R‘I’UDIES ON T H E CHEMICAL TREATMENT O F TUhIORS
IT.
THEEFPECT
OF DISTURBANCES
IN FLUID
EXCHANGE:
ON
TRANSPLANTED
MOUSETUMORS
M. J. SHEAR
Office of Cancer Investigations, 27. S. Public Health Service, Harvard Mediwl School
I n undertaking studies.in the chemistry of cancer in this laboratory,
attention was first directed t o the simple, common constituents of tissue.
A considerable amount of work has been done on the r6le of sodium,
potassium, calcium, and magnesium in cancer ; a survey of this literature (Shear, 1933, a ) revealed that sodium appeared t o be of no especial
significance, that potassium might possibly have a stimulating eff ecat
on tumor growth, that the allegations for the beneficial effect of magnesium were not well founded, and that calcium appeared to have a
retarding effect on tumor growth. The first experimental study (Shear,
1933, b ) was therefore devoted t o an investigation of the effect of calcium on transplanted mouse tumors. However, previous reports as i o
the beneficial action of calcium could not be confirmed.
Water was considered next for, from the point of view of percentage
composition, the most important constituent of tissue is water. According t o a number of investigators, the water content of tumors is
greater than that of normal tissues. Neoplastic tissue is also said to
be more sensitive t o dehydration than are normal tissues. There is
also some evidence that tumor tissue has an imbibition capacity different from that of normal tissues. These various reports * suggested
that a study of fluid exchange in tumors might prove fruitful.
Accordingly, the osmotic behavior of tumor cells on immersion in
solutions was investigated (Shear and Fogg, 1934; Shear, 1935). Concurrently with these u
i z vitro experiments, attempts were made to affect
the growth of transplanted tumors in mice by procedures which were
designed t o disturb fluid exchanges in the living animal. This report
deals with the in vivo experiments.
Mice bearing transplanted tumors, chiefly sarcoma 180 or carcinoma
63, were subjected t o a variety of treatments and were given a wide
variety of substances. I n the first experiments, the mice were deprived
of all drinking water. I n some experiments diuretics were administered. I n others, the effect of massive bleeding was tested. Essentially negative results were obtained, as f a r as affecting tumor growth
was concerned, by these types of disturbances in fluid equilibria.
Ahtempts were next made t o disturb the permeability of cells and
1 The paper entitled “The Effect of Calcium on Transplanted Mouse Tumors” (Shear,
1933, b ) is the first paper in this series.
2 For bibliography see Bhear and Fogg (1934).
See also a recent paper by Okuneff and
Nasarbekowa (1934).
66
STUDIES ON THE CHEMICAL TREATMENT OF TUMORS
67
of capillary walls in the hope of so influencing tissue and cellular fluids
that the tumors might be adversely affected. Histamine was tried, as
well as a number of bacterial metabolites such as tyramine, trimethylamine, indole, skatole and choline. The tumors in a few of the histamine-treated mice were affected, apparently by the treatrncnt ; in general, however, the results were negative.
With pneumococcus antibody and pneumococcus soluble specific carbohydrate, some of the tumors were affected and hemorrhagoe was
observed in some of the tumors. The most striking results, however,
were obtained with bacterial filtrates. Severe hemorrhages were produced in the tumors, a number of which receded; some tumors began
to grow again after the temporary recession; others, however, receded
completely.
Attempts were also made to affect permeability by the administration of substances other than bacterial products. Since lipoids play
an important r81e in cellular permeability, lipoid solvents such as ethyl
alcohol, ether, chloroform, and carbon tetrachloride, were given to
tumor-bearing mice. These were without detectable effect on tumor
growth. However, other lipoid “solvents” such as bile salts, lecithin,
and sodium oleate were also employed. With sodium oleate, some of
the tumors showed hemorrhagic areas, some became soft and bloody,
and others became liquefied.
I n the in vitro experiments it had been found that fibrin, upon clotting around cells and explants, had an inhibiting effect on the swelling
and destruction of tumor cells which resulted from excessive intake of
water. Tests were therefore made of the effect of the administration
of fibrin, sodium citrate and heparin on tumor growth in vivo. Negative results were obtained.
The in vitro experiments also showed that an increase in the colloid osmotic pressure of the fluid surrounding tumor cells counteracted
the intake of excessive amounts of water. Consequently, proteins and
other hydrophilic colloids were given t o tumor-bearing mice t o see
whether they would have any effect on tumors in vivo. High protein
diets were without effect. Injection of agar, acacia and egg albumin
resulted in recessions in a few instances. Gelatin was without effect.
Acacia appeared to have affected a few of the tumors in that they
became soft or contained fluid. Serum albumin appeared to have
caused recession in a few of the mice. I n one of the experiments, in
which repeated injections of horse serum were made, hemorrhage and
breaking down of the tumors were noted in the majority of the 17
treated mice ; complete recession occurred in 3 mice, whereas none of
the 15 control tumors was affected.
Thus, although most of the treatments were without apparent effect
on tumor growth, some of the substances did appear to have a destructive effect on the tumors in some of the mice. The gross effect produced
by different substances appeared to be similar, at least superficially,
in that hemorrhages were produced in the tumors, and in that the tumors became soft and liquefied. However, except for bacterial filtrates,
68
M. J. SHEAR
the positive results were obtained in only a small percentage of the
cases, and could not be reproduced a t will.
I n subsequent experiments with 23. coEi filtrates, performed in collthoration with Dr. H. B. Andervont, 100 per cent of tumors under
certaiii conditions were severely affected by the administration of thcl
filtrate itself aiid of active fractions obtaiiied from the filtrates. Cornplete destruction of transplanted tumors was obtained in a high pwcentage of cases, ranging upwards to 100 per cent. The active fractions
were purified chiefly with the aid of inorganic precipitants and suitable
adjustment of the pH. These experiments will be described in separate
communications.
The present report deals with the earlier experiments, in which positive results were obtained only occasionally. The reasons for the irregularity of the positive results are a t present obscure. The esperimental protocols are therefore given in some detail.
EXPERIMENTAL
STUDIES
W a t e r Depriuatiouz: Eighty mice were divided into three groups.
Two groups of 30 mice each were deprived of water f o r the durat'1011
of the experiment, whilc the third group of 20 mice received water
nd lib. One week after withdrawal of the water, sarcoma 180 was
inoculated into the mice of one of the groups deprived of water. At
the same time the mice that were receiving water were also inoculated.
Of the 30 tumor-bearing mice deprived of water, only 2 were alive
twelve days after inoculation, whereas 10 of the 30 non-inoculated micc
were alive. Most of the tumor-bearing mice which had received water
ad lib. were alive at this time.
Thus, water deprivation did not spare the host at the expense of
the tumor. On the contrary, the presence of the tumor apparently
hastened the death of the host deprived of water.
Guastalla (1931), in an analogous experiment, deprived 7 rats
completely of water. Since only 4 tumor-bearing rats and 3 non-inoculated rats were employed, no conclusion may safely be drawn from this
experiment. In another experiment, 9 tumor-bearing rats were placed
on a restricted water intake while 9 other tumor-bearing rats received
water freely. Guastalla found that the tumors in rats on a restricted
water intake grew more slowly than those in the controls, and that the
experimcntal rats lived longer than the controls. The water content
of the tumors in the rats on a restricted water intake was found to be
about the same as in the animals which received water ad lib. The
water content of the other tissues, however, was distinctly less in the
test group than in the controls, showing the tenacity with which neoplastic tissue conserves its water content.
Diuretics: Attempts were next made to deplete the body's stores of
water by diuresis and thus perhaps to affect tumor cells. CaCl, and
NH,Cl were used, since they not only produce diuresis but also cause an
extrusion of potassium from tissue cells.8 The elimination of potassium
3
For a discussion of diuresis, see Peters and Van Slyke (1931).
STUDIES ON T H E CHEMICAL TREATMENT O F TUMORS
69
from the body was considered all the more desirable since potassium
has often been reported as being present in tumors in increased amounts
and has had ascribed to it the properties of stimulating tumor growth.
Urea also was employed as a diuretic.
