[CANCER RESEARCH 33, 1957-1965, August 1973]
Humoral and Cellular Immune Factors in the Systemic Control of
Artifically Induced Metastases in C3Hf Mice'
Jan Vaage
Department of Radiation Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114
SUMMARY
Active and passive immune resistance to implanted syn
geneic sarcoma cells was expressed more effectively in vis
ceral organs reached by intravascular implantation than in
i.m. and s.c. implantation sites. Passive transfer of serum
from tumor-cured as well as tumor-bearing
donors gave
resistance to unsensitized sublethally radiated recipients
partially restored with transferred normal lymph node cells.
unsensitized mice at the time of challenge injections of living
tumor cells via intravascular and s.c. routes.
MATERIALS
AND METhODS
Mice. All the animals used in these experiments were
12-week-old female mice ofinbred strain C3Hf/Bu from the
defined-flora,2 pathogen-free breeding colonies maintained
by the Section of Experimental Radiotherapy of the Uni
versity of Texas, M. D. Anderson Hospital and Tumor
INTRODUCTION
Institute at Houston, and by the Department of Radiation
Medicine, Massachusetts General Hospital.
Tumors. The fibrosarcoma had been induced in a female
A previous publication (32) presented evidence that in the
C3H/He mouse by methylcholanthrene
(27). It had been
C3Hf mouse there are differences in the level of antitumor
kept in liquid nitrogen and was reintroduced into syngeneic
immune resistance between different anatomical locations.
mice to be used in these experiments in the 3rd and 4th
In sensitized mice, the organs reached by tumor cells in
transplant generations for sensitizing implantations and for
jected via the portal vein and the tail vein were almost to
preparation of tumor cell suspensions.
tally resistant to numbers of tumor cells that caused growths
The mammary carcinoma had developed spontaneously
in a high percentage of unsensitized control animals. The
liver appeared to be more resistant to “inducedmetastases― in a multiparous C3H/He mouse (25). It had also been kept
in liquid nitrogen and was reintroduced into syngeneic mice
than the lungs, although the liver seemed to filter out more
of the injected tumor cells. The s.c. injection sites were less to be used in the 3rd and 4th transplant generations.
Tumor Implantation. Implantation s.c. of I-cu mm pieces
resistant to tumor growth than the liver and the lungs.
Immunological factors in the control of dissemination of Of living tumor tissue was used to initiate tumor growth for
the purpose of immunization. An incision was made in the
tumor growth have been demonstrated earlier by Kim (15),
skin of the right flank, and a tumor piece was placed under
who showed that among methylcholanthrene-induced mam
the skin with a trocar. The incision was closed with a wound
mary carcinomas in rats, the ability to metastasize was in
clip.
versely proportional to the immunogenicity of the tumors.
Removal of tumors implanted s.c. was done under pento
Deodhar and Crile @9)demonstrated
that the nonmetas
barbital anesthesia. A circular incision was made in the skin
tasizing Sarcoma 180 was made able to establish metastases
around the edge of the tumor, and the tumor was removed
in hosts that had been immunologically suppressed with
by blunt dissection. Large blood vessels were cauterized.
antilymphocyte
serum. Vaage and Weiss (33) found that
The incision was closed with wound clips.
mice responded to tumor-specific immunotherapy with in
Portal vein injections were made through a ventral mid
creased resistance to the growth of cells of autochthonous
line incision with the use of a fine glass needle drawn from
mammary carcinomas that had been injected i.v. or i.p. to
750-@tm glass tubing attached to a 1-mi syringe by a length
simulate metastatic extension oftumor growth.
of polyethylene tubing.
The present investigation has compared active antitumor
Intracardiac injections were made with the use of a 30immune resistance in different anatomical location and has
gauge needle attached to a l-ml syringe by a length of poly
tested the effectiveness of humoral and cellular immune
ethylene tubing. To reach the right and left ventricles, the
factors in suppression of neoplastic growth. The tests used
skin was deflected from a midline incision to expose the ribs.
passive transfer of humoral and cellular immune factors to
The right ventricle was reached by inserting the needle to a
1 This
work
was
supported
in
part
by
USPHS
Grants
CA05047,
CAII138,andCA13O18andby a CancerResearch
Scholar
Award from depth of 5 to 6 mm in the 2nd right intercostal space close
the American Cancer Society, Massachusetts
Division. This work was
begun at the University of Texas, M. D. Anderson Hospital and Tumor
Institute, and completed at the Massachusetts General Hospital.
Received December 4, 1972; accepted April 27, 1973.
to
the
2 The
sternum.
mice
canny
The
left
only
the
ventricle
following
was
entenic
reached
bacteria:
by
inserting
Clostridium
sp.,
Peptostreptococcus sp., Bacillus sp., and Bacteroides sp.
