The Effects of Heat and Radiation on Cancers
Implanted on the Feet of Mice
GEORGE CRILE, JR.
(Department
of General Surgery,
The Cleveland Clinic Foundation,
Cleveland, Ohio)
SUMMARY
Heating of S91 melanoma in DBA/1 mice or 5-180 in SWR mice to 44°C. for from
30 to 40 minutes destroyed a high proportion of the tumors without damage to the feet
on which the tumors were implanted. Heat and radiation were synergistic or at least
additive in their effects on tumors when treatments with heat and radiation were given
within a few hours of each other regardless of which agent was administered first.
Moderate exposures to heat and radiation when given close together cured some
radioresistant
tumors that could not be cured by larger doses of radiation without
destroying the mice's feet. Small tumors were more radiosensitive than large ones, and
large ones were more heat-sensitive than small ones. After exposure to one-third of a
lethal dose of heat, tumors and normal tissues became heat-resistant
and were not
damaged by exposures that normally destroy them. Tumors were transplantable
im
mediately after exposures to heat that are lethal to them when they are left in situ.
Injection of serotonin into tumors enormously increased their susceptibility to heat.
Repeated injections of serotonin without heating resulted in inhibition of growth rate
of 5-91 melanomas.
The chemical changes of the inflammatory reaction that follows heating should be
studied in the hope of developing cancerocidal compounds whose injection might repro
duce the destructive effects of heat on cancer.
Fifty years ago Doyen (8) observed, and then
MUller (5), that some human tumors could be
selectively
destroyed
by prolonged heating at
temperatures
below 55°C. and that the effects of
irradiation were potentiated
by heating. Between
1921 and 1955 Rhodenburgand Prime (6), Wester
mark (10), and Warren (9) published studies on
the effects of heat and of heat combined with
radiation on tumors of animals and of man. Since
1955 little significant work has been published on
the reaction of tumors and normal tissues to pro
longed exposures to temperatures
between 42°C.
and 55°C.
In 1958, Selawry, Carbon, and Moore (8) pub
lished a review of the world's literature on the
response of tumors to ionizing rays at elevated
temperatures,
and in 1961 I published a report on
the biologic effects of heat (1), concluding that:
“1.Some cancers, in both man and animals, are
moresusceptible
todestruction
byheatthanare
the tissues they grow in. 2. Heat acts synergistical
ly with radiation in controlling the growth of many
Received
for publication
August 15, 1962.
cancers. 3. The mechanism by which heat kills
cells is poorly understood
and deserves further
study.―
In 1962 I reported (2) on the clinical use of heat
alone, and also in conjunction with irradiation in
the treatment of human cancers, and included the
report of an infant in whom multiple subcutaneous
metastases of a neuroblastoma
disappeared com
pletely following immersion first of the back and
several days later of the abdomen in a water bath
at 45°C. for 1—2hours. Regression did not take
place in unheated areas, and biopsy specimens 3
weeks after treatment showed no viable tumor. On
the basis of these observations in animaLs and in
man, it seems that the use of heat as an adjunct
to the treatment of cancers should be explored.
NATURAL
TUMORS
HISTORY OF VARIOUS
IMPLANTED
ON THE
FEET OF MICE
Physical agents such as heat or cold are difficult
to apply under controlled conditions to cancers im
planted on the bodies or thighs of mice; but it is
372
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CHILE—Effects of Heat and Radiation
easy to apply heat, cold, or radiation to tumors
transplanted
to the webs of the feet without
treating
the body. With either cytosieved
or
minced cells used for the transfer, T241 sarcoma
in C57BL/6 mice (4), 591 melanoma in DBA/1 or
B6D/2F1 hybrids, and S-180 in SWR mice all
form round, progressively growing tumors on the
webs of the feet. Mice from 8 to 6 weeks old were
used in these experiments.
Since most laboratories
do not implant tumors on the feet of mice I shall
describe the natural history of tumors in this
location.
T@241
sarcoma in CÔ7BL/6.—Thistumor, when
minced in an equal volume of Hanks solution and
injected through a 20-gauge needle in an amount
sufficient to make a visible swelling of the foot,
“takes―
in nearly 100 per cent of the mice, appears
in from 5 to 7 days after injection, grows rapidly
and progressively to a diameter up to about 2 cm.,
but usually ulcerates. It kills the majority of mice
in about 6 weeks. The tumor is isologous, and
spontaneous
regression has not been observed.