These substances were administered to tumor-bearing mice in various amounts and under various circumstances. These particular experiments have already been discussed (Shear, 1933, b ) from the point
of view of the effect of calcium on tumor growth. It will therefore
suffice to state here t h d some 500 mice, inoculated with sarcoma 180
or carcinoma 63, were used in these experiments and that a regularly
retardin5 effect on tumor growth was not obtained.
Bleedmg: Fluid equilibria in the body were also disturbed by rcmoving a large part of the circulating blood. Since the blood constitutes about one-fourteenth of the body weight, a 21-gram mouse coiitaiiis about 1.5 C.C. blood. Therefore a loss of 0.5 C.C. of blood in such
a mouse removes about one-third of the circulating blood.
Ordinarily after bleeding, fluid and proteins are rapidly restored
to the blood stream from the various tissues. However, as Peters and
Van Slyke (1931) stated: “If the hemorrhage equals or exceeds onethird of the blood volume new factors enter which prevent the restorative reactions described. . . . The sudden loss of blood volume after
massive hemorrhage leads to an extreme fall of blood pressure, inefficient circulation and stagnation of blood in the peripheral vascular
bod. This, i n turn, results in loss of fluid and sometimes protein from
the blood stream.’’ Bleeding not only removes water but also reduces
the amount of colloids in the blood stream. This in turn results i n ;I
lowering of the water-binding properties of the blood.
With the possibility in mind that such pronounced disturbances iii
the circulating blood might have some effect on tumor growth, a prcliminary experiment was performed in which 3 mice were bled 0.5 C.C.
from the heart. The next day these mice, together with controls, were
inoculated with sarcoma 180. A t the end of a month, 2 of the 3 experimental tumors had shown poor growth.
Thereupon 10 mice were bled 0.5 to 0.8 C.C. from the heart the day
before inoculation with sarcoma 180. Two weeks later, they were
again bled in the same way. Each time that blood was removed, 0.5 C.C.
of an inorganic serum solution (Shear and Fogg, 1934) was injected
intravenously. While this replaced, at least temporarily, the fluid and
inorganic substances that had been removed, it did not, of course,
replace any of the proteins necessary for the maintenance of the oncotic
pressure (colloid osmotic pressure) of the blood. One month after
inoculation there were some large tumors among the test mice; most
of them, however, were found to be definitely smaller than the 10 control
tumors.
I n another experiment, 0.5 C.C. blood was removed from the heart
of each of 10 mice that had been inoculated with sarcoma 180 on the
preceding day. I n each case, 0.5 C.C. of the inorganic serum solution
was injected intravenously after the bleeding. Half of the mice sur-
70
M. J. SHEAR
vived three weeks; 3 of the tumors were smaller than the 10 control
tumors.
Repeated bleeding from the heart was found to be too risky a procedure, for many of the mice succumbed. Consequently, a technic was
developed for bleeding from the tail. This consisted of injecting 1 c.c.
of the inorganic serum solution intraperitoneally a few minutes before
bleeding. Then the tip of the tail was snipped off, and the entire tail
was kept immersed in a 5 per cent solution of sodium citrate held at
a temperature of about 45" C. The warm solution drew blood into
the tail and the citrate kept it from clotting. Unless this was done,
the blood clotted on the tail after slight bleeding.
Eighty mice were inoculated with sarcoma 180. The next day half
of them were bled from the tail in this fashion, The bleeding was rcpeated five and nine days after inoculation. No effect on tumor growth
was noted three and four weeks after inoculation, on comparison with
the 40 control tumors.
PermeabiZity : Since the results obtained in the preceding experiments were essentially negative as f a r as affecting tumor growth was
concerned, other attempts were made t o disturb fluid exchange in the
animal. In vitro experiments with tumor tissue (Shear, 1935) showed
that a high content of protein in the external medium opposes the increase in cell volume which occurs in solutions of low protein content.
Accordingly, a n attempt was made t o increase the protein content of
tissue fluid by the administration of histamine, since it causes capillary
injury with consequent escape of plasma from the blood vessels into the
surrounding tissue spaces.
Aftcr tletcrminiug, in preliminary experiments, the maximum
amount of histamine that the mice could tolerate, 9 mice bearing large
tumors (sarcoma 180) were given 0.5 to 1.0 c.c. of a 0.5 per cent solution
of histamine phosphate subcutaneously. The next day 1.0 c.c. of a 2
per cent solution was given, and on the following day this dose was repeated. At autopsy three days later n o pronounced effect was observed on examination of the tumors.
I n the next experiment, 6 mice received a 2 per cent solution beginning one week after inoculation with sarcoma 180. The histamine
was given three times in intravenous doses of 0.3 C.C. each and five times
in subcutaneous doses of 1C.C. each. The tumors were smaller than the
control tumors twenty-four days after inoculation.
Fifteen mice bearing eleven-day-old sarcoma 180 were then given
0.3 c.c. of the 2 per cent histamine solution intravenously. This treatment was repeated until a total of five injections was given. When
intravenous injections could no longer be made because of the condition
of the tail, 1 c.c. was given subcutaneously instead; the solution was
administered by the subcutaneous route five times. One month after
inoculation, one of the tumors had receded completely and one was receding rapidly; the rest of the tumors, however, were as large as the 14
control tumors.
STUDIES O N THE C H E M I C A L TREATMENT O F TUMORS
71
Bactwial E’dtrates: Gratia and Liiiz (1931) found that intravenous
injection of B. coli filtrate produced hemorrhage and liquefaction in a
llormally non-hemorrhagic liposwrcoma of the guinea-pig. R h w ~ r t z man and Michailovsky ( 1932) obtained similar results with mo11se sal-coma 180 by means of “agar washings” filtrates of meningococcus; the
cixtensive hemorrhage which was produced mas followed by recession of
the tumor in some cases.
The findirip of Shwartzman and Michailovsky were confirmed and
extended in this laboratory. Dr. Shwartzman kindly furnished us with
some of his meiiiiigococcus filtrates for these tests.
That the filtrate was highly toxic was shown in preliminary experiments. Intravenous injection of 0.5 c.c. into 7 mice and of 0.25 C.C. into
9 mice (these were the doses that had been employed by Shwartzmaii
and‘Michailovsky) proved fatal in all cases within twenty-four hours.
Mice bearing sarcoma 180 and spontaneous adenocarcinoma were
cyually affected. Doses of 0.1 and 0.05 C.C. killed 5 out of 10 mice bearing sarcoma 180 within twenty-four hours. Most of the mice receiving
intravenous injections of 0.025 arid 0.01 C.C. survived.
Thereupon 14 mice bearing large sarcoma 180 tumors, four weeks
old, were given three doses of 0.01 C.C. each, intravenously, over a
period of eight days. That severe hemorrhage had been produced was
obvious from inspection of the intact tumors. Two tumors receded,
one remained stationary, and one receded and grew again. The other
mice did not survive the treatment; autopsy showed hemorrhagic
tumors.
I n the next experiment 8 mice were given 0.01 C.C. of the meningococcus filtrate, by the intravenous route, four days after implantation of
sarcoma 180. The same dose was repeated two weeks later. On
oomparing the test mice with untreated controls, this milder treatment
was seen to be without effect.
A larger number of injections was given in the next experiment
One week after implanting sarcoma 180 into 18 mice, 8 were set aside as
controls. The 10 test mice were given, intravenously, 0.01 C.C. of the
rneningococcus filtrate. Four days later the same dose was repeated.
Since the mice had all survived, the dose was increased three days later
to 0.025 C.C. The injection of 0.025 C.C. was repeated seven and nine
days later.
The control tumors grew as usual. I n the treated mice, hemorrhages were noted in 7 of the 10 mice. Two of the tumors receded, one
receded and grew again, and the rest were unaffected o r died before any
offect on tumor growth became evident. The filtrate was also toxic
for non-tumor-bearing mice, but larger closes (0.2 and 0.3 c.c.) were required t o produce death in normal mice.
In all of the preceding experiments albino mice had been employed.
I n the next experiment, 16 agouti mice were inoculated with sarcoma
180, of which 7 were set aside as controls. Seven and eleven days later
the test mice received intravenous injections of the meningococcus fil-
72
M. J. SHEAR
trate of 0.01 C.C. each. Seveii and riiiie days after that, they received
0.025 C.C. each. Of the 9 test mice, 2 died; no effect on tumor growth
was obtained that could be ascribed to the treatment.
