[CANCER RESEARCH 43, 138-145, January 1983]
0008-5472/83/0043-0000$02.00
Resistance of Tumor-bearing Mice to a Second Tumor Challenge
Elieser Gorelik '
Laboratory of Immunodiagnosis,
National Cancer Institute, NIH, Bethesda, Maryland 20205
much attention. It required about 50 years to prove that the
specific immune response can be evoked during tumor growth,
Antitumor resistance of mice bearing 3LL, M109, or Meth A resulting in increased resistane - to the second tumor challenge
tumors to a second tumor challenge was studied. The degree (23). In this experimental model, the antitumor resistance of
of inhibition of a second tumor growth in the tumor-bearing
the host was analyzed after removal of the first tumor graft.
mice was a function of both the size of the primary tumor mass The antitumor resistance in the animals following excision of
and the number of reinoculated tumor cells.
the growing tumor became a favorite model for measuring the
Antitumor resistance of tumor-bearing mice is tumor nonspe
antitumor immune response, immunogenicity, and the antigen
cific and is observed only in the presence of the growing first specificity of the investigated tumors (18, 21, 23). In 1970,
tumor. Following removal of 3LL tumor, the resistance to the Fisher ef al. (7) proposed to distinguish concomitant immunity
second challenge with 3LL or T-10 tumor cells completely (resistance to a second tumor graft in the presence of the first
disappeared.
growing tumor) from sinecomitant immunity (resistance of tu
Antitumor resistance of tumor-bearing mice does not appear mor-excised animals to a second challenge). In many respects,
to be mediated by T-cells, natural killer cells, or macrophages, the resistance to a second tumor challenge shown by the
since similar results were observed in immunologically com
tumor-excised host has different characteristics from the re
petent and nude mice as well as in beige mice and in mice with sistance observed in the tumor-bearing host.
functions of macrophages and natural killer cells suppressed
In previous studies (15), we have found that the resistance
by silica treatment. Growth inhibition of the reimplanted tumor of mice to a second tumor graft in the presence of the growing
cells does not appear to be a result of their elimination. Radio- tumor can be very strong and may completely prevent the
labeled M109 tumor cells survived to the same degree at the growth of high doses of the reinoculated tumor cells. This
local site of inoculation in tumor-bearing and non-tumor-bear
resistance was a function of the volume of the primary tumor
ing control mice. The number of surviving radiolabeled tumor
mass and was not tumor specific. Indeed, mice bearing 3LL
cells inoculated into the footpad of ¡mmunocompetentand tumors were resistant to the reinoculation of 3LL, B16, or EL4
nude mice was similar and did not depend on the presence or tumors. Similarly, the growth of 3LL tumor cells was arrested
absence of growing first tumor. Despite the similarity in the when they were reinoculated into mice bearing B16, EL4, or Tsurvival of reinoculated tumor cells, their growth was inhibited
10 tumors. It appears that this resistance is not mediated via Tonly in the tumor-bearing mice. In mice bearing ¡mmunogenic cell-mediated immunity because in T-cell-depleted animals (BMeth A tumors of different sizes (1 to 3 cm), the clearance of mice or nude mice) bearing the first tumor graft, the inhibitory
the reinoculated Meth A cells was significantly higher than in effect on the subsequently implanted tumor cells was as effec
non-tumor-bearing mice. The number of surviving tumor cells tive as in normal tumor-bearing mice (15).
was similar in mice bearing small (<1 cm) or large (>3 cm)
The tumor-nonspecific character of the resistance to a sec
tumors. However, complete prevention of the second tumor ond tumor challenge and its independence from T-cell-mediated immunity suggest that either NK2 cells or macrophages
growth was observed in mice bearing huge tumor mass (^3
ABSTRACT
cm in diameter). These data may indicate that resistance of
mice bearing immunogenic and nonimmunogenic tumors is
mediated by different mechanisms. The resistance to a second
tumor challenge in mice bearing nonimmunogenic tumor is due
mainly to nonimmunological mechanisms. In contrast, the antitumor resistance in mice bearing immunogenic tumor is a
function of antitumor immune reactions, although additional
effects of nonimmunological mechanisms were also observed.