AUGUST 1973
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1957
Jan Vaage
the needle to a depth of 4 mm in the 2nd left intercostal
space about 3 mm from the sternum. The color of the blood
entering the polyethylene tubing indicated the proper posi
tion of the needle. Intracardiac and portal vein injections
were done under pentobarbital anesthesia.
Challenge implantation oftreated and untreated mice was
by injection of viable (trypan blue-negative) tumor cells sus
pended in TC Medium 199 (Difco Laboratories, Inc., De
troit, Mich.) and consisting predominantly
of single cells
and a few clumps of up to 5 cells each.
The proportion of trypan blue-negative cells in the tumor
cell suspension was usually about 25%. The mechanical
preparation of single cell suspensions from tumor tissue has
been described elsewhere (29). The tumor cell suspensions
and syringes were kept on crushed ice during the injection
procedures, and the cell suspension was shaken frequently
to keep the tumor cells from clumping.
Immune Lymphocytes. The inguinal, brachial, and axillary
lymph nodes were removed from each unsensitized mouse
and from mice from which bilateral s.c. sensitizing tumor
implants had been surgically removed 2 days previously.
The sensitizing tumors were on the average 10 x 10 mm at
the time of removal after a growth period of about 20 days.
The lymph nodes were minced with scissors in cold Me
dium 199, and the cells were dislodged from the tissue by
gently drawing the pieces into a I-mi syringe 15 to 20 times.
After letting the larger pieces settle out, the supernatant sus
pensionthat could passthrough a 20-gaugeneedlewaswith
drawn and washed once in cold Medium 199. The cell sus
pensions were kept in crushed ice during counting and dilu
tion procedures and then brought to 37°and injected i_p.
Immune Serum. Blood was collected by cardiac puncture
and the serum was separated after 2 hr at room tempera
ture. The serum was kept in the refrigerator in crushed ice
and was used within 48 hr. The serum was brought to 37°
before it was injected i.p.
Immune serum was collected from mice that were carry
ing 10- to 15-mm bilateral s.c. sensitizing tumor implants.
Hyperimmune serum was collected from mice from which
bilateral s.c. tumor implants had been surgically removed
and then had received I i.p. injection of 50 mg of radiation
killed tumor cells in 0.2 ml of Freund's complete adjuvant
plus 3 s.c. injections of 5 mg of killed tumor cells in 0. 1 ml
of 0.9% NaCI solution given at weekly intervals. The mice
were bled 1 week after the last injection.
Radiation Procedures. Animals received whole-body ra
diation on a 250-kVp, 30-ma Maxtron X-ray machine with
a 0.5-mm copper and 1-mm aluminum filter. The dose rate
was 90 rads/min at 50 cm from the source, calibrated with a
Victoreen chamber. Five mice were radiated at 1 time in a
rotating compartmentalized
plastic box. The total dose to
the midplane of the mice was 400 rads.
Tumor cell suspensions used for sensitizing injections were
killed by exposure to 10,000 rads on a parallel opposing
dual-source ‘37Csmachine with a dose rate of 1,052 rads/
mm.
Statistical Analysis. The effect of treatment is described
in terms of differences in tumor incidence following chal
1958
lenge injections and in terms of differences in the amount of
tumor growth following challenge. For comparison of tumor
growth, Student's t test was used.
Differences between groups were considered significant
only when thep value ofcomparison was 0.05 or smaller.
RESULTS
Active Systemic Tumor Immunity. Table 1 presents the
results of a preliminary study to determine the comparative
frequencies of tumor growth in presensitized; in normal, un
sensitized; and in sublethally radiated, unsensitizedmice.
The mice were challenged with 3.3 x l0@or I x 10' viable
tumor cells suspended in TC Medium 199, injected via I of
the following routes (a) the hepatic portal vein; (b) the left
ventricle; (c) the right ventricle; (d) i.p.; and (e) s.c., with
equal inocula both at the left shoulder and at the left hip.
Table 1 represents data from 2 identical experiments,
which followed the same procedures to test the reproducibil
ity of the results. Since the results of the 2 experiments were
similar, the data have been combined. In each of the 2 ex
periments the presensitized mice were given s.c. implants of
I-cu mm pieces of living tumor tissue in the right flank to
initiate tumor growth for the purpose of immunization.
After a growth period of 20 days, when the immunizing im
plants measured about 10 x 10 mm in diameter, the im
plants were removed by surgery 4 days before the challenge
injections. The immunologically depressed groups had no
prior exposure to the tumor and received 400 R whole-body
radiation about 24 hr before challenge.