It thus appeared that small doses did not affect tumor growth and
that large doses killed too large a proportion of the animal^.^
Dr. H. H. Andervont of this laboratory performed analogous experiments ' with this meningococcus filtrate, employing some 50 mice with
spontaneous mammary carcinomas. No effect was obtained on the
growth of spontaneous tumors, although the usual positive results were
obtained with control mice bearing sarcoma 180.
Other Bacterial Products: Some experiments were performed ill
which the soluble specific carbohydrate (S.S.) and the antibody of the
Type I pneumococcus were employed. The S.S. solution contained 1
mg. carbohydrate per C.C. arid was active t o a dilution of 1 mg. irk 2
million C.C. The antibody solution contained a concentration of antibody sufficient to precipitate, almost completely, the S.S. contained in
an equal volume of the carbohydrate solution. These experiments were
made possible through the kindness of Dr. 1,. D. Felton, who supplied
these solutions and the above information.
Since the specific carbohydrate combines with the antibody with the
formation of a precipitate in solutions that are not too dilute, it was
hoped that administration of the carbohydrate to mice bearing growing
tumors might result in the incorporation of some of the S.S. by the
tumor; if then the antibody were injected, it might possibly affect the
tumor. Accordingly 43 mice were inoculated with sarcoma 180 and
clivided into two test groups of 14 mice each and a control group of 15
mice.
Two weeks later the mice in one of the test groups were given 0 . 5
C.C. of the S.S. solution subcutaneously ; this dose was repeated seven
more times in the course of the next eight days. Two days later 0.25
C.C. of the antibody solution was injected intravenously ; this proved
immediately fatal to 2 of the mice. The mice were given 0.25 C.C. intravenously on each of five successive days. No effect on the tumors was
noticeable by the time the experiment was terminated.
The mice in the other experimental group were treated similarly
except that the order of administration of the solutions was reversed.
They received cight subcutaneous injections, of 0.25 c.c. each, of the
antibody solution. Then 0.25 C.C. of the S.S. solution was given intravenously. This resulted in the death of 5 of the mice by the next day.
Two days later another 0.2 C.C. of the S.S. solution was given; this
proved fatal to one mouse. The next day a final intravenous injectioll
of 0.2 C.C. of S.S. was given. I n the surviving mice the tumors seemed
to have been affected somewhat, judging by their physical characteristics ; they continued t o grow, however.
(i
4 These results were briefly summarized in the Annual Report of the Surgeon General of
the Public Health Service of the United Rtates, Washington, I). C., 1932, pp. 28-26.
5 Ibid., 1933, p. 18.
0 F o r further inforniation regarding mch solutions, see L. D. Felton: J. Immunology 21:
367-373, 1931. Also, L. D. Felton, G. Kauffmann, and H. J. Stalll: J. Bacteriology 29:
149-161, 1935.
STUDIES ON T H E CHEMICAL TREATMENT O F TUMORS
73
Because some of the tumors seemed to have been affected, the experiment was repeated. Sarcoma 180 m7as implanted into 48 mice, of
which 16 were set aside as controls. Beginning nine days after inoculation, the antibody solution was injected, in 0.25 c.c. doses, every other
t h y into half of the test mice. The solution was given by the subcutaneous and by the intraperitoneal route, alternately.
The other test group of 16 mice received similar treatment, but
larger doses were administered. After two subcutaneous and two
intraperitoneal injections of 1 c.c. each of the antibody solution had
resulted in the death of 4 mice, the dosage was cut to 0.5 ex., of which
five injections were given at intervals of two days each.
Two weeks after tumor inoculation, each of the test groups was subdivided into two sets. One set in each group received four intravenous
injections of the S.S. solution, while the other set received four intravenous injections of the antibody solution. No effect on tumor growth
was noted; hemorrhage was observed in some of the tumors, however.
Bacterial Metabolites .- Since some bacterial filtrates are capable of
producing damage to the capillaries in the tumor, a number of compounds known to be produced in the metabolism of bacteria' were
tested for their effect on tumor growth. Of the results obtained with
compounds of this sort, those given by histamine have already been
described. Tyramine, which is produced by B. coli and by streptococci,
and which causes damage to the smaller blood vessels (Wolf, 1923),
was tried, also with negative results. I n agreement with Wolf's findings, tyramine was found to be quite toxic, killing 10 out of 20 mice
bearing a transplant of one of our own spontaneous tumors (No. 29,
225). No retardation of tumor growth was observed in the 10 mice
which survived sixteen injections of a 1 per cent solution, which was
administered alternately by the intravenous, subcutaneous, and intraperitoneal routes.
Similar treatment with a 1 per cent solution of trimethylamine
was given 20 mice bearing the same tumor. A total of 33 injections
was given by the three routes as above. Another 20 mice were given 35
injections, in the same way, of a 0.1 per cent solution of indole. On
comparison with the tumors in the 20 control mice, no effect could be
ascribed to the treatment with tyramine or trimethylamine. The
tumors of the iiidole-treated mice were somewhat smaller than the controls ; this, however, may well have been due to chance variation.
I n another experiment, with sarcoma 180, 10 mice received eight
intravenous injections of 0.5 per cent choline solution. A t the same
time, another 10 mice received nine intravenous injections of a 10 per
cent aqueous solution of "peptone. " The peptone treatment appeared
to result in tumors that were larger than the 10 control tumors, whereas
the choline seemed to produce smaller tumors.
This experiment was therefore repeated with a larger number of
7 A survey of the subject is given i n Vol. 111 of Physiology and Biochemistry of Bacteria
by R. E. Buchanan and E. I. Fulmer, Baltimore, Williams &. Wilkins, 1930.
8Unless otherwise stated, the solutions were made up in 0.9 per cent NaCl and the pH
was adjusted to 7.5 before using.
74
M. J. SHEAIt
mice, using 75 animals divided equally into three groups. One group
received eleven intravenous injections of the peptone solution, and the
other group received eleven intravenous injections of the choline solution, This time there was no difference between the tumors in the test
and control groups.
I n another experiment, employing the fourth generation of a tumor
originally produced by dibenzanthraceiie,’ 20 mice received 24 treatments with a saturated solution of skatole; 1 C.C. was given subcutaneously and 1 C.C. intraperitoneally a t each of the 24 treatments.
Rexamethylenetetramine, although not a bacterial product, was also
tried ; 20 mice received 24 treatments with a 5 per cent aqueous solution,
administered in the same way as the skatole. On comparison with the
tumors in 20 control mice, it was seen that no retardation of tumor
growth had been eff’ected with either substance.
Ligoid Solvents: According to current concepts, the cell membrane
probably consists of lipoids, proteins, and perhaps other substances
bound in more or less loose chemical combination. It was therefore
considered possible that the lipoid constituents of the cell wall might
be sufficiently affected by lipoid solvents to disturb the permeability
and might consequently have some effect on tumor growth.
According to Adolph (1931), “The general influence of anesthetics
upon permeability has been much discussed, but the investigations that
have to do directly with the rate of passage of water under their influence are limited t o the diverse experiments of Loeb (1922), Winterstein (1916), I d l i e (1918) and Heilbrunn (1925). Loeb demonstrated
that Furidulus embryos when placed in hypertonic sea water shrank
more slowly arid when placed in hypotonic sea water swelled more
rapidly if in the presence of ether; and Winterstein and Heilbrunn
found that freshly isolated frog muscles when placed in distilled water
swelled more slowly if in the presence of a narcotic. It was obviously
impossible to gain from these various observations upon diverse living
materials any notion as t o what would happen t o the rate of osmosis
into a frog if anesthetics were present.” It was, however, apparent
that anesthetics could influence fluid exchange in some types of cells.
I n the first experiment on the effect of lipoid solvents on tumors, I1
mice were given a 1 per cent ethyl alcohol solution in lieu of drinkillg
water. The concentration was increased gradually to 5 per cent. S i x
clays after the alcohol rkgime was started, these mice together with all
equal number of controls were inoculated with sarcoma 180. No retarding effect on tumor growth was obtained.
