CHEMICAL STUDIES ON TUMOR TISSUE
VI.
COMPARATIVE EFFECTS OF SERUM COLLOIDS AND OF SALTS ON THE
SWELLING OF MOUSE TUMOR CELLS IN VITRO
MORRIS BELKIN
AND
M.
J. SHEAR
(From the Office of Cancer Investigations, U. S. Public Health Service,
Harvard Medical School, Boston)
INTRODUCTION
The swelling of mouse tumor cells in salt solutions has been described in
previous communications (Shear and Fogg, 1934; Shear, 1935). The swollen
cells usually exhibit blisters, or bulges, of clear fluid underneath the cell membrane. These accumulations of fluid form within a few minutes and frequently attain a volume equal to that of the cell initially (for photographs, see
Shear, 1935). As previously stated, " This swelling was independent of the
salt concentration and occurred in salt solutions which were hypertonic, as well
as isotonic and hypotonic, to salt solutions having the same inorganic composition as blood plasma." The only factor which was found to control the swelling was the presence of colloids, such as proteins.
The numerous data obtained by various investigators on osmotic properties of free cells such as marine ova, erythrocytes, and plant cells have usually
been considered as showing that for such cells the salt content of the environment is, in general, the determining factor in osmosis; i.e., in certain salt concentrations (isotonic) the cell volume remains unchanged, whereas in more
concentrated salt solutions (hypertonic) the cells shrink, and in more dilute
salt solutions (hypotonic) the cells swell. In the case of cells of normal and
malignant solid tissues of mice in our experiments, however, the oncotic (colloid osmotic) pressure, rather than the osmotic pressure of the salts, seems to
play the predominant rOle.
The present paper reports experiments designed to evaluate the relative
importance of salt concentration and of colloid concentration on the bulging
phenomenon in mouse tumor cells. The comparative significance of these two
factors was ascertained by observations upon cell swelling in solutions of varying salt and protein concentration.
The greatest amount of swelling occurred in protein-free salt solutions.
Increasing the salt concentration did not produce a decrease in the number of
cells exhibiting bulges; even when the salt content was raised from a point considerably below the amount present in normal mammalian blood plasma to a
point considerably above, repression of the swelling was not obtained.
Repression of the swelling occurred only when colloid was contained in
the medium. Solutions were then employed in which the colloid content was
such as to permit swelling to occur in a significant proportion of the cells; the
salt content of these solutions was then varied. With the colloid content kept
constant, it was found that increasing the salt concentration did not further
62
CHEMICAL STUDIES ON TUMOR TISSUE
63
repress swelling. When the osmotic pressure of the solution was increased by
addition of equivalent amounts of sucrose, instead of salt, similar failure to
curb bulging was observed.
Quantitative data of satisfactory reproducibility were obtained not only as
regards the swelling but also as regards the viability of the cells studied. The
latter information was obtained by including neutral red in the media employed. These results confirm the conclusion reached previously (Shear and
Belkin, 1937), that the swelling phenomenon is not a post-mortem change but
occurs in cells apparently alive and in good condition.
METHOD
Tumor tissue was minced with fine scissors in about 5 C.c. of solution contained in a small Petri dish. When colloid-free salt solutions were used, the
concentration of neutral red employed was 1 to 40,000. When solutions containing protein or other colloid were used, higher concentrations of neutral red
were found necessary to give comparable staining. Neutral red was added to
such solutions in a concentration of 1 to 5,000. Although in some cases the
dye precipitated slowly, this concentration did not appear to be deleterious to
the cells and did not interfere with the observations.
The tumor used in this investigation was sarcoma 37, carried in Strain D
mice. The spindle cells, the only ones considered in this study, were grouped
into six categories as follows: (1) colorless cells with bulges; (2) colorless
cells without bulges; (3) vitally stained cells with bulges; (4) vitally stained
cells without bulges; (5) diffusely stained cells with bulges; (6) diffusely
stained cells without bulges.
