sent in the presence of other structural evidence of
cell damage, two conditions differed from those of
the other studies: (a) the cell suspension was pre
pared from solid tissues, and (6) the antisera used
were both homologous to the cells tested (17, 26).
Zollinger (31), in a study of this phenomenon,
called "potocytosis" by him, found that distilled
water greatly accelerated the rate of formation of
blisters. Ascites cells are more resistant to blister
formation in isotonic saline than were the cells he
used, which were all prepared from solid tissues,
but we have also found that hypotonie solutions
cause intense blistering of the cells. Many normal
tissues are known to swell when suspended in
sahne or more complex isotonic fluids (22, 24). It
seems then that if cells suspended in fluid environ
ment are caused to swell by a number of means,
much of the swelling is irregular and produces lo
calized protrusions rather than a simple, regular in
crease in volume. At present, no definite evidence
may be quoted to choose between the possible rea
sons for this—namely (a) local intrinsic weak
nesses in the cell membrane, or (o) localized
changes in the cytoplasm beneath the visibly af
fected areas of the cell membrane.
That there are differences in the qualities of the
blistered membrane caused by antiserum and by
hypotonie solutions is evident, since, for equiva
lent increases in cell volume due to these causes,
there is a far greater loss of the intracellular con
tents with antiserum than with hypotonie solu
tions (7).
Surface activity of the antiserum and comple
ment may be presumed for at least the early part
of the changes, since diffusion of the antibodies
into the cell must be a fairly slow process, if it oc
curs at all, and the observed changes begin within
1 or 2 minutes. That surface attachment of the
antibodies occurs is a deduction from the aggluti
nation, which has been observed elsewhere (6, 14)
and in this laboratory, and also the alteration of
cell fragility mentioned above (7).
The described cellular responses to antiserum
and complement appear to be all-or-none in nature
and not a graded response from each cell in the
population. Preliminary evidence shows a close
correspondence between the magnitude of the
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1184
Cancer Research
changes in packed cell volume occurring with vari
ous dilutions of antiserum and complement and
the number of cells affected by blistering.
The ascites cells do not lyse completely at 20°C.
in less than an hour. Morphologically,
they retain
a distinct outline. There remains a concentration
gradient of ASOP between cells and medium at
either 20°C. or 37°C. There is a very much larger
difference in the concentrations
between the acidextractable IP of the cells and the IP of the me
dium, marked at 20°C., and much less marked at
37°C.
The observed disturbances of the functional in
tegrity of the cell membrane may account for the
biochemical inadequacies of the antiserum-damaged cells, which have been reported by Flax (10)
and Colter et al. (3). Diffusion of endogenous sub
strates and co-factors out of the cells could result
in the disturbances of respiration and the deficien
cies of synthesis of large molecules that they de
scribed.
SUMMARY
1. The effects of antiserum and complement on
the volume and permeability of the Ehrlich ascites
tumor were studied.
2. The cells quickly underwent marked swelling
concomitant with an increased permeability to in
organic phosphate. Following this there was a loss
of acid-soluble organic phosphorus from the cells
to the suspending medium.
3. At 20°C., within 30 minutes, significant
binding of inorganic phosphate to the acid-soluble
organic fraction occurred. At 37.5°C. there was an
over-all degradation
of some of the acid-soluble
organic phosphorus fraction to inorganic phos
phate.
4. Despite the increased permeability
of the
cell membrane at 20°C., there was still a marked
concentration gradient of inorganic phosphate be
tween cell and suspending fluid. At 37.5°C. this
effect was largely abolished.
5. Dilution of either antiserum or complement
led to a diminution of all these effects. Both com
ponents were necessary.
6. A possible mechanism for the swelling of the
cells was suggested.
ACKNOWLEDGMENTS
Grateful acknowledgment is made to Professor P. M. de
Burgh for his continued interest in this work.
REFERENCES
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1957.
Vol. 18, November,
1958
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H. N., and RIGGS,T. R. Concentrative Up
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1957.
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Some Aspects of the Ascites Tumor Cell Response to a
Heterologous Antiserum
K. A. O. Ellem
Cancer Res 1958;18:1179-1185.
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