The 2 experiments were terminated 17 and 19 days after
challenge, respectively, for the animals challenged intra
vascularly and i.p., and after 24 and 26 days, respectively,
for the animals challenged s.c. The incidence of tumors at
the s.c. injection sites was checked at weekly intervals, and
their sizes were measured with calipers and recorded from
the time they became palpable. Each experiment was termi
nated when some of the mice in any group became cachectic
because of progressive s.c. tumor growth. The values per
group at the last recording of tumor sizes are presented in
the data. Frequent palpations to detect the development of
i_p. growth decided the termination time for the animals
challenged i.p. and this also determined the termination
time for the animals challenged intravascularly. The ani
mals were killed by asphyxiation in 100% CO2 gas, and all
parts of the mice except the head were carefully searched
for the extent and location of “metastatic―
tumor growth.
The sarcomatous
nature of “metastatic―visceral tumor
growth was determined by histological examination of ran
domly chosen samples.
To arrive at a common denominator
describing the
amount of tumor tissue growing s.c. where measurements
were easily made and in the viscera where accurate meas
urements were not practical, values from 0 to 3 were given
according to the number and size of growths found at au
topsy. The values were determined at blind readings and
were derived according to the following grading system:
CANCER RESEARCH
VOL. 33
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Immunological
Table I
on the growth of intravascularly,
The effect of activeimmunization
cellsChallenge°No.
i.p., and s.c. transplanted
fibrosarcoma
ofNo.
ofAv.miceAv.cellsmice
ofNo.
withtumor
routeinjectedtumonsbgrowtW
cellswithtumorGroupImmune
injectedtumorsbgrowthcISensitizedPortalvein3.3
statusInjection
10'9/200.592SensitizedLeft
10°11/171.103SensitizedRightventnicle3.3
ventricle3.3
10°10/141.144Sensitizedi.p.3.3
10°14/201.405Sensitizeds.c.3.3
l0@1/200.10
l0@5/160.38
l0@3/150.26
l0@5/200.35
l0@0/400
167Normal,
unsensitizedPortal
vein3.3
10°18/182.508Normal,
unsensitizedLeft
ventricle3.3
10°19/192.709Normal,
unsensitizedRight
ventricle3.3
10°20/203.0010Normal,
unsensitizedi.p.3.3
10°32/401.84IIRadiated,unsensitizedPortalvein3.3
unsensitizeds.c.3.3
x
x
x
x
x
l0@1
1/201
10°12/191.45
l0@11/171.62
l0@16/201.68
l0@8/400.44
10°20/202.6012Radiated,
10°14/142.6013Radiated,
unsensitizedLeft
10°18/182.8014Radiated,
unsensitizedRight
10°20/203.0015Radiated,
unsensitizedi.p.3.3
unsensitizeds.c.3.3
x
x
x
x
x
l0@12/201.32
l0@16/201.84
lO@15/192.
l0@15/202.03
l0@20/401
a The
mice
were
challenged
with
3.3
x
10° or
ventricle3.3
vencticle3.3
1 x
10° live
tumor
cells
in
single
No. of
1
1
1
1
x
x
x
x
x
10°18/400.776Normal,
Control of Induced Metastases
x
x
x
x
1x
. 10
1
1
1
1
1
1x
1x
1x
1x
1 x 10°36/402.25
13
.05
intravascular
or
i.p.
x 10°18/202.
x
x
x
x
inocula
or
were
challenged
with
s.c.
tumor
cell
inocula both at the left shoulder and at the left hip. The figures represent the combined data from 2 separate but identical experiments.
bOnly the differences in average tumor growth have been evaluated statistically. Each of the sensitized groups (I to 5) differ significantly from
their normal (6 to 10) and radiated (I I to 15) counterparts.
C Values
from
0
to
3
expressing
the
total
amount
of
tVmor
tissue
found
in
the
various
anatomical
locations
each group was derived by dividing the sum of the values of the number of mice, on, for the mice challenged
plantations in each group.
0 = No tumor growth found by gross examination
or by
histological examination of visceral organs from ran
domly chosen samples.
I = One to 5 tumors
from
1 to 3 mm in diameter
and/or
1 tumor of not more than 5 mm in diameter found in
visceral organs or in the peritoneal cavity; s.c. tumors
3 to 7 mm in diameter.
2 = More than 5 tumors of from I to 3 mm and/or up to 5
tumors of not more than 5 mm in diameter found in
visceral organs or in the peritoneal cavity; s.c. tumors
8 to 14 mm in diameter.
3 = Multiple tumors larger than 5 mm in diameter found
in visceral organs or in the peritoneal cavity (solitary
large tumors were never seen; they were invariably
part of massivetumor growth); s.c.tumors larger than
14 mm in diameter.
Following injections of tumor cells via the portal vein,
tumor growth was found only in the liver, with the exception
that, in 18 of the 120 mice implanted via this route, tumor
growth was also found in the peritoneal cavity. This prob
ably occurred because some of the cell suspension was
spilled during the process of injecting the portal vein.