When the feet are amputated before the 9th day,
while the tumors are still oval swellings (not
rounded), metastasis develops in less than 10 per
cent of the mice. When amputation
is deferred
until the 14th day, at least 75 per cent of the mice
die of pulmonary metastasis. The 5 days between
the 9th and 14th days are a good period to study
the effect of treatment on metastasis.
S91
melanoma
in
DBA/1
mice
or
F1 hybrids
(B6D/@).—When mice are caged on wire, the 591
melanoma is difficult to implant on the feet when
the temperature
of the air is below 21°C. To pro
mote growth, the implantation
preparation
was
concentrated
to a ratio of not more than 1 : 1 be
tween volume of cells and volume of Hanks solu
tion. The tumor “took―
better and at first grew
more rapidly when the mice were incubated at
temperatures
near 32°C. At 21°C. it was from
2 to 6 weeks before the tumor appeared ; it then
grew progressively and usually killed in about 3
months. Sometimes the tumor attained a diameter
of 2.5 cm., but there was considerable variation
in its rate of growth in individualmice.
In DBA/1
mice spontaneous regression was extremely rare,
since the tumor is isologous, but in as many as 10
per cent of the hybrid mice the tumor showed re
gression or very slow growth.
S91 rarely metastasized to the lungs before the
tumor was 1.5 cm. in diameter, and in more than
half of the untreated animals did not metastasize.
Early amputation
protected against metastasis
and seemed not to increase metastasis, contrary to
reported results when this same type of tumor was
implanted in the thigh (7).
on Implanted
Cancers
373
Sarcama-180 in SWR mice.—Sarcoma 180 in
SWR mice took promptly in nearly 100 per cent of
the mice, appearing 3 or 4 days after transfer. It
transferred easily in dilutions of cytosieved cells
up to 1 in 100 parts of Hanks solution. Tumors in
mice given inoculations of greatly diluted prepara
tions did not appear for 3 weeks.
The tumor grew rapidly, and when it was 1.5
cm. in diameter it often ulcerated or was partially
eaten by the mouse. Since the tumor is homolo
gous, spontaneous
regression occurred in 15 per
cent of the mice. The tumor did not metastasize
and remained well localized to the feet, but when
spontaneous
regression did not occur the mice
sickened and died in from 3 to 4 weeks. Amputa
tion was curative when the mouse was not too
sick.
TECHNIC
OF HEATING
When tumors were about 7 mm. in diameter,
the tumor-bearing
feet were immersed in a ther
mostatically controlled water bath. To hold only
the tumor in the bath, the mouse was restrained
in stocks made from sheet lead (5 X 13 X 0.1
cm.), with a slot 5 cm. long and 0.3 cm. wide
through which the tumor-bearing
foot was passed
and was firmly held by adhesive tape.
TECHNIC OF IRRADIATION
A lead shield similar to that on which the mouse
was
restrained
in the
water
bath
was
used
as a
cover of a mouse-sized box in which the mouse was
kept during irradiation.
Through the slit in the
lead the foot was brought out and was irradiated
while the body was shielded beneath the lead.
With this protection the body received 7 per cent
of the dose the foot was receiving. Addition of a
second thickness of lead reduced total-body
ir
radiation to 3 per cent, and three thicknesses re
duced it to less than 1 per cent. Usually two layers
of lead were used. Six mice arranged radially with
the tumors at the center were irradiated simul
taneously. After practice it took only a minute or
two to restrain the six mice for treatment.
The
equipment operates at 200 kv. and 20 ma. The
HVL is 0.4 cm. of copper. Target-skin distance is
20 cm., and dosage rate is 350 r.p.m. calculated in
air.
RESPONSES
OF TUMORS TO
HEAT OR RADIATION
The biologic effect of heat both on tumors and
on normal tissues can be expressed by an exponen
tial type of curve in which approximately the same
biologic effect of heat is obtained by doubling the
length of exposure for each degree centrigrade that
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1963 American Association for Cancer Research.