F a t solvents were next given parenterally. Since, when undiluted,
they produce precipitates in body fluids, they were first carefully dissolved in serum. Five per cent solutions of ethyl alcohol and of ether,
and saturated solutions of chloroform and of carbon tetrachloride were
prepared in horse serum. Sarcoma 180 was inoculated irlto 50 mice,
which were then divided into five groups: four test groups and a control group. The mice received, subcutaneously, three injections of 1
@
Unpublished experiments.
STUDIES O N T H E CHEMICAL TREATMENT O F TUMORS
75
each and thirteen injections of 1.5 C.C. each of the respective preparations, beginning eleven days after tumor inoculation. No proiiounced difference between the test and control tumors was obtained.
Our few experiments with alcohol thus are in agreement with the
more extensive work of Marsh (1933), who could not confirm the favorable results with ethyl alcohol reported by Thursz (1928).
Other lipoid “solvents” that were tried were sodium oleate, bile
salts, aiid lecithin. I n studying the chemical behavior of cell memlbranes in vitro, it was observed1‘)that sodium oleate had a solvent effect
011 tumor cells ; similar behavior was exhibited by bile salts.
Since all
three substaiices appear to have important effects on membraiie permeability (see, f o r example, Bayliss, 1927 ; Peters arid Van Slyke, 1931),
it was hoped that administration of these substances might have some
selective action oil the membranes of tumor cells or on the walls of
tumor capillaries.
Accordingly, a 1per cent solution of sodium oleate was iiijected into
30 mice bearing sarcoma 180. The mice were given two iiitraveiious,
five subcutaneous aiid five iritraperitoneal injections. Several of the
tumors showed hemorrhagic areas, 3 of them became soft arid bloody,
aiid 3 tumors became liquefied t o a coiisiderable extent. Only 3 of the
mice survived all the injections, whereas the tumors in an equal number
of control mice were unaffected.
The effect of oleate was then tested on a larger number of mice.
Sarcoma 180 was implanted into 36 mice, of which 12 were set aside as
controls. Half of the test mice were given a 0.25 per cent solution of
sodium oleate (adjusted to a p H slightly above 8) in lieu of drinking
water. They took this for five days, when they lnegaii to show distress ;
the oleate solution was then withdrawn. Water was offered for three
days, aiid then the oleate solution was restored until the end of the
experiment.
Concurrently, the mice received a total of fourteen injections of
a 1per cent solutioii of sodium oleate in water ; the injections were given
intravenously, subcutaneously, and intraperitoneally, in turn. The
other group of test mice received the same treatment except that no
oleate solution was given by mouth. Of the test mice, 13 died in the
course of the experiment, whereas only 1 of the control mice died. I n
the test mice that survived, 2 had tumors that were soft or bloody,
whereas this effect was not observed in the control tumors.
I n the next experiment, 39 mice inoculated with sarcoma 180 were
divided into three groups of 13 each. One group received thirteen injections of 1 per cent sodium oleate as in the preceding experiment;
the second group received three injections of the oleate solution and
then eight injections, two intravenously and six subcutaneously, of a 2
per cent solution of lecithin dissolved in 1per cent sodium oleate. The
treatment proved too drastic for a large proportion of the mice but
again liquefaction occurred in 3 of the tumors. The controls did not
show this effect.
C.C.
10 Unpublishcd
cxpcriinents.
76
M. J. SHEAR
A mixture of bile salts was next tested. A solution containing 1per
cent sodium taurocholate aiid 1per cent sodium glycocholate was given
to 25 mice inoculated with sarcoma 180. Only half the mice survived
four days after receiving two intravenous injections ; half of the remaining test mice failed to survive two subsequent subcutaneous injections. Similarly poor results were obtained with 25 mice that had
received parallel injections of a solution containing 0.5 per cent sodium
olcate in addition to the 3 per cent mixed bile salts. No effect on the
tumors of the surviving mice was observed on comparison with the 25
control mice.
A 3 per cent sodium oleate solution was next substituted f o r the
drinking water of 14 mice bearing twenty-five-day-old sarcoma 180
tumors. I n 6 cases, the tumors became soft and bloody ; autopsy showed
considerable fluid in the tumors.
I n the next experiment 20 mice were given a 1 per cent sodium oleate
solution to drink. This W H S increased to 3 per cent and then to 3 per
cent in the course of two weeks. These mice, together with 20 controls,
were then inoculated with sarcoma 180. The concentration of the
sodium oleate was increased in increments of 1 per cent until the mice
were receiving a 6 per cent solution. The mice tolerated this dose hettcr than mice in prcceding experiments had tolerated a 3 per cent solution. I I o ~ ~ e v eonly
r , one of the treated tumors became soft and full
of fluid in this experiment.
On repetition of the oral administrution of sodium oleate with 20
test mice ( a d 18 control mice), completely negative results were ohtained. The experiment was repeated still another time, again with 40
mice, and again negative results were obtained.
Fibrin E z p r r i m m t s : The iri vitro experiments (Shear and Fogg,
1934) showed that the destruction of tumor cells by imbibition of water
could be offset by a protective clot of fibrin. When sodium citrate was
present in the solution iii sufficient amount, the clot did not form and
destruction of the tumor cells proceeded as usual. If tumor tissue in
the body were protected by a thin film of fibrin clot, then the addition
of an anticoagulant might p ~e v e i i tsuch clot formation and might exert
a n inhibiting effect on tumor growth.
Sarcoma 180 was accordiiigly inoculated into 50 mice, of which 20
were set aside as controls. The test mice were given a 0.5 per cent
solution of sodium citrate tlie next day in place of tlrinking water. The
concentration was gradually iiicreased to 5 per cent. No difference
betweeii the test and coiitrol tumors was obtained.
Larger amounts of sodium citrate were then given to other mice,
starting oiie month before inoculation. Ten mice were given a 2.5 per
cent solutioii arid the concciitratioii was increased to 9 per cent before
toxic effects were visible. At the end of two weeks, 4 mice were dead,
aiid the eoncentration was tlicrefore reduced i o 7 per cent. The mice
app,carcd t o do well oii this strong solution aiid two weeks later, along
with an equal iiumber of coiitrols, were inoculated with sarcoma 180.
However, no retarding e&ct on tumor growth was produced, even by
STUDIES ON THE CHEMICAL TREATMENT O F TUMORS
77
the continued administration of the 7 per cent solution for another three
weeks.
In another experiment 10 mice, bearing six-day-old tumors of sarcoma 180, were’given a 2.5 per cent solution of sodium citrate to drink.
r i
1lle concentration was increased stepwise to 7 per cent. These mice
did not do well. They were in worm condition than mice receiving the
same amount of sodium citrate but not bearing tumors. The tumors
were slightly smaller than the control tumors, but this may well have
been due to the poor condition of the mice.
Heparin also was administered because of its anticoagulant action.
Eleven days after inoculation of 36 mice with sarcoma 180, they werc
divided into two groups, a test group and a control group. The mice
in the test group each received four intravenous injections of 0.1 C.C.
each of a 1per cent heparin solution within a period of five days. No
difference between the test and control tumors was noted when the
experiment was ended twenty-f our days after inoculation.
Des Ligiieris (1930), after prcbvious failure, successfully transplanted goat melanoma by using plasma as a protective medium for
the tumor fragment; clotting was found to occur around the implants
h i the host.
This appeared to fit in with a tentative hypothesis, suggested by our i?z vitro experiments, 011 the protection from swelling
afforded to tumor tissue by fibrin clots. The hypothesis was tested by
i ~v ,i m experiments as follows: 24 mice were inoculated with carcinoma 63 as a control group ; aiiotlier portion of the same tumor was cut
up in mouse plasma and the clot-covered fragments of the tumor were
inoculated into 24 other mice. However, the treatment did not result
in an increased number of takes or in larger tumors. This experiment
was repeated with 60 mice, also with negative results.
To determine whether fibrin, when parenterally administered, had
a protective action on tumors, 12 mice bearing sarcoma 180 were given
intravenous injections of dog fibrin and of chicken fibrin. The coiicentration of the fibrin was made 2.5 times that of plasma. Half of
the mice received two injections of the dog fibrin solution and the other
half received four injections of the chicken fibrin solution; both solutions were given in 0.5 c.c. doses. On comparison with aii equal number
of untreated tumor-bearing mice, n o effect of the fibrin treatment was
evident.