Following injection of tumor cells into the left ventricle,
tumor growth was found in a great variety of locations. The
incidence of tumor growth in the various organs and tissues
is listed in Table 2.
AUGUST
at
value
for
s.c., by the number of s.c. challenge
autopsy
(see
text).
The
average
im
Following injection of tumor cells into the right ventricle,
tumor growth was found mainly in the lungs. In the 76 mice
that developed tumors of the 102 injected into the right yen
tricle, tumors were found only in the lungs in 5 1 mice; in the
lungs and in the heart in 9 mice; in the lungs, heart, and I
adrenal gland in 2 mice; in the lungs, heart, and 1 kidney in 1
mouse; in the lungs, heart, and in I ovary in 1 mouse; in the
lungs, heart, adrenals, and kidneys in 5 mice; in the lungs
and adrenals in 3 mice; in the lungs, kidneys, and adrenals in
2 mice; in the lungs, 1 adrenal, and I ovary in I mouse; and
in I adrenal only in 1 mouse.
When injected i.p., the tumor cells grew attached to the
peritoneum only.
When injected s.c., the tumor cells grew only at the in
jected sites.
The results presented in Table 1 show that the challenge
dose of 3.3 x l0@viable tumor cells was suitable for testing
of specific resistance against the fibrosarcoma in mice chal
lenged intravascularly or i.p. but was apparently too low for
testing by s.c. challenge implantation. The dose of 1 x l0@
viable tumor cells was too high for intravascular or i.p.
challenge but suitable for s.c. challenge. Consequently, in
subsequent experiments, 3.3 x l0@cells were given in intra
vascular injections and I x l0@cells were given in s.c. injec
tions.
The data further show that the mice cured of their sen
sitizing tumor implants had acquired a significant level of
1973
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1959
Jan Vaage
Table 2
The effect of active immunization on the growth of fibrosarcoma
cells transplanted intraarterially
in16of3330of3730of34normal,normal,radiated,sensitizedunsensitizedunsensitizedLocation
Tumors foundb
sitization, while the degree of resistance attained in such
areas or tissues as the retroperitoneal
space, the myocar
dium, the diaphragm, the axillae, the chest wall, and the
abdominal wall did not cause a significantly lower incidence
of
tumor
growth
in sensitized
mice.
Passive Transfer of Systemic Tumor Immunity. The next
experiments were designed to determine whether humoral
and cellular immune factors could affect the growth of tu
withtumôns°tumors°pCtumorstumorsLungsIc89Kidneys036Adnenals6c1719Ovaries3d912Mesenteny2c1012RetropenitonealI23spaceMyocardium6II12Mediastinum2c99Diaphragm366Axillae2
withmice
withmice
ofmice
mor cells implanted via different routes into unsensitized
normal mice and into unsensitized mice made immunologi
caily unresponsive by sublethal whole-body radiation.
Table 3 represents the data from 2 separate but similar
experiments. In each of the 2 experiments, the presensi
tized mice (group 19) had their sensitizing s.c. tumor im
plants surgically removed 4 days before challenge after a
growth period of 20 days. The mice that received sublethal
whole-body radiation (Groups 10 to 18) were radiated 4
days before challenge. The mice called “normal―
(Groups I
to 9) in Table 3 received no treatment 4 days before chal
wall344Abdominal
lenge.
wall242Thigh
On the 3rd day before challenge, the mice were treated
muscle010Liver000Pancreas000Spleen000
with i.p. injections of lymph node cells and/or hyperim
mune serum as indicated in Table 3. The mice given in
jections of lymph node cells each received the equivalent of
one-half of the quantity of cells obtained from 2 axillary,
a This
table
represents
a breakdown
by specific
tumor
location
of the
figures given in Groups 2, 7 and 12 ofTable 1.
2 brachial, and 2 inguinal lymph nodes. Since the pooled
b The
differences
in numbers
of positive
mice
and
the
differences
in
lymph node suspension contained pieces of tissue as well
incidence of tumor growth in specific locations have been evaluated
as single cells, a rough estimate indicated that each re
statistically.
CThe p values of comparison between sensitized and unsensitized mice cipient was given an injection of about S x 10@lymph
and between unsensitized and radiated mice are indicated between the ap
node cells. The mice given injections of hyperimmune
propniate columns: c, p < 0.05; d, 0.05 < p < 0.1; no mark, no signifi
serum received 0.5 mI/inoculum.