Cancer Research
374
the temperature of thewater bath is reduced (Chart
1). Water at 44°C. was usually used, and varia
tions in the biologic effect of heat were obtained by
varying the time of exposure. To regress, most
tumors required from 20 to 40 minutes' exposure
at 44°C. When not completely destroyed by heat,
tumors growing on the feet of mice recurred
promptly, usually within 2 weeks, but never after
3841
*
J
Time in
I
£
minutes
200
180
160
140
120
@
In the case of heat the reverse was true, for im
mediately after implantation of the cells, and until
they became a clearly visible tumor, they were
resistant to heat. As soon as the tumor was well
established it was heat-sensitive and became more
so as it grew larger. When the tumor was 2 cm. in
diameter, its sudden destruction by heat often re
sulted in the death of the mouse, and even if the
mouse survived, the foot was destroyed together
with the tumor. The effect of a few days of growth
on the curability of 5-180 is shown in Table 1. As
the tumor grew it became increasingly radiore
sistant
Thenurne,aFo,of
froclions re,o@'esenIs
number of mice
Ihol lost feel.
i
6
0
100
Mojority
I
increasingly
CURABIIrn
feel
4
a Mojority
a
60
OF
20
45
44
43
42
41
40
Temperoture, C.
CHART. 1.—Exponential curve expressing relation of length
of exposureand temperature for destruction onthe majority of
5-180 tumors implanted
on the feet of SWR mice, and for de
struction of normal feet of SWR mice. Each point represents
an experiment on from five to eight mice.
morethan3weeks;
whereas,
afterirradiation,
they
sometimes at first appeared
recur long after 3 weeks.
Com@r@TIoNs
(control)
Heat (tumor barely
visible)
Heat (well established,
rounded tumors)
Radiation (very small
tumors)
Radiation (well estab
lished, rounded tu
mors)
Radiation+heat
Radiation+heat
0
15
2
5
16
10
66
0
15
6
40
5
15
15
6
2
14
6
153
1420@
0
S
515
Radiation+heat—
Cured@CWLE4
13j@
iSfi
95
100
93
to be cured, yet would
BETWEEN HEAT
SENSITIVITY
OF Tu@oi@s,
&@
SHIP OF Cua@uinrrr
TO SizE
TIME
(pm
TREAT
MENTNuMsasTotal
CENT)None
46
BY
or MICE
HEAT
0
TREATMENTDAYt
47
1
OF S-iSO IN SWR MICE BY RADIATION,
a
48
the
Showing correlation of results with the size of the tumors
at the time of treatment.
0
recurred
49
with
HEAT, AND BY RADIATION AND HEAT COMBINED
40
@
heat-sensitive,
TABLE
cured
. Mojorily cared
but lost
and
result that combined treatment was equally effec
tive for tumors of various sizes.
Despite the di.fficulties that the size of the tumor
introduces into interpretation
of results, there is a
The denominclor
represents number of
n@cein experimenl
80
@
Vol. 23, March 1963
a Heat = 440 C. for 15 mm. Radiation
= 750 r.
t Day 0 = 6 days after implantation of tumor.
@wzRADIATION
THE RE@&TIoN
OF TUMORS
AT
Free of tumor S weeks after treatment.
§
Spontaneous
regression.
# Tumors too small to be heat-sensitive.
IITumors too large to be radiosensitive.
OF TREATMENT
Curability of tumors by treatment with physical
agents is difficult to evaluate because the rate of
cure depends greatly on the size of the tumor at
the time of treatment.
In regard to radiation, a
dose of 750 r cured many mice with S-180 when
the radiation was given soon after implantation
of the tumor, or up to the time that a definite tu
mor appeared; but later, when the tumor was 1
cm. or more in diameter, from 1500 to 2000 r was
required to destroy it.
striking correlation between heat sensitivity and
radiation sensitivity of tumors from 5 to 10 mm.
in diameter, those that are heat-sensitive
being
radiosensitive,
and those that are heat-resistant
being radioresistant
in the five types of mouse
tumors that have been studied.' The same correla
tion applies to several types of spontaneous tu
mors of dogs and of humans (2).
1 Ehrlich's
tumors
sarcoma
and
8-37
in
SWR
mice
in
addition
to
the
described.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1963 American Association for Cancer Research.
CRILE—Effects
of Heat
SYNERGISM
OF HEAT AND IRRADIATION
GIvm@ WITHIN A FEW Hoims
OF ONE ANOTHER
and
RadiatiOn
WHEN
Effect of heat followed by irradi@itionon normal
feet.—When normal feet of mice were heated at
44°C. for 30 minutes no permanent damage oc
curred. Likewise, radiation in a dosage of 2000 r
on
Implanted
375
Cancers
effect of heat on radiation.