Lustig and Wachtel (1934) reported some experiments in which
blood-coagulating substances arid hirudiii mere tested for their effect
on the growth of mouse tumors ; they, too, obtained negative results.
Treatment with Proteiias and Similar Colloids
The in vitro studies ~ i t tumor
h
cells (Shear, 1935) showed that with
increasing protein coilcentration of the external medium there was a
decreasing tendency of mater t o enter the cells ; with the highest protein concentrations swelling was inhibited. I n the hope that a higher
protein coiiteiit of blood and tissue fluids might affect fluid exchange in
tumor cells i n vivo sufficiently t o be reflected in the rate of tumor
78
1
M. J. SIIEAIl
growtli, attempts werc' matlc to iiicreasc the protein concentration of
h d y fluids. In some cxpcr~imeiitsthe proteins werc' giveii iii the diet
and in others t h y were in,jected parentcrallp. (:olloids other than
proteins were also used hccausc of their high water-binding properties.
H i g h Protein Diets: Various high protein diets were given to tumorbearing mice for varying lengths of time. Those experiments iii which
cdcium salts were given in coiijuiictioii with the high protein diets liave
beeri described elsewhere (Shear, 1933, b ) . No effects on the tumors
were noted, whether calcium salts were added to the protein or riot;
in the latter experiments some 160 tumor-bearing mice were employed.
Otlwr Hyclrophilic Colloids : Sarcoma 180 was implanted into 56
mice, which were then divided into four groups. Beginning nine days
after inoculation, solutions containing 0.2 per cent agar, 6 per cent
acacia, aiid 14 per cent egg albumin, respectively, were injected intraveiiously into cach of three groups; the fourth group served as the
control. One group received six iiijections of 0.2 C.C. each of the agar
solution; another group received six injections of 0.2 C.C. each of tlie
acacia solutioii; and the third Froup received four injections of 0.3 c.c.
each of the egg albumin solution. The injections were given at intervals of three or four days.
I n the 14 control mice there were no recessions one month after
tumor inoculation. I n the agar group 1 tumor was receding and 1 was
growing poorly. In tlie acacia group 3 tumors had receded, arid in the
egg albumin group 1 had receded and 2 others were small and stationary. I n the control and agar group all the mice survived; in the
acacia group there were 3 aiid in the egg albumin group there were 7
fatalities.
I n the next experiment, 15 mice inoculated with carcinoma 63 were
divided into two groups two weeks after tumor implantation; all of the
implants were growing well. The 14 per cent egg albumin solution
was injected iiitravenously into 8 of the mice; 0.2 0.c. was injected into
cach mouse on the fifteenth day, and 0.3 C.C. was injected two days later.
No retardation of tumor growth was noted.
Sarcoma 180 was inoculated into 35 mice. One week later 5 per cent
gelatilie was injected subcutaneously in 17 of the mice. A total of
fifteen injections of 1C.C. cach was given. A retarding effect oil tumor
growth was not obtained.
Aiiother group of 60 mice was iiioculated with sarcoma 180; half of
them received iiijections of 15 per cent gclatine and the other 30 were
kept as controls. The test mice received an intravenous injection of
0.5 ex., then a subcutaneous injection of 1 ex., aiid next an iiitraperitoneal iiijectioii of 1 c.c.; this series of injections was repeated four
times. The mice were then given nine injections subcutaneously of 1
C.C. each. The treatment was beguii 011 the day of tumor inoculation
mid was continued f o r two months; it did not retard tumor growth
appreciably.
hi the iiext experiment, 20 mice were inoculated with sarcoma 180;
half of them received injections of 30 per cent acacia in 4.5 per cent
STUDIES ON T H E CHEMICAL TREATMENT O F TUMORS
79
NaP1 ( E l i I d l y Co.). The mice were given four intravenous injections, each of 0.02 (a.c, per gram of body weight, a i d seveii subcutaneous
each. The tumors seemed to have bee11 affected
injections of 0.5
I.)? the treatment, which was thereupon repeated on a larger number of
mice.
Sarcoma 180 was inoculated into 24 test mice and into an equal
number of controls. The 30 per cent acacia solution was injected in
the same doses as in the preceding experiments, the four intravenous
iiijections alternating with 5 subcutaneous ones. The treatment was
so drastic that only 15 animals survived after three weeks, whereas all
the controls were living. After one month only 6 of the acacia mice
were alive, as compared with 22 survivors in the control group. I n
the treated mice one tumor became quite soft and two others contained
considerable fluid, apparently as a result of the treatment.
Srrurri Albumin: A 30 per cent solution of serum albumin was prepared by filtering a 6 per cent solution through a Berlrefeld filter and
then concentrating the solution by evaporation in a cellophane sac.
The day after inoculation of 32 mice with carcinoma 63, half of them
were given 0.2 c.c. of this 30 per cent solution intravenously; eleven
days later 0.1 c.c. was given intravenously. No effect on tumor growth
was noted.
Thirty mice were given four intravenous injections of 0.1 c.c. each
of the 30 per cent serum albumin solution beginning the day after
inoculation with sarcoma 180. The injections were given at two and
three day intervals. Fo u r weeks after inoculation, there were no negatives, no recessions, and no poorly growing tumors among the 20 controls, whereas among the 30 treated mice there were 1 negative, 2
i*eccdingtumors, and 2 poorly growing tumors.
Serum: Each of 20 mice received 2 C.C. of horse serum intraperit oneally and 2 c.c. subcutaneously nineteen days after inoculation with
sarcoma 180. I n the course of the next four days, each received an
additional 2 C.C. intraperitoneally and 7 C.C. subcutaneously. The tumors were no smaller than those in 20 control mice one month after
tumor inoculation.
After iiioculation with sarcoma 180, 45 mice were divided into three
q u a 1 groups of which two groups received subcutaneous injections of
horse serum. The serum was given daily for twenty days in 2 C.C.
doses, beginning with the day of inoculation. One of the experimental
groups was also given a salt mixture, containing 2 parts CaC1, to 1part
NH,Cl, for reasons discussed in the first paper of this series (Shear,
1933, b ) ; 3 grams of this mixture were incorporated in each 100 grams
of the ration, beginning with the day of inoculation. The tumors of the
mice in the experimental groups were no smaller than the control tumors one month after tumor iiioculation. 111 fact, the group that had
received serum alone had more large tumors than had the control group.
To see whether the administration of serum really stimulated tumor
growth, the serum treatment was repeated, bearing in mind the report
of Aleyer (1931) that mice inoculated with a lymphosarcoma and
(>.(a.
80
M. J. SHEAR
treated with horse globulin Fave a larger percentage of takes, and
larger and more rapidly growing tumors, than did the controls (Meyer,
however, apparently used oiily a few mice). In the next experiment,
13 mice were given four daily injections of 2 C.C. each before tumor
inoculation, arid sixteen daily injections after inoculation ; but this time
the tumors in those that received serum alone were not larger than the
14 coritrol tumors.
Thus the admiiiistratioii of serum did not have a stimulating effect ;
the greater number of large tumors obtained in the serum group in
the preceding experiment was therefore to be attributed to “biological
variation” and not to the serum treatment.
The serum-salt experiment was also repeated. Sixteen mice were
placed on the CaCl,-NH.,Cl rkgime and were injected with 2 C.C. serum
the day before tumor inoculation. These mice received twelve subcutaiicous aiid two iiitraperitoneal injections, of 2 C.C. each, of the horse
serum after tumor inoculation. The tumors were the same size as
the 16 coritrol tumors wheii the experiment was terminated.