Quantitative data were obtained by making cell counts in each of which
some 300 cells were examined individually and classified as above. For this
study, only isolated cells and marginal cells of explants were considered appropriate material from the point of view of satisfactory observation, of adequate
exposure to the medium, and of absence of tissue pressure (Burch and Sodeman, 1937). Two counts were made in each experiment, one beginning fifteen
minutes and the other seventy-five minutes after mincing. Each count took
about thirty minutes. A fresh spread was prepared, as described in a previous
paper (Belkin and Shear, 1937), for each count. All experiments were performed in triplicate at room temperature (about 220 C.). A fresh tumor was
used for each experiment.
The cell counts were made in balanced salt solutions containing various
concentrations of base. The following is the composition of a typical solution
containing, in this instance, 154 milliequivalents of base.
Constituents
Concentration of Base (mEg.)
NaCI ....
..
139
..........
5
KCl
...
3
CaC!,
MgCl, .
...
3
...........
4
Na,HPO.
Total Base ..... . . . . . . .. .... 154
64
MORRIS BELKIN AND M.
J.
SHEAR
These values were selected on the basis of the data given by Peters and
Van Slyke (1931; Vol. I, pp. 752-762) for the milliequivalent concentrations
in normal human blood plasma. Although normal plasma contains 5 mEq. of
calcium, about half of it is present in combination with protein and hence is
osmotically inert; the concentration of ionic calcium in plasma is about 3 mEq.
For simplification of technic, bicarbonate was omitted inasmuch as it had previously been found (Shear and Fogg, 1934) to be without noticeable effect on
the bulging. The phosphate present adequately buffered the systems used.
The chloride was increased, as NaCl, to compensate for the omission of
NaHCO a , thus maintaining the total base at 154 milliequivalents. This value
was selected as representative for normal or "isotonic" solutions since the
concentration of total base in normal human serum varies, according to Peters
and Van Slyke, from 150 to 160 milliequivalents. The other salt solutions
TABLE
I: Bulging and Viability of Tumor Cells in Salt Solution
Cell: mouse sarcoma 37 spindle cell. Solution: balanced salts with total base of 154 mEq.
15 minutes after mincing
75 minutes after mincing
Categories
%
1.
2.
3.
4.
5.
6.
Colorless cells with bulge
Colorless cells without bulge
Vitally stained cells with bulge
Vitally stained cells without bulge
Diffusely stained cells with bulge
Diffusely stained cells without bulge
TOTAL =
7. Total of living cells with bulge
(1 + 3)
8. Total of living cells without bulge
(2 + 4)
9. Total of all living cells (7 + 8)
%
%
Av.
% %
%
%
Av.
%
- - - - - - - - - --- - - - - -
35
22
a a
0.4
2.1
1.1
i.a a 0.4 a
0.1
17
24 24
23
22
21 21
12
15
19
- - ---- - --- -- - -- ---100 100
100
100 100
100
100
100
- -- ------ - --43
28
1.7
0.8
16
11
47
24
2.0
1.6
16
11
42
21
2.6
1.5
19
14
44
24
28
27
36
19
40
21
--
45
48
44
46
28
35
42
35
29
74
25
73
23
67
26
72
27
55
20
55
21
63
23
58
employed had the same composition except for NaCl, which was varied so as
to give solutions of the desired total base concentrations.
The protein-containing solutions were prepared, in one set of experiments,
by diluting normal horse serum with either distilled water or with NaCl solutions of various strengths; in all cases in this set of experiments the serum was
diluted 20 per cent. These solutions had a protein content of 6.4 per cent.'
In another set of experiments, solutions containing 10.1 per cent serum
albumin were employed.' The albumin was obtained by dialyzing normal
horse serum against distilled water until the dialysate was negative for chloride. Solid NaCI was dissolved in this protein solution to give the desired salt
concentrations.
All solutions were adjusted to about pH 7.4 before using.
1 The analyses were performed by Mr. John H. Weare, through the kindness of Professor E. J.
Cohn of the Harvard Medical School.
CHEMICAL STUDIES ON TUMOR TISSUE
65
EXPERIMENTAL
The data obtained from cell counts in the solution containing 154 mEq. are
shown in Table 1. The percentage distribution of the cells among the six
categories mentioned above is given in this table. In addition, the percentages
of living cells with bulges, living cells without bulges, and the total of these two
categories are also tabulated. In accordance with criteria discussed in an
earlier paper (Belkin and Shear, 1937), the colorless cells as well as those exhibiting vital staining were considered to be alive.