The incidence of intact
feetafterexposures
to heat(44°
C.forfrom20to
30 minutes) followed by radiation (2000 r) at van
ous intervals of time is shown in Table 2.
Accelerated reaction to radiation after heating.—
When heat was given with 1500-2500 r the time of
appearance of the radiation damage was earlier
than in mice subjected to from 2500 r to 4000 r
TABLE 2
without heat (Table 3). Approximately
the same
THEEFFECT
OFTHETr@izINTERvAL
BETWEEN
HEATAND proportion of mice in each group eventually in
curred damage, but the damage became evident
RADIATIONON THE DEsmUCTWE EFFECTSOF RADI
AflON ON FEET OF NoR@tAL SWR MICE
about 1 week earlier in those mice treated with
combined heat and radiation.
Since mice receiving 4000 r showed damage no
FEET
TREATMENT*MIcE,
TABLE
TOTAL NO.INTACT
centHeatalone
No.
Radiation alone
Heat first, and radiation:
Immediately
4hourslater
ldaylater
7dayslater
l4dayslater19
* Heat
28
25
89
28
20
80
1
2
4
10
so
so
4dayslater
1919
— 440
C.
for
20
to
SO
Per
12
40
28
77
21
70
19100
68
minutes.)@Summary
of
PROPORTION*
DIATION D@&oE
. IOUS
TIMES
S
OF MICE
RADIATION
RA.
AND
A.FrER COMBINED HEAT AND RADI
ATION
No. DAYS
r
AFTER
TREATMENTTREATMENT2500—4000
plus heat
riooo—ssoo
(44 C. for
mlxi.)8
so
10
14
0/20
4/20
17
* Proportion:
diation
8/28
15/28
17/20
17/200/28
190/20
Number
damage/total
radiated.
Radiation
given first. Summary
of two experiments
-
FEET
TREATMENTNo.
MICEINTACT
centHeatforSOmin.
No.
Per
65
100
Si
20
10
10
10
6
Immediately
20
S
15
idaylater
10
10
6
7
60
70
9
Heatfor60min.
Radiation (700 r)
Radiation (2000 r)
Radiation (700 r)+heat
60
for
60 mm:
SHOWING
TO FEET AT VAR
AFTER
RELATIONSHIP OF THE LENGTH OF TIME BETWEEN RADI
ATION AND HEAT (44°C.) TO DEsmUcTIvE
EFFECTS
ON FEET OF NoRMAL SWR MICE
three
Radiation —2000 r.
f experiments.
Final reading 40-60 days after irradiation.
TABLE
4
16/28
16/28
of
mice
number
with
ra
of mice ir
caused little visible damage to the feet. However,
when the feet were irradiated inimediately before
or immediately after being heated, the destructive
effects of radiation were greatly increased. Sepa
rating the applications of heat and of radiation by
more than a few hours reduced the potentiative
Sdayslater
7dayslater
14 days later
Radiation (2000 r)+heat
SO mm. immediately20
7
78
10
8
80
1020
1100
10
for
Note: Damage appeared several days earlier when 700 r was
followed immediately by 44@C. for 60 mm. than it did when
2000 r was followed by exposure to 44°C. for SO rain. or by no
heating at all. This again shows that the time of appearance
of radiation reaction is accelerated by strong heating.
earlier than those receiving 2500 r, the acceleration
of the reaction does not seem to be the result of an
increase in the effective dosage of radiation but
may be related to a biologic change induced in the
tissues by the heating. In this connection it is in
teresting that the damage to radiosensitive tumors
always appeared several days before the damage
to normal tissues was perceptible.
Effect of radiation followed by heat on normal
feet.—There was no difference in results whether
heat preceded or followed irradiation as long as the
interval was only a few hours. When irradiation
was applied first, the effect was greatest when heat
followed within a few hours (Table 4). Even a
small dose of radiation, that normally causes no
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1963 American Association for Cancer Research.
Cancer Research
376
damage to the feet, was potentiated to the point of
destruction
when the irradiation
was followed
closely by an exposure to heat that is not sufficient
in itself to be lethal to the feet.