However, in still another experiment, in which 17 mice were given R
total of fourteen injections (eight subcutaneously and six intraperitoiieally) of 2 C.C. each of horse serum, begiiiriing two days prior to
irioculatioii with sarcoma 180, a quite unusual result was obtained. On
the iiiiieteeiith day after tumor inoculation, 8 of the tumors suddenly
“opened,” leaving only a hollow shell of good tumor tissue where the
day before there had been large intact tumors. None of the 15 controls
behaved in this way. The next day 2 more of the treated tumors
“opened.” Orie of the intact test tumors showed a bloody area visible
through the skin; autopsy showed that the hemorrhage had occurred
within the tumor. Another intact test tumor was strikingly bloody;
autopsy showed hemorrhage in the active portion of the tumor. Thc
open tumors began to heal over, but subsequently new tumor tissue
developed from the margins in some cases ; in 3 mice, however, no new
tissue arose aiid the mice were free from tumors at the time of death.
None of these effects was noted i n the tumors of the 15 control mice.
Serum Comefitrates: Horse serum was concentrated by evaporation in cellophane sacs to one-fourth the original volume. This preparation was injected intravenously, in three 0.5 C.C. doses, into 20 mice
eleven days after inoculation with carcinoma 63. Following this, eight
subcutaneous injections were given, also in 0.5 C.C. doses, of a horse
serum that had been concentrated by evaporation to one-sixth its
original volume.
All of the 20 control mice were alive one month after tumor inoculation, whereas 6 of the experimental mice were dead. Among the
controls there were 1 negative and n o poorly growing tumors, as compared with 3 negatives arid 2 poorly growing tumors in the test group.
The stronger concentrate (6 X ) was thereupon given subcutaneously
in six 0.5 C.C. daily doses to 10 mice, beginning the day after irioculatiort
with sarcoma 180. After twelve days they were given subcutaneously
arid iiitraperitoiieally a total of 9 C.C. of the weaker concentrate (4x )
STUDIES ON T H E CHEMICAL TREATMENT O F TUMORS
81
over a period of a week. A t first the tumors in the test group were
larger than the controls, but at the end of the month the tumors in
both groups were of approximately the same size.
I n another experiment the stronger concentrate was given subcutaneously in five 0.5 C.C. daily doses to 20 mice, beginning the day after
inoculation with sarcoma 180. After twelve days, they were given
another 8 C.C. subcutaneously and intraperitoneally in the course of a
week. Three weeks after inoculation, the experimental mice began
to die off as a result of the resumption of the treatment, whereas all
of the control mice survived until the experiment was terminated one
month after inoculation.
DISCUSSION
I n this investigation, in which some 2,000 tumor-bearing mice were
employed, the results were negative for the most part. I n some experiments the treatment was so drastic that a hiFh percentage of the
mice failed to survive the experimental manipulations ; in cases where
the general condition of the animal was poor as a result of treatment,
slowing down of the rate of tumor growth is more likely to have been
due to the poor condition of the animal than to a specific effect of the
therapeutic agent.
I n a significant number of instances, however, a definite effect on
the tumor seems to have been produced. This was notably the case
with bacterial filtrates. A somewhat similar effect seemed to have been
produced i n isolated instances by the use of some of the other substarices tested. With the exception of bacterial filtrates, the positive
results were obtained only occasionally and could not be reproduced
with regularity. Even in the case of bacterial filtrates, the results
were not satisfactorily consistent. The reasons for these irregularities
are at present obscure.
I n other experiments now under way, several modifications in technic
have been introduced in an attempt to minimize the disturbing effect
of accidental “biological variations. ” Among the modifications ’’ are :
the employment of genetically pure strain mice instead of ordinary
stock mice ; the use, in transplantation, of tumors that arose originally
in these same pure strains of mice; the use, for the transplants, of
homogeneous tumor suspensions instead of tumor fragments.
It is, of course, possible that the treatments which affect tumors in
the manner described do not exert their effect through disturbances
in fluid exchange; the fluid exudation and the edema noted in affected
tumors may be only adventitious. However, as a tentative working
hypothesis, the concept of disturbed fluid equilibria may be of some
value.
It is conceivable that disturbances in fluid exchange, if carried out
over longer periods of time than were possible in the experiments with
sarcoma 180 and carcinoma 63, might be able to influence the behavior
11
Cf. Shear, M. J.: J. Biol. Chem. 109: lxxxi, 1935.
82
M. J. SHEAll
of tumors. With such tumors, the growth ordinarily is so rapid that
the mice are usually dead five or six weeks after tumor inoculation.
Any treatment, to be effective in such a situation, must produce its
effects rapidly. Hence the measures employed in these experiments
were drastic f o r the most part.
One of the advantages of employing dilute tumor suspensions is
that it prolongs the length of time available for treatment. A tumor,
of which small fragments will give appreciable g r o w t h seven clays
after implantation, requires twenty-eight days for growths of a comparable size to appear after injection of a dilute tumor suspension.
Under such conditions, treatments so drastic as to result in the death
of a n appreciable number of the mice may be moderated; the longer
period of administration of the less rigorous measures may bring out
i ' e ~ u l t sriot detectable in shorter experiments.
The negative results obtained in this laboratory with high protein
diets are iii a y e e me n t with those of Sugiura and Benedict ( l % 5 ) , who
fed various diets to mice aiid rats, including a meat protein diet and R
milk protein diet. They found that "The malignant character of caiicer
cells can not be influenced by the ingestion of high protein or proteinfree diets." Caylor, Raldes and Mann (1931) did not obtain retardation in growth of the Flexner-Jobling rat carcinoma by feeding frcsh
liver or muscle and, in fact, reported that liver has a stimulating effect.
Woglom (1932) found that a liver diet had no demonstrable effect upon
the growth of r a t sarcomas 8, 10 and 39 and the Flexner-Jobling rat
carcinoma. Watson (1933), however, confirmed earlier reports as to
the stimulating effect of liver feeding on the development of tumors
in tarred animals. Hill (1931) fed various diets, including a low-protein diet aiid a meat-containing diet, to mice having a high incidence of
spontaneous tumors; the diets were found to be without effect on the
incidence. On the other hand, Hirokawa (1932) has reported that tlie
percentage of takes aiid the rate of growth of mouse carcinoma 36 were
distinctly diminished in mice fed with a gluten preparation, as coiltrasted with tumors in mice fed rice dough or wheat bread.
While the oral administration of proteins had no effect on tumor
growth, it was thought that p a r e n t e d administration of proteins or
other colloids with high water-binding properties might have some
cffect. During the last decade, work on the fate of parenterally administered proteins has indicated that proteins can pass from the tissue
spaces to the lymphatics and to the blood stream, and in the reverse
direction. The occasional instances, therefore, where tumors failed to
grow, or grew poorly, may possibly have been due to the effect of the
injected colloids on coming in direct contact with tumor cells. It is
also possible that these colloids may have caused damage to the capillaries of the tumors and thus interfered with their growth.
The most clefinitc results were those obtained with meningococcus
filtrate, and, in more recent experiments conclncted in conjullctioll wit21
Dr. H. B. Andervont, with H . coli filtrates. Although interest ill this
12
C f . Rhwrr, M. J. : J. Riol.
('lieiii.
109 : lxxxi, 1935.
STIJI)IICS ON T H E CHEMICAL TREATMENT O F TUMORS
83
direction has recently been revived by the work of Gratia and Linz
(1931), the study of the effect of bacterial preparations on malignant
tumors goes back a t least half a century to Fchleisen (1882). P rior
to that time it had been observed by clinicians that certain infectious
diseases occasionally exercised a modifying or even curative effect on
malignant tumors ; a number of infectious diseases were alleged to have
this property, but the reports iiidicated that erysipelas was the most
effective. After Fehleisen had isolated a streptococcus as the cause
of erysipelas, he injected cultures of living organisms in the treatment
of human caiicer aiid, according to Coley (1893), obtained cures in two
cases. Lassar (1891), in order to avoid the obvious objections to the
use of live organisms, employed sterile filtrates of erysipelas streptococcus ; the results obtained with two patients were not encouraging.
The first attempt to treat, in this way, spoiitaiieous malignant tumors
of animals appears to have been made by Spronck (1892). With the
collaboration of Thomassen he injected sterile filtrates of erysipelas
st reptococcus into 7 dogs bearing spontaneous tumors. Repeated doses
of the filtrate were injected at a distance from the tumors ; some tumors
were unaffected, but changes of a degenerative nature were obtained
in others. Ti1 the most striking case, a hard tumor became bluish-red
a i d , in the course of several days, became quite soft and liquefied.