The mice given both
cant difference.
lymph node cells and serum received mixed aliquots of
cells and serum in a single inoculum. These injections were
resistance to challenge and that, among the organs reached
by tumor cells injected intravascularly,
the liver appeared to
repeated about I hr before challenge and again 3 days
after challenge.
have developed a particularly
high degree of resistance
All of the mice were challenged with 3.3 x l0@ viable
(Groups 6 and 11 versus Group I , Table 1). This observa
tion agrees with previously reported results (32). On the tumor cells injected into the right ventricle. To provide an
whole, the anatomical areas reached by intravascular and indication of when the experiment might be ready for ter
i_p. injections revealedthe immune resistanceof the hosts mination, 1 group of 5 radiated, untreated mice were given
injections of I x l0@ viable tumor cells into the right yen
more clearly than the s.c. injection sites.
The incidence of tumor growth following the injections of tricle. When these mice showed signs of dyspnea and a
tumor cells into the left ventricle is presented in Groups 2, slightly unhealthy appearance, 16 days after challenge, all
7, and 12 of Table I and has also been presented in Table 2 the test animals were killed by asphyxiation in 100% CO2
as a listing of specific incidences of tumor growth in the gas, and their lungs were examined for the extent of tumor
various major organs and tissues examined. The values in growth.
Table 2 suggest that the liver, pancreas, and spleen are un
The results presented in Table 3 show that the transfer
common sites for the localization of growth of tumor cells of lymph node cells from mice sensitized against the fibro
disseminated with arterial blood, and this may be due to sarcoma had transferred a significant degree of resistance
factors other than specific immune resistance, since these to both normal and radiated recipients (Groups 1 to 3 versus
organs were also not the site of tumor growth in unsensi
Groups 7 to 9, and Groups 10 to 12 versus Groups 16 to
tized animals or in animals depressed by sublethal whole
18). The transfer of resistance was assisted by the simulta
body radiation. Equally noteworthy is the very high mci
neous transfer of antifibrosarcoma
serum (Group 2 versus
dence in unsensitized mice of tumor growth in such small Groups I and 3, and Group I I versus Groups 10 and 12).
Transfer of lymph node cells from mice sensitized against
organs were also not the site of tumor growth in unsensi
cidence in the much larger lungs. In all of these organs, as an antigenically unrelated mammary carcinoma was not ef
fective in unradiated mice, except in the group that also
well as in the mesenteries and the mediastinum, statistical
received injections of immune serum from mice immunized
evaluation of the differences in tumor incidence between
sensitized and unsensitized mice indicated that there was against the fibrosarcoma (Group 5 versus Groups 4 and 6).
Transfer of lymph node cells from mammary carcinoma
particularly effective resistance to tumor growth after sen
1960
CANCER RESEARCH
VOL. 33
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Immunological
Control of Induced Metastases
Table 3
The efftct of transferred lymph node cells and serum on the growth of intravascularly
transplanted
FS cells°
GroupImmune
growthdI
statusTreatmentbChallengerNo.
of
mice with
tumorsAv.
tumor
11/20
0.88
2
Normal,
174
3Normal, Normal,
unsensitized
unsensitized
unsensitizedFS
sensitized lymph node cells
FS sensitized cells + FS serum
FS sensitized cells + MC serum12/20
. 14
S
Normal,
1.767
Normal,
6Normal,
unsensitized
unsensitized
unsensitizedMC
sensitized cells
MC sensitized cells + FS serum
14/20
16/202.04
1.46
MC sensitized cells + MC serum17/20
8
Normal,
1.7610
9Normal, Normal,
unsensitized
unsensitized
unsensitizedFS
serum
MC serum
No treatment15/20
16/20
16/201
1.68
13/20
1.50
17/201.90
1
16/20
1.60
9/201
1.
.80
II
Radiated,
12Radiated,
.621
Radiated,
unsensitized
unsensitized
unsensitizedFS
sensitized cells
FS sensitized cells + FS serum
FS sensitized cells + MC serum15/19
3
14
Radiated,
I2.5016
5Radiated,Radiated,
unsensitized
unsensitized
unsensitizedMC
sensitized cells
MC sensitized cells + FS serum
MC sensitized cells + MC serum20/20
20/202.20
17
Radiated,
18Radiated,Radiated,
2.7019SensitizedNo
unsensitized
unsensitized
unsensitizedFS
serum
MC serum
No treatment20/20
20/20
20/202.80
2.70
treatment4/200.26
a FS,
fibrosarcoma;
b The
mice
challenge,
C The
were
MC,
treated
mammary
with
i.p.
carcinoma.
injections
again at the time of challenge,
mice
were
challenged
with
3.3
of
immune
lymph
node
x
10°
live
tumor
cells
injected
represent the combined data from 2 separate but identical experiments.
antiserumaresetinitalics.
d Values
from
0 to
3 expressing
the
cells
and/on
antiserum
3 days
before
and 3 days after challenge.
total
amount
of
tumor
tissue
into
the
right
ventricle.
The
figures
The values for groups treated with FS
found
in
the
lungs
at
autopsy
(see
text).