The damage that followed overheating of nor
mal feet occurred promptly
and was maximal
within S or 4 days, whereas the reactions to radia
tion, even when accelerated by heating, did not
TABLE
5
EFFECT OF HEAT FoLLowED BY RADIATION
ON GROWTH OF S.180 IN SWR MICE
Relationship
treatments.
of control of tumors to length of time between
Vol. 23, March 1963
ing, even to a degree that is not lethal to a tumor,
usually resulted in the disappearance
of mitotic
figures
and in the cessation
of growth
for several
days. When irradiation
followed heating by 2
days or more, the depression in the growth rate of
a heat-treated
tumor resulted in tumors that were
smaller and hence more radiosensitive
than if
they had not been heated. If there was delay be
tween radiation and heating, the tumor continued
to grow for a few days after radiation and hence
became larger and more heat-sensitive.
These
changes in the size of tumors after the first phase
of divided treatments,
and the rapid growth rate
of many of the tumors, make it difficult to plan an
experiment in which the sizes of the tumors at the
Cu@mtTREATMENT*No.
TABLE
6
MICENone
cent
EFFECT
OF HEAT
FOLLOWED
BY RADIATION
AND OF
RADIATION FOLLOWED BY HEAT ON GROWTH
15Heat (controls)27No.
4Per
OF
Relationship
(day 0)
87Radiation
Heat
(day 7)27
277
1026
2711
341
S-180
IN
of control
SWR
MICE
of tumors
to time between
treat.
ments.
(day 0)
8Heat
Radiation
(day
7)27
LOST
—@
(day 0) radiation
TREATMENT*No.
MICECuaastFEET
(day 0) (immediately)
(day 8) (immediately)
27
18
67
(day 8) (immediately)
Heat (day 7) radiation
27
20
74
74Heat
(day 7) (immediately)27
2722
2082
Heat (day 0)+radiation:
(day 0) (immediately)
(day 1) (1 day later)
(day 3) (3 days later)
(day 5) (5 days later)
(day 5) radiation
(day 7) (2 days later)
Heat (day 8) radiation
Heat (day 0) radiation
956
1715
(day 7) (7 days later)27
18
67
3
14
52
1
4
12
12
44
44
1
0
4
0
(day 0) (immediately)
53
17
13
75
2
12
(day 1) (1 day later)
17
17
17
8
88
9
47
47
58
0
1
0
0
6
0
27
5
19
1
4
271
.54
190
20
7
(day 0)+heat:
(day 8) (3 days later)
(days)(Sdayslater)
C Heat
=
Radiation
440
C.
for
15
—750-1000
to
20
r.
min.@Summary
f
of
three
11
27
27
27
27
Radiation
52
14
27
Per
(controls)
Radiation (day 8) heat
(day 7) (4 days later)
,
No.
centNo.Per
centNone
Heat (day 8) radiation
ex•
periments.
t Freeoftumorfor21days.
appear for at least a week. Since the damage that
followed heat combined with radiation occurred
not early, but late, at the expected time for an ac
celerated radiation reaction, it would seem that
the synergism of heat and radiation is directed
solely toward increasing the effects of radiation
and that treatment with radiation before heating
does not increase the biologic effects of the heating.
Thus, the feet of 26 mice were given 3000 r in
divided doses over a period of 2 weeks, from 3 to 6
weeks before the same feet were heated at 44°C.
for 60 minutes. These feet showed no increased
vulnerability
to heat as a result of the previous
irradiation, nor was there any later stimulation of
latent radiation damage.
Effects of heat and radiation on tumors .—Heat
Radiation
alone (day 0)
Heatalone(day0)27
*
Heat
=
44°
Radiation
C.
for
15
to
20
min.@Summary
= 500—1500r.
J
of
three
ex
periments.
t FreeoftumorSweeks
afterlasttreatment.
time
of
radiation
or
heating
are
comparable.
Nevertheless,
we have studied the efficacy of ra
diation given to tumors at various times after
heating and the efficacy of heating at various times
after radiation, and believe that tumors responded
in the
creased
same
the
way as do normal
effects
of radiation,
tissues : heat in
especially
when
the heat and radiation were administered
within
a few hours of one another. It seemed to make
little difference which treatment
was given first
(Tables 5 and 6).
In two other types of transplantable
tumors, the
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1963 American Association for Cancer Research.
CRILE—E.ffects
of Heat
and
Radiation
T241 sarcoma in C57 mice and 591 melanoma in
DBA/1 mice, the heat and radiation treatments
were much more curative when given within a few
hours of one another than when they were sepa
rated by more than 24 hours. No cures of the slow
ly growing 591 melanoma (46 cases) were effected
by heating for 20 minutes at 44°C., by exposure to
from 500 to 1000 r, or when the heat and radiation
treatments
were separated by 3 or more days, re
gardless of whether heat or radiation was given
first. However, when heat was immediately
fol
lowed by radiation, four of ten mice were cured,
and when radiation was immediately followed by
heat three of ten mice were cured.