Table I shows the results of three separate experiments, each with a different tumor. A cell count was made after fifteen and another after seventyfive minutes in each experiment. The columns of bold-faced type give the
averages of the results obtained in the three experiments. The variation in
the results from experiment to experiment was less than had been anticipated;
this held true for experiments done with all solutions studied.
These data are presented graphically in Chart I, Figs. 2a and 2b. In these
figures the percentages of cells stained vitally are indicated by the dotted portionsof the rectangles; the percentages of colorless cells are given by the colorless portions. As indicated, the left-hand rectangle in each figure represents
the results after fifteen minutes, and the right-hand rectangle the results
after seventy-five minutes. Fig. 2a summarizes the data on living cells with
bulges, and Fig. 2b the data on all living cells, both with and without bulges.
Similar data were obtained for tumor tissue minced in analogous salt solutions having total base concentrations of 124, 185 and 204 milliequivalents
respectively. The composition of these solutions was identical with the one
described above, except for the NaCI content. These results are also shown
on Chart I. As in the case of Figs. 2a and 2b (154 milliequivalents), each set
of figures represents the average of 3 different experiments.
On comparing the results obtained after fifteen minutes, as regards the
number of living cells with bulges (see Chart I, Figs. la, 2a, 3a and 4a), it is
seen that increasing the salt concentration did not repress cell swelling. In
fact, the percentage of cells with bulges increased as the salt concentration was
increased from" hypotonic" to " hypertonic."
In the course of the next hour some of the cells died, as shown in the bottom line of Table I; the total number of living cells at 154 milliequivalents decreased in this time from 72 to 58 per cent (Table I; Chart I, Fig. 2b). As a
consequence of the death of some cells, and of the pinching-off of bulges in
other cells (a phenomenon described in a previous communication), the number of living cells with bulges after seventy-five minutes was, in each experiment, less than after fifteen minutes. This is shown by the left-hand and
right-hand rectangles in each pair of figures (Chart I, Figs. la, 2a, 3a and 4a).
The picture obtained after seventy-five minutes (right-hand rectangles in
Chart I, Figs. la, 2a, 3a and 4a) was analogous to that obtained after fifteen
minutes, i.e., the proportion of living cells with bulges did not decrease as the
salt concentration was increased; in fact, the bulging increased progressively
with increasing salt concentration.
At the end of fifteen minutes the maximum number of bulged living cells
66
MORRIS BELKIN AND M.
J.
SHEAR
'111. 2a
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CHART
I.
.......
.....
PROTEIN-FREE SALT SOLUTIONS
This chart summarizes the data on the bulging and viability of mouse sarcoma 37 spindle cells
in protein-free solutions of various salt concentrations. Chart IA gives the number of living cells
with bulges in "hypotonic" (124 mEq., Fig. la), "isotonic" (IS4 mEq., Fig. 2a), and" hypertonic" (ISS mEq., Fig. 3a; and 204 mEq., Fig. 4aj balanced salt solutions fifteen minutes and
seventy-five minutes, respectively, after immersion. Chart IB gives the number of living cells, with
and without bulges, in these same solutions at these same times. Dotted areas represent vitally
stained cells. Colorless areas represent colorless cells. Numbers are expressed in per cent of all cells
examined.
(50 per cent) was obtained at 185 milliequivalents. This was an unexpected
finding. It had been supposed that if increase in the salt concentration exerted
any effect, it would be in the direction of retardation of cell swelling. The percentage of bulged cells was greater, however, at 154 milliequivalents than at
124, and greater at 185 than at 154 milliequivalents. While the percentage of
bulged cells fell off somewhat from 185 to 204 milliequivalents, yet the extent
of bulging at the latter concentration was greater than at both 124 and 154
milliequivalents.