Sproiick also tried the filtrate o n 25 patients bearing inoperable tumors
and obtained hemorrhages and inflammation i n the tumor in some
instances. I n discussing the mechanism involved, Spronck expressed
the opinion that the toxins produced capillary dilatation and serous
exudation locally in the tumors.
111 this country Coley (1891, 1893, 1906) independently had his attcntion drawn to this subject by a remarkable cure, of a recurrent sarcwma, following two attacks of erysipelas. As a result of Fehleiseii’s
work, Coley began treating patients with injections of live erysipelas
streptococci, with favorable results in some instances. F r o m that time
to the present, he has steadily persisted in the study of the effect of
this treatment. About thirty years ago he began using sterile preparations from cultures of erysipelas streptococcus and B . prodigiosus,
which he continues to use a t the present time (Coley, 1933) with favorable results ill a significant percentage of cases.
The first use of such bacterial products in the treatment of transp1antc.d tumors appears to have been made by Beebe and Tracy (1907),
who employed dogs bearing transplants of a lymphosarcoma. They
used B. coli commzmis, Staphylococcus pyogenes aureus, Streptococcus
~ , I J O ~ ~ W and
S ,
B. prodigiosus. They used cultures sterilized by heating
at 75’ and also by boiling; the active agent was not destroyed by heat.
Thciy liberated the endotoxiiis by various methods aiid fractionated
active filtrates by alcohol precipitation. The alcohol-insoluble fract ioii was toxic aiid the alcohol-soluble fraction contained a hemolytic
siibstancc. 111 order to have some metisure of the poteiicy of their
1)rcpartitioiis, Heebc aiid Tracy employed the nitrogen content as a
c ~ u d eestimate of the coiiccritration of the active toxic agent.
84
AI. J. S H E A R
Bcebe and Tracy inoculated multiple tumors in 11 dogs and tested
the efficacy of the killed cultures and of the filtrate fractions. The
material was injected into some tumors and at a distance from others.
T n several tumors softeniiig was obtained, with accumulation of fluid.
Some of the tumors regressed and some tumors receded completely.
Somewhat similar results appear to have been obtained with bacterial preparations by Uhlenhuth, Haendel and Steffenhagen (1910)
onsRashford’s rat sarcoma, and by Beck (1911) on Bashford’s mouse
carcinoma ; the positive results, howeGer, were obtained only on injection of the material directly into the tumors.
The clinical use I d of such bacterial preparations has continued since
then, so that there are now a considerable number of cases listed in the
literature reporting favorable results. The study of such products in
experimental cancer, however, seems t o have received little attentioil
until Qratia and I i n z (1931), employing filtrates of B . coli, obtained
hemorrhage and liquefaction in transplanted liposarcomas in guineapigs. Shwartzman and Michailovsky (1932) obtained similar results
oil iiijectirig meningococcus filtrate into mice bearing sarcoma 180 ; some
of the tumors receded completely. This WMS coiifirmed by Shear and by
Aiitlervont iii this laboratory.’”
Duran-Rcyiials (1933) has made furthcr contributions to this subject. H e employed filtrates of B. roli and found that “Whereas rapidly
growing transplantable malignant tumors in rats and mice a r e very
susceptible to blood-carried B. coli toxin, slow-growing malignant spoiitaneous or transplantable tumors, malignant tumors rapidly growing
in heterologous hosts, embryomas, and granulomas are practically nonsusceptible. ”
Ire stated, further, that “mice and rats are unsusceptible to the
ordinary Shwartzman phenomenon, and it would seem that special conditions existing in the tumor rerider its vessels, supplied by the host,
apt to react with the blood-carried bacterial toxins. ”
Apitz ( 1 9 3 ) studied the hemorrhagic reaction produced in Ehrlich
mouse carcinomas by means of B . coli filtrates and other substances.
Ilis histological observations in 197 mice led him to conclude that the
walls of the tumor capillaries were damaged, that this led to dilatation
arid rupture of the walls of the blood vessels and that the resultant
bleeding obliterated the capillary wall. Occasionally there was dilatation without hemorrhage, but never the reverse.
Apitz found that the striking results obtained in the tumor werc
confined characteristically to the tumor and were iiot part of a general
vascular rcaction to the administration of a toxic substance: tlie tumor
hemorrhages were not one aspect of a general hemorrhagic diathesis.
Intravenous injection .uf snake venom produced the usual multiple
1.3 ‘ E r y ~ i p e l a sand Prodigiosus Toxiiis (Coley) I’ waR iiicludrd ill thc list of‘ New :tl~tl
Non-Official Remedies (J. A. M. A. 54: 290, 1910). The Council 011 Pharmacy and Chemistry
has retained it iii its rcecntly revised lilit “ w i t h a view t o Lacilitating further studics with the
product . .” (J. A . M. A. 103: 1067-69, 1934). This report of thc Council gives 17 referv ~ i e wto the l i t e w t u i c ~on tlic cliiiicd use of such prrparntions.
1 4 Sre footnotrs, page 72.
.
STITDIES ON T H E CHEMICAL TREATMENT O F TUMORS
85
hemorrhages in the skin and internal organs of normal and tumor1)eariiig mice hut the tumors were free from hemorrhage. On the other
hand, the substaiices that produced hemorrhage in the tumors left the
riormal tissues unaflected. Furthermore, Apitz found, as did DuranBeynals, that young tumors were usually not affected. This observation was also made in our experiments.
Torrey and Kahri (1927) carried out numerous experiments with
B. s p o r o g m c s and B. histolyticus in an attempt to cause recession of the
Flexner-Jobling r a t carcinoma. These organisms were selected because of their ability to digest proteins. Filtrates, as well as the cultures themselves, were employed in an attempt to secure selective
proteolysis of tumor tissue. The proteolytic filtrates did not contain
toxic substaiices aiid hence could be given parenterally in large doses.
The proteolytic material was administered by various routes, but negative results were obtained. Favorable results were obtained only by
irijectiori directly into the tumors.
The hemorrhages occur, according to Gratia and Linz and to Apitz,
in the zone lying between the necrotic area and the youngest area of the
tumor. Apitz often noted R high-grade edema in the tumor ; furthermore, the individual tumor cells were dropsical. This appears to be a
primary edema of the tumor cells, independent of the hemorrhage, according to Apitz. The edema is widespread arid occurs in cells all
over the tumor and is not restricted to the areas where bleeding has
occurred. Apitz concluded : “Diese Tatsacheii werden erst dann verstiindlich, ~ e i i man
n
auch eine primare, nicht zirkulatorische Schadigung
cler Carcinomzelleri aiinimt . . . bei einem Teil der Falle lrommt es zu
cinem schweren odem des Tumors ; diese Dhcheinung wird durch
Zirkulationsstiirurigen nicht hinreichencl erklart ; vielmehr muss eine
primare Schadigung des Carcinoms durch die Behandlung vorliegen. ”
I n some experiments where hemorrhage and edema were both obtaiiied
with large doses, smaller doses gave only edema, recognizable microscopically.
R~RUM~
I n these experiments on fluid exchange, a variety of substances arid
procedures were employed in empirical attempts to alter the water content of tumor cells or to damage them either by excessive intake of fluid
or by excessive loss of fluid from the tumor. It is interesting to note
that Apitz, i n studying the Shwartzman phenomenon in tumor-bearing
animals, used the same agents in a number of instances; he selected
these substances because they were endothelial poisons. He found that
NaAuC1, and snake venom (neither of which substances was used in
this study) failed to produce tumor hemorrhages. Histamine was
given by Apitz in very large doses, with negative results as regards
bleeding in the tumor. Similarly, in the present experiments with histamine no hemorrhagic reaction was noted, but ‘> tumors (sarcoma 180)
receded and the slight retardation that occurred in the growth of the
treated tumors may have been due to the treatment. Apitz found pep-
86
M. J. SHEAR
tone to be without effect; the results with peptone in this stiidy n ~ ~ * e
also iicgtitive.