In summary, it appeared that tumors were best
controlled when treatments
with heat and with
radiation were separated by only a few hours.
Possibly, when radiation is administered first, the
two treatments can be separated more widely and
on
RESISTANCE
their combined
377
Cancers
TO HEAT
INDUCED
EXPOSURE
BY PREVIOUS
TO HEAT
When the left hind feet of normal mice were cx
posed to 44°C. for 90 minutes, almost all the mice
lost their exposed feet as a result of the intense
inflammatory
reaction that follows heating. Pre
heating the foot for 30 minutes at 44°C., the day
before the main treatment of 90 minutes at 44°C.,
protected against this reaction. In all three experi
TABLE
PROTECTIVE
EFFECT
7
OF PREVIOUS
EXPOSURE
TO HEAT ON DESTRUCTIVE EFFECT OF A
SUBSEQUENT
EXPOSURE
TO HEAT
Feet of normal SWR mice
DESTROYED
TREATMENT*No.
MICEFEET
centHeat
be effective, but an interval of more than 4 hours
diminished
Implanted
No.
Per
effectiveness.
Preheat+heat:
4 hours later
ldaylater
Sdayslater
7 days later21
CURABILITY
OF RADIORESISTANT
TUMORS
BY
TREATMENT
WITH HEAT AND RADIATION
C0MBINED, AS COMPARED
WITH LARGE DOSES
OF
22
S
21
1495
25
23
25
1820
88
15
84
78
RADIATIoN ALONE
Although it is clear from the foregoing experi
ments that heat potentiates the destructive effect
of radiation both on tumors and on normal tissues,
there would be no advantage in following radiation
with heat if heat did not sensitize tumors more
than normal tissues to radiation. In the treat
ment of S-180 it is difficult to prove that treatment
with heat and radiation was better than with
radiation alone, because the tumor is so radio
sensitive that from 1500 to 2000 r cures most of
the tumors without damage to the feet; but in the
treatment
of the isologous T241 sarcoma in C57
mice the situation is different, this tumor being so
radioresistant
that it can rarely be cured by radia
tion alone without destruction of the foot.
When 24 mice with T241 sarcoma were exposed
to single doses of from 2000 to 2500 r, there were
only two cures, except in mice whose feet were
destroyed by the radiation (of 24 mice ten were
cured, but eight of them had their feet destroyed).
When the same type of tumor was treated by a
combination of heat (44°C. for 80 minutes) and
radiation (800—1500r), seventeen of 24 were cured
S Heat
=
Preheat
440
C.
for
90
minutes.
= 440 C. for 50 minutes.
TABLE 8
EFFECT OF PREVIOUS ExPosuRE
HEAT-SENSITIVITY
OF
8-180
TO HEAT ON THE
IN
SWR
MICE
SIZE OF
TREATMENT*No.
TUMORS
MICECuximf
No.
1—4)Heat
Per centAVERAGE
(INDEX
47
Preheat 1 day
3215
beforeheat32
S Heat
=
44°
C.
for
SO
1
31.2
2.7
mlii.
Preheat = 440C. for 10 min.
t Noevidence
oftumor2—S
weeks
aftertreatment.
mentsofthisseriesthemajorheatingwasdoneat
the same time in the same bath. Only 13 per cent
of the mice preheated 24 hours before the main
treatment
lost their feet, compared with 95 per
cent of those that had only the main treatment
(Table 7).
Preheating
induced heat resistance in tumors
as well as in normal tissues. Exposure of 591
melanoma for 15 minutes at 44°C. protected for 2
days against subsequent treatment for from 30 to
40 minutes at the same temperature. The effect
was clear also in 5-180 (Table 8). The main
(noevidence
oftumor
2months
after
treatment),
and only five of these seventeen suffered destruc
tion of their feet. Thus, it appears that heat has a
specific
effect
in increasing
the
radiosensitivity
both of normal and of tumor tissues, but that the
effect was more pronounced on T241 sarcoma than
on the tissues in which it grew.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1963 American Association for Cancer Research.
Cancer Research
378
treatment was always given at the same time in
the same bath.