This increase in the number of bulged cells with increasing salt concentra-
CHEMICAL STUDIES ON TUMOR TISSUE
67
tions does not appear to be correlated with cell injury. The total number of
living cells at 185 and 204 milliequivalents (Chart I, Figs. 3b and 4b) was
considerably greater and slightly greater, respectively, than at 124 and 154
milliequivalents (Chart I, Figs. lb and 2b). The salt content which was most
favorable for cell survival under these conditions was 185 milliequivalents. At
this concentration the percentage of living cells was considerably greater than
at 124 or 154 milliequivalents , both after fifteen and seventy-five minutes.
Furthermore, conditions for survival at 185 milliequivalents were relatively so
favorable that the proportion of living cells after seventy-five minutes was
somewhat greater than after fifteen minutes at 124 and 154 milliequivalents.
And yet, at 185 milliequivalents, the most favorable concentration for survival,
there also occurred the greatest amount of cell bulging.
It is reasonable to suppose that in those solutions in which cell survival is
higher, serious cell injury is less. Since it was found that the highest percentage of bulged cells occurred at the concentration yielding the highest proportion of living cells, it does not seem likely that injury is the cause of the
bulging.
When salt solutions more concentrated than 204 mEq. were employed,
definite signs of injury were noted. Observations were made with solutions
containing the same salts in the same concentrations as described above, except
that the NaCI content was increased to give total base concentrations of 238,
272, 306 and 340 mEq., respectively. In these more concentrated salt solutions, many of the" round" cells were markedly distorted, and the spindle cells
in many cases exhibited hyalinization of the nucleus with complete disappearance of the nucleoli. These changes were more pronounced the higher the salt
content. Because of the obvious injury produced by high salt content, quantitative observations were not made with concentrations above 204 mEq. Even
at that concentration, however, measurable injury due to excessive salt content
may have occurred, as indicated by the decrease in the percentage of total living cells (Chart I, Fig. 4b) as contrasted with 185 mEq. (Chart I, Fig. 3b).
Serum Colloid Solutions: In earlier reports from this laboratory it was
stated that cell bulging was controlled by the addition of protein; i.e., the
presence of protein in the experimental solutions reduced the number of cells
showing bulges, and also reduced the size of the bulges; furthermore, addition
of protein to salt solutions containing bulging cells produced a reversal of the
swelling phenomenon. The quantitative data obtained in the present study
confirm and extend these observations. The experiments with protein-containing solutions were done with normal horse serum diluted 20 per cent, and
with serum albumin prepared from normal horse serum as previously described.
Dilution of the horse serum served a two-fold purpose: first, it reduced
the protein content from 8 to 6.4 per cent, thus permitting a considerable proportion of the cells to bulge; and secondly, it allowed of convenient variation
of the salt concentration below as well as above the normal value. Analysis 2
of the specimen of normal horse serum used in these experiments gave a value
of 164 mEq. of total base; this is comparable with the values given by Peters
2 The authors are indebted to Dr. M. A. Logan, of the Department of Biochemistry of the
Harvard Medical School, for this analysis.
68
MORRIS BELKIN AND M.
J.
SHEAR
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CHART
II.
6.4 PER
..
.
.
.. . .:
CENT SERUM PROTEIN SOLUTIONS
This chart summarizes the data on the bulging and viability of mouse sarcoma 37 spindle cells
in salt solutions of various concentrations, each solution containing 6.4 per cent protein. Chart IIA
gives the number of living cells with bulges in "hypotonic II (131 mEq., Fig. 1a), "isotonic II (164
mEq., Fig. 2a), and" hypertonic" (195 mEq., Fig. 3a) balanced salt solutions fifteen minutes and
seventy-five minutes, respectively, after immersion. Chart lIB gives the number of living cells,
with and without bulges, in these same solutions at these same times. Dotted areas represent vitally stained cells. Colorless areas represent colorless cells. Numbers are expressed in per cent of
all cells examined.
and Van Slyke (1931) for normal human serum. The horse serum was diluted with distilled water or with NaCI solution so that the resultant mixtures
all had the same protein content and had total base contents of 131, 164 and
195 mEq., respectively. This gave a series of protein solutions which were
"hypotonic," "isotonic," and "hypertonic" with respect to salt content.
This set of solutions was comparable to the analogous protein-free series of
salt solutions described in the' preceding section.
With the technic already described, tumor tissue was minced in these solu-
69
CHEMICAL STUDIES ON TUMOR TISSUE
So
....