With agar, Apitz ohtiiiiietl hernorrhayes in the tumors ; the results
were irregular tiiitl were obtained especially when lethal doses werc
given. In the mice treated with agar in the present experiments, oiic
tumor receded completely and several tumors retrogressed. Apitz
also used antigen-antibody preparations (horse serum arid rabbit aiitihorse serum; arid anti-mouse serum of the rabbit) a d obtained bleeding and edema in the tumors. I n the experiments with piieumococcus
antibody and prieumococcus soluble specific carbohydrate reported in
this paper, hemorrhages were observed in several of the tumors, but no
clear-cut effect 011 tumor growth was obvious.
I n addition, a number of substances and procedures were used in
the therapeutic experiments, most of which were without perceptible
effect on tumor growth. It should perhaps be emphasized here that
Apitz was interested in the immediate histological effects produced in
the tumor aiid did not attempt to carry out therapeutic experiments,
whereas, in the work described in this paper, attempts were made to
affect tumor growth and hence the animals were permitted to live instead of being sacrificed for histological study.
I n tumor-bearing mice, complete withdrawal of drinking water
resulted in earlier death than in non-tumor-bearing control mice. Massive bleeding did not have a pronounced effect 011 tumor growth, although it seemed in some experiments to result in smaller tumors;
this may well have been due to poor general condition of the mice and
not to a specific effect on the tumors. The various diuretics employed
did riot seem to influence the growth of the tumors.
Tyramine, trimethylamine, choline, arid skatole were without any
apparent effect on tumor growth; iiidole appeared t o have had some
slight restraining effect, but this may have been due to adventitious
factors. Alcohol, ether, chloroform aiid carbon tetrachloride were
without effect. Fibrin, sodium citrate, and heparin did not affect
tumor growth perceptibly ; neither did high-protein diets. Gelatin was
found to be without effect. With acacia, however, recession of the
tumor occurred in a few instances ; in a few other instances, liquefaction
occurred apparently as a result of the treatment. With egg albumin
and with serum albumin, a retarding influence was noted in tt few instances. I n one experiment with horse serum, hemorrhage or striking
destruction of the interior of the tumor was obtained in 12 out of 17
mice ; i n some cases the tumor grew again but in 3 mice there was cornplete recessioii.
With sublethal doses of meningococcus filtrate, hemorrhages were
obtained in the tumors. Many of the mice died but, in the survivors,
some tumors receded arid grew again while others receded completely.
Sodium oleate also produced hemorrhage or liquefaction in a11 tillparently sigriificarit number of cases. This is of interest in the light of
the reports of Nakahara (1921,1925) on the inhibiting effect of sodium
oleate and oleic acid 011 traiisplanted and spontaneous mouse tumors,
STUDIES O N T H E CHEMICAL TIlEATMENT O F TUMORS
87
Rpitz, in his histological iiivestigatioii of the effect on tumors of K.
c d i filtrate a i i t l of ti v a ~ i e t yof other preparations, fomitl that i i o effect
o i l thc tumor is usually ohtaiiietl iuiless a lethal or suhlcthal tlosc is
giveii. h7en then the effect is not always obtained consistently. This,
we have fouIid, held true in our earlier therapeutic experiments. Oiily
some of the mice responded, and repetitions of experiments frequently
gave negative results. Work is now in progress in which efforts are
bciiig made to ohtain positive results with greater regularity.
SUMMARY
1. Attempts were made to affect the growth of transplanted tumors
in mice by procedures which were designed to disturb fluid exchange in
the living animal.
2. Water deprivation, diuretics, massive bleeding, fibrin, anticoagulants, high protein diets, parentcral administration of proteins
aiid other hydrophilie colloids, meningococcus filtrate, bacterial metabolites, aiid lipoid solvents were some of the substances arid measures
tested in these experiments, in which some 2,000 tumor-bearing mice
were employed.
3. Most of these measures were without effect. Ilowerer, in some
iiistaiices the tumors became soft and liquefied; in others they became
hemoi*rliagic; in a few instances the tumors receded completely.
Tt is a pleasnrr to record the valnahle technical assistance rendered by Mr. Adrien
I'errnult.
REFERENCES
ADOLPH,E. 11.: Am. J. Physiol. 96: 569-86, 1931.
RPITB,K.: Ztschr. f. Krebsforsch. 40: 50-70, 1933.
W. M. : Principles of General Physiology, 4th ed. 1924, London.
BAYLISS,
HECK,31.: Ztschr. f. Kiebhforsch. 10 : 149-54, 1911.
BEEBE,S. P., AND TRACT, M.: J. A. M. A. 49 : 149:3-8, 1907.
H. D., BALDES,E. J., AND MANN,3'. C.: Arch. Path. 11: 854-6, 1931.
CAYLOR,
COLEY,W. B.: Ann. Surg. 14: 199-220, 1891.
COLEY,W. B.: Ann. Surg. 97: 434-60, 1933.
COLEY,W. €3.: Am. J . M. Sc. 105: 48'7411,1893.
('OLEY, W.13.: Am. J. M. Sc. 131: 375-430, 1906.
I)ES LIGNEHIS,
&I. J. A. : Brit. J. Expel.. Path. 11: 249-51, 1930.
F.: Pro(.. Soc. Expei-. Diol. & Metl. 31 : 341-4, 1933.
I)IJRAN-REYNALS,
~ ' ~ ~ L E I S F: ;Ueutsche
~Y
metl. Wclinschr. 8 : 553-4, 1882.
GRATIA,A., A N D LINZ,H. : Compt. rend. Soc. de biol. 108: 427-8, 1931.
( ~ J A S T A L L A , R.: Caiicro 2 : 98, 1931. (Ahst. in Am. J . Cancer 16: 286, 1932.)
HILL,LEONARD:
Lancet 1: 966-9, 1931.
HIROKAWA,
M. : Trans. J a p . Path, SOC.22 : 989-91, 1932. (Abst. in Am. J. Cancer 19 :
405, 1933.)
0. : Deutsche med. Wchnschr. 17 : 898-9, 1891.
LASRAR,
LIJSTIG,B., A N D WACHTEL,H. : Biochem. Ztschr. 271 : 345-66, 357-69, 1934.
MARSH,M. C.: Am. J. Cancer 19: 847-52, 1!333.
~ I E Y E R ,J. R.: Arch. 1114.Biol. Defesa Agric. e Animal 4 : 285-90, 1931. (hbst. in Biol.
A h t . 6 : 2'290, 1932.)
NAKAHAKA,
W.: J . Exper. Med. 40: 363-73, 1924; 41: 347-66, 1925.
N., A N D NASARIX~KOWA,
T. : Ztwhr. f . Krebsforsch. 41 : 28-37, 1934.
OKUNEBF,
88
M. J. SHEAIL
l’wrms, J. P.,AN]) V A N SLYKF;,
1). 11.: Quantitative Clinical Chemistry, Vol. 1, 1931,
Baltimore.
HH EAR, M. J . : Am. J. Ctincw 18: 924-1024, 1933. a.
SHEAR,
M. J . : ti. S. Pub. Health Report:, 48: 1103-14, 1933. I).
SHEAR,
M. J . : Am. J . Curicer 23: 771-83, 1935.
SHEAR,
M. J., A N D Fow, 11. C. : U. 8 . 1’111). Health lleports 49 : 225-40, 1934.
S n W A R T z & L t N , G., A N D MICHAILOVSKY,
N.: I’roc. h e . Exper. Biol. & Med. 29: 73-42,
1932.
SPRONCK,
C. 13. H. : Ann. Inst. Pasteur 6 : 683-707, 1892.
SIJGIURA,
K., mi) BENEIWT,S. It. : J. Cancer Iles. 9 : 204-15, 1925.
THVRSZ,I). : Ztschr. f. Krehsforsch. 26 : 260-2, 1928.
TORREY,
J. C., A N D KAHN,M. C.: J . Cancer Res. 11: 334-76, 1927.
U H L E N I I U T H , IIAENDEI,, A N D STEB’PENHAGEN : Ztschr. f . ImInUIlit
Therap. 6 : 654-64, 1910.
WATSON,A . F.: Am. J. Cancer 19: 389-95, 1933.
WOGLOM,
W. H.: Am. J. Cancer 1 6 : 564-7, 1932.
WOLF, P:. P. : J. Exper. Metl. 37 : 511-24, 1923.