The time between treatments
which was re
quired to induce resistance was the same for 5-180
as for normal tissues, the greatest resistance oc
cunning when the second treatment followed 1 day
after the first (Table 9).
Vol. Q3, March 1963
tumor grew to a visible size did it become heat
sensitive, and the larger it grew the more heat-sen
sitive it became (Table 11). Heating the foot be
fore the tumor was transplanted did not decrease,
but in fact slightly increased, the incidence of
takes of a diluted suspension of tumor celLs (51
takes on the preheated feet compared with 39 on
the unheated contralateral
feet).
TUMORS ARE NOT DESTROYED
BY HEAT ITSELF
BUT BY THE INFLAMMATORY
REAcTION
THAT OccuRs AFV@ER
HEATING
When S-180 implanted on the foot of a Swiss
mouse was heated at 44°C. for from 30 to 45
minutes (an exposure that is almost always lethal
to a large tumor), and when the tumor was im
mediately minced and transplanted
to another
mouse, it grew almost as though it had not been
heated. When the tumor remained on the mouse
TABLE
TRANSPLANTABILITY
TREATMENTNo.
EFFECT OF TIME BETWEEN TREATMENTS ON
THE
RESISTANCE
TO THE
SECOND
HEAT TREATMENT
No.
TreatmentNo.
miceNo.
10
2
8
8
6
2
10
10
10
101@
Preheatdayl,heatdayS
day 2, heat day S9
—
440
C.
for
15
33
31
2943
1
183
366
015
9
108
8
0100
87
S
4hours
8hours
24hours8
of
compared
mm.
and left at
room temperature:
S Time
S Preheat
38
4hourslater
8hourslater
3Tumor
24hourslater52
amputated
(controls)
Preheat
transfer:
heat201785Tumor
4 hours, no
.
PreheatdayO
HeatdayS
Preheatday0,heatdayS
Per
(controls)504998Tumor
heated with tourniquet,
transfer:
Immediate
0Tourniquet
4hourslater41
S-isoinSWR mice
curestNone
MICE“TaxES―
centNone
Immediate
9
AYI@ER HEATING
AT 44@C. FOR 80-45 MINuTEs
heated,
TABLE
10
OF S-iSO
appearance
of
these
tumors
with those transferred
was
greatly
88
0
prolonged
immediately.
Heat = 440 C. for SO miii.
t Free of tumor S weeks after treatment.
@
Spontaneous
regression.
for 4 hours after heating, the tumor appeared 1—2
weeks later than those tumors that were trans
planted at once. Eight hours after heating the tu
mon transplants
rarely grew (Table 10). Applica
tion of a tourniquet
to the mouse's leg during
heating increased the damage to the tumor cells
and resulted in loss of transplantability.
Minced
tumor tissues or cytosieved tumor cells in vitro
also were resistant to exposures to 44°C. for 45
minutes
(eighteen
of 24 tumors
took
well after
being so heated).
When the process was reversed, and a minced,
unheated tumor was injected into the feet of a
group of mice and the feet were then heated at
various times after the injections, heating the
recently injected feet to a degree that would be
lethal to most established tumors had little or no
effect on the growth of the tumor. Not until the
TABLE
11
RELATIONSHIP OF HEAT SENSrrIvrrY
OF S-180 TO THE
SIZE OF THE TUMOR AT THE TIME OF HEATING
S-180 transferred to the feet of SWR mice and the feet
then heated at various times after the implantation.
TREATMENT5No.
MICECuasatNo.Per
centNone
(controls)
Transferred and heated:
Immediately
idaylater
S days later
9
0
0
0
10
1
10
9
6
66
18i
170
94
28
5 days later (tumor
visible)
7 days later (rounded
tumor)so
S Heat
=
440
C.
for
80-45
minutes.
t No evidence of tumor 4 weeksafter treatment. Previous
dilution experiments
indicated
that if S-ISO did not appear in
4 weeksit did not appearat all.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1963 American Association for Cancer Research.
CRu@E—Effects of Heat and Radia&n
Possmi@
Ror@ OF ENDOGENOUSCOMPOUNDS
IN THE DESTRUCTION
OF Tu@ioi@s
AFTER HEATING
Injection of serotonin enormously increased the
destructive effects of heat both on neoplastic and
on normal tissues. When 2.5 mg. of serotonin
creatinine sulfate was injected into the tumors of
mice with S91 melanoma or 8-180 the majority of
tumors
were “cured―
by exposures
to heat which
were only half of those normally required to effect
such control (Table 12).