I'l
...
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CHART III.
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II
••
II II II
lJ,Yll11 ..11• •1U1 ... wlUlolat
ftlc•••
PROTEIN-CONTAINING" ISOTONIC" SALT SOLUTIONS
This chart summarizes the data on the bulging and viability of mouse sarcoma 37 spindle cells
in "isotonic" salt solutions containing various concentrations of protein. Chart IlIA gives the
number of living cells with bulges in such solutions fifteen minutes and seventy-five minutes, respectively, after admission. Chart IIIB gives the number of living cells, witb and without bulges,
in these same solutions at these same times. Dotted areas represent vitally stained cells. Colorless areas represent colorless cells. Numbers are expressed in per cent of all cells examined.
tions and cell counts were made as before. The experiments were done at
least in triplicate. The results are shown in Chart II.
The most striking finding is the very high percentage of total living cells
obtained at 131 mEq., in the presence of 6.4 per cent protein (Chart II, Fig.
1b). This value (92 per cent) was the average of 4 different experiments,
done with 4 different tumors in which the values ranged between 90 and 93
per cent. With increasing salt concentration (Chart II, Figs. 2b and 3b) the
percentage of total living cells decreased. It is also to be noted that the presence of protein resulted as well in an increase in the percentage of living cells
that were vitally stained (cf. dotted areas in Charts I and II).
With regard to the bulging phenomenon, it is seen that the presence of
70
MORRIS BELKIN AND M.
J.
SHEAR
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CHART
IV.
PROTEIN-CONTAINING" HYPERTONIC" SALT SOLUTIONS
This chart summarizes the data on the bulging and viability of mouse sarcoma 37 spindle cells
in " hypertonic .. salt solutions containing various concentrations of protein. Chart IVA gives the
number of living cells with bulges in such solutions fifteen minutes and seventy-five minutes, respectively, after immersion. Chart IVB gives the number of living cells, with and without bulges,
in these same solutions at these same times. Dotted areas represent vitally stained cells. Colorless
areas represent colorless cells. Numbers are expressed in per cent of all cells examined.
6.4 per cent protein (Chart II, Figs. la, 2a and 3a) reduced the bulged living
cells to 33, 43, and 38 per cent from 42, 46, and 50 per cent, respectively, at
comparable salt concentrations with no protein (Chart I, Figs. la, 2a and 3a).
The effect of a higher concentration of protein was studied in similar experiments with solutions containing 10.1 per cent serum albumin. The preparation of this serum albumin solution has already been described. The results are summarized in Charts III and IV. NaCI was added to the albumin
solution so that the total base content was 154 mEq. for the II isotonic" solution (Figs. 3a and 3b of Chart III), and 185 mEq. for the II hypertonic" solution (Figs. 3a and 3b of Chart IV).
CHEMICAL STUDIES ON TUMOR TISSUE
71
The repressive effect on cell bulging produced by the presence of protein
in the medium is clearly shown by the data in Chart III (Figs. la, 2a and 3a)
and in Chart IV (Figs. la, 2a and 3a). With the total base in the region of
" isotonicity," increasing the protein content resulted in a pronounced decrease
in the percentage of living bulged cells. Analogous results were obtained with
" hypertonic" solutions.
The effect on cell swelling of increasing the colloid content of the solutions
was even more marked than the figures indicate. In protein-free solutions the
bulges are quite large (see Shear, 1935, Figs. 1 to 14). When protein is
added, not only is there a diminution in the number of cells exhibiting bulges,
but there is also a reduction in the size of the bulges. In the concentrated protein solutions the bulges were, for the most part, quite small. In Charts III
and IV the figures show that the number of bulged cells decreased with increasing protein concentration; in addition, it is to be borne in mind that the size of
the bulges also decreased with increasing protein content.
Miscellaneous: When the total base content of the 6.4 per cent serum protein solution was increased from 131 to 164 mEq., there was a decided increase
in the percentage of bulged cells (Chart II, Figs. 1a and 2a) . This increase in
total base was obtained by addition of NaCI. To see whether this increase in
cell bulging was due to ionic imbalance, analogous experiments were performed
in which the total base was brought to 164 mEq. by the addition of balanced
salt solution, instead of NaCI alone. Here, too, a decided increase in the percentage of bulged cells was obtained, showing that the effect of increased salt
content was not due to imbalance between NaCI and the other constituents.