The average value for each group was derived by dividing the sum of values by the number of mice in each
group. Only the differences in average tumor growth have been evaluated statistically. Groups I to 3 versus
Groups 7 to 9, p < 0.001; Groups 10 to 12 versus Groups 16 to 18, p < 0.001; Group 2 versus Groups I and 3,
p < 0.05; Group I I versus Groups 10 and 12, p 0.05 < p < 0.1; Group 5 versus Groups 4 and 6, p > 0.05;
Groups 13 to 15 versus Groups 16 to 18, p < 0.05; Group 14 versus Groups 13 and 15, p < 0.01; Group 19
versusGroup9,p < 0.01.
sensitized mice partially restored normal reactivity to radi
ated mice (Groups 13 to 15 versus Groups 16 to 18); this
reactivity was assisted by the simultaneous transfer of anti
fibrosarcoma serum (Group 14 versus Groups 13 and 15).
Antifibrosarcoma
serum injected alone into normal and
radiated mice had no effect (Group 7 versus Group 9, and
Group 16 versus Group 18). Actively sensitized mice were
strongly resistant to challenge (Group 19 versus Group 9).
To examine further this weak but suggestive protective
dures were otherwise as described for the previous experi
ment. All of the mice were challenged with 3.3 x l0@viable
tumor cells injected into the right ventricle. The experiment
was terminated and evaluated 16 days after challenge by
the procedure described for the previous experiment.
The results in Table 4 show that the transfer of lymph
node cells from mice sensitized against the fibrosarcoma
transferred a significant degree of protection against chal
lenge implantation of fibrosarcoma cells to radiated, unsen
effect by unsensitizedlymph node cells and immune serum sitized mice (Group 1 versus Group 10). The transfer of
in radiation-depressed mice, groups of animals were pre
normal lymph node cells plus serum from mice immunized
treated with whole-body radiation or with live tumor im
against the fibrosarcoma also gave the radiated recipients
plantation followed by surgical resection or were left un
significant protection against challenge (Group 4 versus
treated as in the previously described experiment. On the Group 10). The transfer of serum alone (Groups 7 to 9),
3rd day before challenge, the mice were treated with i.p. control lymph node cells alone (Groups 2 and 3), or control
injections of lymph node cells and/or immune serum (pre
lymph node cells plus control serum (Groups 5 and 6) had
pared differently than hyperimmune serum; see “Materials no significant effect. Actively sensitized mice were strongly
and Methods―) as outlined in Table 4. The further proce
resistant to challenge (Group I 1 versus Group 12).
AUGUST
1973
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1961
Jan Vaage
Table 4
The effect of transferred lymph node cells and serum on the growth of intravascularly
transplanted
FS@cellsChallengecNo.
ofAv.mice
withtumo,GroupImmune
statusTreatmentbtumorsgrowthdIRadiated,
unsensitizedFS
unsensitizedMC
unsensitizedNormal
.022Radiated,
102.003Radiated,
104Radiated,
sensitized lymph node cells6/
101
sensitized cells10/
cells10/102.
unsensitizedNormal
serum8/101.285Radiated,
unsensitizedNormal
106Radiated,
unsensitizedNormal
serum10/102.007Radiated,
cells + FS
cells + MC serum10/102.
cells + normal
unsensitizedFS
serum10/102.708Radiated,
unsensitizedMC
serum10/102.409Radiated,
unsensitizedNormal
serum10/102.8010Radiated,
unsensitizedNo
treatment9/92.80I
ISensitizedNo
treatment0/10012UnsensitizedNo
treatment10/102.
a FS,
fibrosarcoma;
b The
mice
were
MC,
treated
mammary
with
carcinoma.
i.p.
injections
of
normal
on
immune
mune serum 3 days before challenge, again at the time ofchallenge,
C The
d
Same
mice
were
as
Table
challenged
3,
Footnote
with
3.3
d.
tically. Group I differs significantly
significantly from all other groups.
10
x
Only
10°
the
live
tumor
differences
cells
in
lymph
node
cells
and/or
normal
injected
into
average
tumor
the
right
growth
im
ventricle.
have
been
evaluated
from all other groups except Group 4 and vice versa; Group
The last experiment reexamined the protection of radi
ated mice by transferred normal lymph node cells plus im
mune serum, which was seen in the 2 previously described
experiments, and examined the effect in the different ana
or
and 3 days after challenge.
statis
I 1 differs
DISCUSSION
Immune resistance reactions directed against normal and
neoplastic solid tissue are generally held to be mediated pre
tomical locations reached by intraarterial, i.v., and s.c. dominantly by cellular immune factors. Humoral immune
tumor cell implantation. This experiment also followed the factors, while capable of acting with complement to destroy
pretreatment
and treatment procedures described for the normal and neoplastic cells that tend to grow singly, are
previous experiments, giving 3 transfers of normal lymph also generally considered capable of abrogating cell-medi
node cells and/or injections of immune serum. The mice ated immune destruction of solid tumors by processes
were challenged with 3.3 x l0@living tumor cells injected known as immunological enhancement or blocking (12, 13).