When serotonin was injected into T241 sarcoma
before heating, four of 21 mice were free of tumor
without serious damage to the feet from 4 to 8
weeks after treatment,
TABLE
OF HEAT
879
TABLE
13
POTENTIATIONOFHEAT EFFECT ON NORMALFEET BY
INJECTING 2.5 MG. OF SEROTONIN CREATININE
SULFATE INTO EACH FooT
12
EFFECT
ON S-iSO
IN SWR
MICE.
MICE
AND891 MELANOMA
IN F, HYBRIDS(B6D/2) OFSERO
TONIN CREATININE SULFATE INTO TUMOR IMMEDIATE
LY BEFORE HEATING
Heating
Cancers
er degree, increased the efficacy of heat, whereas,
norepinephrine
in as large a dose as could be
tolerated by the mice did not potentiate the reac
tion. Two and one-half mg. of serotonin creatinine
sulfate, injected daily into tumors for from 2 to 3
weeks and accompanied by no heating, “cured―
seven of 55 B6D/2F, hybrid mice with S91 mela
nomas (living without demonstrable
tumor and
without damage to feet from 2 to 5 months after
treatment)
as compared with one “cure―
in 46
mice whose tumors were given injections daily of a
similar volume of saline solution. In the tumors
that did not disappear the rate of growth was
greatly reduced.
whereas in experiments
involving more than 100 mice with this tumor
treated by various exposures to heat alone there
were no cures without loss of feet.
POTENTIATION
on Implanted
HEAT
NO.
(44' C.).
EXPOSURE
TIME,
.
Saline solution,
(Mr@i.)TREATMENT.
.
.
Serotonin, 5.5 mg.
.
in 0.1 ml. water
injected into
footSo
0.1ml.injected
into foot.
45
0/20 lost feet
14/18 lost feet
600/2
5/20 lost feet5/17
18/19
was44°C. for 15 minutes for S-180, and44°C. for 20
minutes for 891 melanoma.
lost feet
lost feet
lost
feet
solu
tion, 0.1 xxii.,
@
injected
tumor;
TumorSaline
into
fol
5.5 mg. in
0. 1 ml. water,
injected
into
mg.
inj@t@
into tumor;
lowed by
tumor; fol
no
heatSerotonin,
lowed by heatSerotonin,
heatS-180:
No. of cures5/
no. of mice
S91:No. of cures5/
no.ofmice4/17
1/912/13
12/13
(3 lost
feet)0/8
0/11
We are currently investigating
the growth-in
hibitory effect of serotonin to ascertain whether
or not we can develop combinations of the endoge
nous compounds involved in inflammation which
will reproduce chemically the cancerocidal effects
of the postheating
inflammatory
reaction. The
so-called “spontaneous regression― of human can
cers, which has been reported to occur following
infections,
S
Cure
=
free
of
disease
2
weeks
after
treatment.
(Experi
meats evaluated at 2 weeks in 5-180 because after this sponta
neous regressions began to occur. Evaluated
at 4 weeks in
891.)
Serotonin potentiated
the effects of heat on
normal tissues as well as on tumors (Table 18).
The effectiveness of serotonin increased with the
dosage up to 10 mg., but little effect was seen from
doses less than 0.2 mg.
In order for serotonin to potentiate the effects of
heat it must be injected immediately before the
heating. When injected immediately after, it was
ineffective (nine of fourteen normal mice lost their
feet when serotonin was injected immediately
before heating as compared with none of eighteen
when injected immediately after).
Histamine, in the same dose, angiotension, and
1-methyl-6-methoxytetrahydrocarboline
to a less
may
have
been
due
to the
cancerocidal
effects of the compounds involved in inflammatory
reactions. Perhaps by using these compounds it
would be possible to produce the cancerocidal
effects of heat without heating.
ACKNOWLEDGMENTS
Gratitude is herewith expressed to Mrs. Aleksandra Mirko
vitch, Mrs. Margo Kiraly, Mrs. George Crile, Jr., and Mr.
Robert Wagar, who have helped in the technical side of this
work and have made many valuable suggestions.
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The Effects of Heat and Radiation on Cancers Implanted on the
Feet of Mice
George Grile, Jr.
Cancer Res 1963;23:372-380.
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