Similar experiments were performed in which the osmotic pressure was increased by the addition of sucrose instead of salt. To the 6.4 per cent protein
solution containing 131 mEq. total base, 30 mM. sucrose were added. The increase in osmotic pressure produced by the sucrose resulted in a pronounced
increase in the percentage of bulged cells. Thus, contrary to expectations, increasing the osmotic pressure increased the cell swelling whether the osmotic
pressure increase was due to NaCI, balanced salts, or sucrose.
An interesting phenomenon was encountered when these tumor cells were
immersed in the salt-free serum albumin solution to which neutral red
(1: 5000) had been added. In this medium, the nuclei of the spindle cells
were uniformly hyaline and were, for the most part, diffusely pink. The cytoplasm also lost its granular appearance and became hyaline. WhHe many of
the spindle cells were swollen, some appeared shrunken; the " round" cells
(see Belkin and Shear, 1937) were so shrunken and distorted as to be almost
unrecognizable.
NaCl, even in small amounts, tended to counteract these abnormal changes.
Improvement was slight but definite with concentrations as low as 60 mEq.
NaCl. s As the salt concentration of the albumin solution was increased, the
8 There is a striking parallelism between injury in solutions of low salt concentration in these
experiments and in the tissue cultures of Brues and Masters (1936; p, 321). In their experiments
no migration of fibroblasts was obtained from explants of sarcoma 180 in NaCI solutions of
51 mEq. or less. At 85 mEq. of NaCI, migration was obtained but the celIs did not survive. It
was only when a concentration of 120 mEq. NaCI was employed that the sarcoma cells migrated
and survived. The concentration ranges in which severe injury, partial injury, and no injury were
observed were quite similar in these tissue culture experiments and in the experiments described in
this paper, although different conditions and different criteria for injury were employed.
72
MORRIS BELKIN AND M.
J.
SHEAR
appearance of the spindle cells improved until, when concentrations of 120
mEq. were employed, the cells exhibited their usual morphology. With analogous increases in salt concentration, the improvement in the appearance of the
" round " cells was striking.
In one experiment done with a salt-free solution containing 10 per cent
gum acacia, pronounced cellular shrinkage and distortion were noted. Here,
too, addition of Nael improved the morphology of the cells, the improvement
in appearance being greater in the case of the " round" cells than in that of
the spindle cells.
DISCUSSION
These quantitative observations on the relative effects of protein and of salt
on the swelling of tumor cells that were alive, as judged by their behavior to
neutral red, are in agreement with earlier results reported from this laboratory
as regards the counteracting effect of protein on cell swelling and as regards the
lack of counteracting effect of salts. It has been found, moreover, that increasing the salt content produced, unexpectedly, an increase in the amount
of the swelling.
Brues and Masters (1936) investigated the effects of osmotic pressure on
normal and malignant fibroblasts in tissue cultures, and the permeability of
these cells to water, the tumors studied being mouse sarcoma 180 and Walker
rat tumor 256. They noted the cell bulges, with which the present paper is
concerned, in about 10 per cent of both normal and malignant cells, and stated
that these bulges occurred in cells which were apparently undamaged, that they
occurred as often in isotonic saline as in hypotonic, and that they were in all
respects similar to those previously described in this laboratory.'
Brues and Masters concluded that " The volume of the entire fibroblast,
whether embryonic or malignant, depends largely upon the osmotic pressure
of salts in the medium, as is the case with the erythrocyte." In the experiments reported here, however, the salts had an osmotic effect opposite to that
usually reported for erythrocytes and marine ova. Brues and Masters were
of the opinion that the swelling which produces the bulges is of a different type
from the phenomenon of swelling of the whole cell homogeneously. While the
former type may be due to changes in colloid osmotic pressure of the medium,
they stated, the latter type is governed by the osmotic pressure of the salts.