into the left or right ventricle or with 1 x l0@cells injected
In the present study, which was originally undertaken as
s.c. at the left shoulder and at the left hip. The experiment
an attempt to determine the immunization procedures and
was terminated 18 days after challenge for the animals chal
conditions that might produce immunological enhancement
lenged intravascularly
and 27 days after challenge for the in a syngeneic, in vivo system, I have found that humoral
animals challenged s.c.
as well as cellular immune factors will, under carefully se
The results presented in Table 5 show that the transfer lected and prepared conditions, transfer resistance and not
of normal lymph node cells plus immune serum gave the growth facilitation to unsensitized syngeneic recipients.
The protective effect of immune serum was most clearly
radiated recipients significant protection against intravas
cular challenge (Group 2 versus Groups 1, 3, and 4, and exhibited in recipients that had been suppressed by radia
Group 8 versus Groups 7, 9, and 10). Normal lymph node tion and partially restored with transferred normal lymph
node cells (Group 14, Table 3; Group 4, Table 4; Groups 2
cells alone or immune serum alone gave no protection
(Groups 1 and 3 versus Group 4, and Groups 7 and 9 versus and 8, Table 5). The protective effect of transferred im
Group 10). The distribution of tumor growth in treated and mune serum was less perceptible in normal unsensitized re
untreated mice challenged via the left ventricle was similar cipients or in adoptively immunized recipients where early
developing (3 1, 32) or existing immune resistance could
to the distribution shown in Table 2.
have obscured the protection transferred
with immune
Radiated mice challenged s.c. did not exhibit any protec
tion by transferred cells and/or immune serum (Groups 13, serum. Immune serum by itself did not transfer detectable
14, and 15 versus Group 16). Actively sensitized mice were resistance to radiated recipients where cellular immune
strongly resistant to challenge (Group 5 versus Group 6, functions were presumably abrogated or impaired.
The protective effect of transferred
immune serum,
Group II versus Group 12, and Group 17 versus Group
which, in contrast to protection by transferred immune cells,
18).
1962
CANCER RESEARCH
VOL. 33
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Immunological
Control of Induced Metastases
Table 5
The efftct of transferred lymph node cells and serum on the growth of intravascularly
growthd1
GroupImmune
route
on no. of
of mice
tumorsChal1enge'@No.
with tumorsAv.
statusTneatmentbInjection
unsensitized
2 Radiated, unsensitized
3 Radiated, unsensitized
4 Radiated, unsensitized
5 Sensitized
6Radiated,
.367 UnsensitizedLymph
node cells
Lymph node cells + FS serum
FS serum
No treatment
No treatment
No treatmentLeft
Left
Left
Left
Left
Left
unsensitized
8 Radiated, unsensitized
9 Radiated, unsensitized
10 Radiated, unsensitized
11 Sensitized
12Radiated,
UnsensitizedLymph
2.3413
node cells
Lymph node cells + FS serum
FS serum
No treatment
No treatment
No treatmentRight
Right
Right
Right
Right
Right
unsensitized
14 Radiated, unsensitized
I 5 Radiated, unsensitized
16 Radiated, unsensitized
17 Sensitized
UnsensitizedLymph
18Radiated,
node cells
Lymph node cells + FS serum
FS serum
No treatment
No treatment
No treatments.c.
s.c.
s.c.
s.c.
s.c.
s.c.17/20C
a FS,
fibrosarcoma.
b The
mice
were
treated
the time ofchallenge,
with
i.p.
and s.c. transplanted FS°cells
injections
of
normal
lymph
node
cells
ventricle
ventricle
ventricle
ventricle
ventricle
ventricle10/
tumor
10
.90
0.96
8/10
10/10
ventricle
ventricle
ventricle
ventricle
ventricle
ventricle10/10
2.40
10/10
3/ 10
8/ 101
2.70
0.60
1
4/10
10/10
0.92
2.60
10/10
0/ 10
9/102.80
2.90
0
18/20
20/20
1.89
2.10
17/20
5/20
16/201
1.96
0.35
1.28
.62
and/or
immune
serum
3 days
before
challenge,
again
at
and 3 days after challenge.
CThe mice were challenged with 3.3 x l0@live tumor cells injected into the left on the right ventricles or were challenged with
1 x 10' live cells injected s.c. both at the left shoulder and at the left hip.
d Same
as
Table
3,
Footnote
d.
Only
the
differences
in
average
tumor
growth
have
been
evaluated
statistically.