The hypothesis that two different cell-swelling mechanisms exist is an
interesting one. An alternative hypothesis is that the bulges merely represent
• These bulges appeared to be formed by distension of the cell membrane by clear fluid and
were obviously inside the cell. In bulged cells there was an irregular but distinct boundary between
the dense, granular cytoplasm and the clear fluid of the bleb which could usually be seen without
difficulty under the conditions of our experiments j no membrane separating the bulge from the rest
of the cell was discernible. In some cases there appeared to be a dividing membrane but this was
found to be due either to optical effects or to interposition of the cell between the eye and the
bulge. These relationships may be seen in the photographs of bulged cells published in a previous
communication (Shear, 1935). When, upon addition of protein to the medium, the bulge was
caused to shrink and disappear, the surface of the bulge resumed its former position as the boundary
membrane of the cell. In a personal communication, Dr. Brues stated that his interpretation of the
nature of these bulges is in agreement with ours, and that his statement that the bulges were" outside" the cell referred to their being outside of the dense, granular cytoplasmic boundary, and not
outside of the cell itself.
CHEMICAL STUDIES ON TUMOR TISSUE
73
one way in which the cell disposes of the excessive influx of fluid. When the
cell disposes of the excess fluid uniformly instead of locally, uniform enlargement of the cell is noted without a localized bleb. There is insufficient evidence at present for determining whether the mechanisms which govern the
ingress and exit of fluid are the same in the two instances.
In several important respects the technic employed by Brues and Masters
differs from that used in this laboratory. In their experiments the cells were
at all times embedded in a plasma clot and were consequently " at a greater
or less distance from the experimental anisotonic medium." Hence, the data
which they obtained were for clot-invested cells, and not for cells immediately
in contact with the solutions studied. In our experiments no plasma clot was
used; the cells, therefore, were directly immersed in the experimental solutions.
The plasma clot affects water equilibria in such cells in two important respects:
first, the clot exerts a mechanically restraining effect on cell swelling and, secondly, the proteins retained in the plasma clot provide an environment which
exerts oncotic as well as osmotic pressure.
The restraining effect of the plasma clot on cell swelling was directly observed in a previous study (Shear and Fogg, 1934). Even in dilute plasma
solutions in which the clot was so thin as to be hardly discernible, this mechanical restraint was evident. In addition to this mechanical interference with cell
swelling, the oncotic pressure exerted by the dissolved proteins present in the
plasma clot should be taken into account. In spite of these two restraints on
cell swelling, Brues and Masters found that, when the clot-enclosed cells were
transferred from fluid plasma to 0.9 per cent Nael solution, the cells increased
in volume. That the salt content alone does not govern fluid exchange, and
that oncotic pressure may play a part in the control of " the phenomenon of
swelling of the whole cell homogeneously" is indicated by their finding that
" fibroblasts in general swell when removed from plasma to 0.9 per cent sodium
chloride solution."
At the present writing, it appears that there are at least three external factors concerned in governing the flow of water into and out of the cells of solid
tissues of mammals in the intact animal. Two of these factors have been studied experimentally in vitro, i.e., the effect of salts and the effect of colloids.
The third factor, namely the effect of tissue pressure on water equilibria, remains to be studied in cells in vitro.
As far as the two factors already studied are concerned, several observations have been made which were unexpected in that they differed sharply from
the conclusions usually reached by numerous investigators studying osmotic
phenomena in erythrocytes and marine ova. With such cells, the osmotic exchanges have generally been reported as being controlled by the salt content,
whereas mouse tissue cells (not encased in clot) swell even in "hypertonic"
salt solutions. Up to the present, the swelling has been counteracted only by
the presence of hydrophilic colloid in the cell environment. It had been
thought that salts might perhaps exert a synergistic effect in restraining cell
swelling. However, the data obtained in this study do not support such an
expectation; in fact, in these experiments increasing the salt content, even in
the presence of protein, promoted rather than retarded cell swelling.
The addition of salt to the protein solution may conceivably promote cell
74
MORRIS BELKIN AND M.
J.
SHEAR
swelling, in part, at least, by reducing the colloid osmotic pressure of the cell
environment. Dodds and Haines (1934) found that addition of NaCI produced a pronounced decrease in the colloid osmotic pressure of 6 per cent gum
acacia solutions. It is possible that salts affect the oncotic pressure of serum
protein solutions in a similar fashion."