Group
Groups 1, 3, and 4, p < 0.01; Group 8 versus Groups 7, 9, 10, p < 0.001; Group 5 versus Group 6, p < 0.05; Group
Group 12,p < 0.001; Group 17 versusGnoup l8,p < 0.01.
2 versus
II versus
is not always conspicuous, was however constant in the re
peated tests reported here. Studies of the development of
active tumor immunity (31) and other studies of passive
the rapid development of the normal primary immune re
sponse stimulated by the challenge injection of tumor cells
(31. 32).
transfer procedures, which will be published elsewhere,
If the “arming―
mechanism functions in antitumor im
have also shown that whenever an effect resulted from ac
mune resistance, an additional process must be proposed,
tive or passive immunization in syngeneic systems, the ef
which can explain how circulating antitumor antibodies
fect was protective, never growth promoting.
may be “arming―
and thereby committing to 1 specificity
The observations reported here resemble those reported
possibly the entire population of 1 class of uncommitted
by Tsoi and Weiser (28), who found a synergistic protec
lymphocytes without creating a persistent state incompati
tive effect by normal macrophages and immune serum ble with the normal immune functions of an intact animal.
against allogeneic sarcoma growth in radiated, unsensitized The known rapid turnover of a great portion of the small
passive transfer recipients. Other investigators have ob
lymphocyte population (8, 17, 24) and the relatively short
servedsynergistic cytotoxic action by cells and serum in al half-life of some immunoglobulins (34) could explain how
logeneic systemsin vitro (5, 16,20, 26) and in allograft re “arming―
of normal lymphocytes by cytophilic antibody
jection (4).
could be a very effective, but temporary, immune defense
Pollack (22) and Pollack et al. (23) reported that antitu
mechanism.
mor serum made normal lymph node cells active in the in
Both active and passive immune resistance to tumor
vitro destruction
of syngeneic tumor target cells. The effect
growth was more clearly expressed in visceral organs
was given the descriptive term “arming―
of lymphocytes by reached by intravascular implantation than in i.m. and s.c.
antibody. This proposed “arming―
mechanismcould have implantation sites. The protective effect of immune serum
caused the protective effect seen most distinctly in radiated
was also most evident when tumor challenge was via vas
recipients of normal lymph node cells and immune serum.
cular routes. One credible explanation for this effect is that
The transferred immune serum may also have acted inde
immune resistance factors are encountered immediately
pendently in a complement-dependent
cytotoxic destruction
and maybe also more abundantly by tumor cells injected
of tumor cells. Either mechanism could have been made
intravascularly.
Separate studies (30, 3 1) dealing with the
quate in lymphocyte-depleted
radiated mice, discernible
effects of tumor antigen on the established antitumor im
in mice partially restored with normal lymph node cells, mune resistance found that depression of immune resistance
and obscured in intact recipients by existing immunity or by by excess antigen, which was clearly evident when the level
AUGUST
1973
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1973 American Association for Cancer Research.
1963
Jan Vaage
of resistance was tested with s.c. challenge, was not evident
when the challenge implantation of tumor cells was made
i.v. These observations may indicate that immune destruc
tion of antigenic neoplastic cells is most effective in the
blood and that antitumor antibodies may be primarily or
particularly effective in preventing metastatic dissemina
tion of tumor growth.
The protection seen with passive transfer of serum from
tumor-cured and tumor-bearing donors may pertain to an
important theoretical question in tumor immunology. In
question is the objection that is repeatedly raised to all
forms of tumor-specific
immunization
procedures on
grounds of the presumed inherent danger of causing anti
body-mediated tumor enhancement instead of the intended
cell-mediated tumor destruction. The presumption is based
on very extensive data derived from investigations of anti
body-mediated
in vivo enhancement in allogeneic systems
(14) and on data from in vitro demonstrations of serum fac
tors that can block cell-mediated destruction of syngeneic
and autochthonous neopiastic target cells (12, 13). How
ever, directly applicable data, demonstrating
immunologi
cal enhancement of tumor growth in syngeneic or autoch
thonous in vivo systems, are relatively few ( I, 2, 6, 7, 18).
In view of the sparsity of directly applicable evidence, and
in view of the present as well as previously published evi
dence (3, 10, 11, 18, 19, 21) that antitumor serum may pro
tect against tumor growth in syngeneic, in vivo systems, the
inference that humoral immune factors are less likely to
protect than to interfere with immune protection may have
been drawn too readily.
There is no conflict between the observations presented
here and the well-documented in vitro phenomenon of se
rum factors that can block cell-mediated destruction of
neoplastic target cells or the results of extensive in vivo in
vestigations of antibody-mediated
allogeneic enhancement.
All are important biological phenomena that need to be
studied and understood.
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1965
Humoral and Cellular Immune Factors in the Systemic Control
of Artifically Induced Metastases in C3Hf Mice
Jan Vaage
Cancer Res 1973;33:1957-1965.
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