The results of Mond and Hoffmann (1929) indicate another possible mechanism. They found that although cartilage cells of the summer frog shrank in
hypertonic balanced salt solutions tb,P."
They
• ,.-·r swelled again in a few minutes.
therefore concluded that frog cartilage cells in contradistinction to erythrocytes, are permeable to salts. Whether the cells studied in this investigation
swell in hypertonic salt solutions because of permeability to salts remains to be
determined.
An analogous permeability to salts has, within recent years, been ascribed
even to erythrocytes. The statements regarding the osmotic properties of the
red blood cell which were made in this paper refer to the generally held opinions on this subject. It is of interest, however, to note that Ponder and Saslow
( 1930) have reported results that are at variance with these widely accepted
conclusions. They measured the volumes of erythrocytes in plasma solutions
of various salt concentrations and found swelling even in hypertonic solutions.
They stated that " our present information is much less satisfactory than is
usually believed . . ." and ascribed previous contrary results to errors of technic. Swelling of the red cells was noted in serum solutions made hypertonic
with KCI and with glucose as well as with NaCl. This increase in volume was
taken to mean that the substances added to the outside medium penetrate into
the cell.
Such conclusions, Ponder (1933) stated, are" not far removed from physiological heresy . . . ." If these views regarding the erythrocyte should become accepted, then the apparent discrepancies between the osmotic behavior
of solid tissue cells and of red blood cells, of mammals, may be found to be
non-existent.
SUMMARY
1. Mouse sarcoma 37 was minced in protein-free and in protein-containing
salt solutions. Data were obtained on the osmotic behavior of the tumor
spindle cells with respect to swelling as shown by the distension of the cell wall
to form bulges.
2. This cell swelling was correlated with viability as judged by reaction
to neutral red. The following conclusions refer .to the living cells.
3. In protein-free salt solutions, increasing the salt content from below
normal (" hypotonic") for mammalian serum to above normal (" hypertonic ") did not restrain this swelling phenomenon. On the contrary, increasing the salt content promoted cell swelling.
4. A restraining effect, both as regards the number and size of the bulges,
was obtained when the solutions contained serum protein. The greater the
protein content, the greater was the restraining effect.
S. Even in the presence of protein, increasing the salt concentration from
5 This question is being studied experimentally in the laboratory of Dr. Cecil K. Drinker,
Harvard School of Public Health.
CHEMICAL STUDIES ON TUMOR TISSUE
75
below normal (" hypotonic") to above normal (" hypertonic") did not restrain this cell swelling. On the contrary, increasing the salt content of a
solution containing 6.4 per cent serum protein promoted cell swelling.
6. This cell swelling could not be correlated with cell injury, in so far as
the latter was shown by the number and survival rate of viable cells.
NOTE: This investigation was aided by the Works Progress Administration.
BELKIN, M., AND SHEAR, M. J.: Am. J. Cancer 29: 483-498, 1937.
BRUES, A. M., AND MASTERS, C. McT.: Am. J. Cancer 28: 314-323, 324-333, 1936.
BURCH, G. E., AND SODEMAN, W. A.: Proc. Soc. Exper. Biol. & Med. 36: 256-258, 1937.
DODDS, K C., AND HAINES, R. T. M.: Biochem. J. 28: 499-503, 1934.
MOND, R., AND HOFFMANN, F.: Arch. f. d. ges. Physlol. 221: 460-468, 1929.
PETERS, ]. P., AND VAN SLYKE, D. D.: Quantitative Clinical Chemistry, Williams and
Wilkins, Baltimore, 1931.
PONDER, K: Cold Springs Harbor Symposia on Quant. Biol, 1: 170-177, 1933.
PONDER, E., AND SASLOW, G.: J. Physiol, 70: 169-181, 1930.
SHEAR, M. ].: Am. J. Cancer 23: 771-783, 1935.
SHEAR, M. J., AND BELKIN, M.: Am. ]. Cancer 29: 499-502, 1937.
SHEAR, M. J., AND FOGG, L. c.: U. S. Public Health Reports 49: 225-240, 1934.