INTRODUCTION
The inhibition of growth of a second tumor graft in mice
bearing the original tumor was described by Ehrlich (6) in
1906. This phenomenon was later attributed to the antitumor
immunological response and termed concomitant tumor im
munity (2). Although the antitumor resistance of tumor-bearing
mice to a second challenge is rather strong, even at high doses
of tumor cell inocula, this experimental model did not attract
1Present address: Surgery Branch, National Cancer Institute, NIH, Bethesda,
Md. 20205.
Received March 24, 1982: accepted October 11,1982.
138
may be responsible for the inhibition of a second tumor growth.
In the present study, the possible involvement of NK cells and
macrophages in the resistance of the tumor-bearing host to a
second tumor challenge was investigated. Inhibition of tumor
growth can result from elimination of the tumor cells or preven
tion of their proliferation. To analyze the survival and growth of
tumor cells reinoculated into normal or tumor-bearing mice, we
used a new radioisotope technique that makes it possible to
determine the survival of tumor cells at the inoculation site (11,
12).
MATERIALS
AND METHODS
Mice. Inbred female C57BL/6-H-2",
beige C57BL/6-H-2",
and
BALB/c-H-2a mice, 2 to 3 months old, were obtained from The Jackson
Laboratory, Bar Harbor, Maine. (C3H/eB x C57BL/6) (H-2VH-2")
F,
mice were supplied by the Animal Breeding Center of the Weizmann
Institute of Science, Rehovot, Israel.
Tumors. The following tumors were used: Lewis lung carcinoma
2The abbreviations used are: NK, natural killer; dUrd, deoxyuridine: i.f.p., into
the footpad.
CANCER
RESEARCH
VOL. 43
Mechanisms of Concomitant
(3LL) that arose spontaneously in C57BL/6 mice; Meth A fibrosarcoma
induced by methylcholanthrene
in BALB/c mice; Madison lung carci
noma (M109) spontaneously developed in BALB/c mice; YAC-1, a
tissue culture cell line of YAC, a Moloney virus-induced lymphoma of
A/J origin. These tumor cells were maintained in vitro in complete
Roswell Park Memorial Institute Medium 1640 supplemented with 10%
fetal bovine serum (11,12). The T-10 tumor was induced by methyl
cholanthrene in (C3H/eB
x C57BL/6)
Fi mice and maintained by
serial transfers in these mice.
Tumor Cell Survival in Vivo. Nonconfluent monolayers of cultivated
M109, Meth A cells, or nonsaturated cultures of YAC-1 lymphoma cells
were incubated for 16 hr with fluorouridine (0.06 mg/ml) and 12Sl-dUrd
(0.4 /uCi/ml; specific activity, 5 Ci/mg; The Radiochemical Centre,
Amersham, England). After washing, tumor cells were resuspended in
complete Roswell Park Memorial Institute Medium 1640. Radiolabeled
tumor cells were inoculated i.f.p. in normal or tumor-bearing mice. The
level of radioactivity in the footpad was measured at 30 min and then
at various times following tumor cell inoculation. The level of radioac
tivity in the inoculated leg was measured with a low-energy y-scintilla
tion counter (Ludlum model 2200; Ludlum Measurements Inc., Sweetwater, Texas) adjusted to detect y-rays between 10 and 40 keV, using
a detector with a 1-mm-thick, 1-inch-diameter Nal (T1 ) crystal with a 7
mg per sq cm aluminum window. The detector was shielded with 2mm-thick lead with an aperture of 1 x 1.5 cm. The inoculated foot was
put into this opening, and the level of the radioactivity was measured
during 20 sec. Mice were marked, making it possible to follow the rate
of clearance of radioactivity in individual mice. Vernier calipers were
used to measure the diameter of i.f.p. established tumors at various
periods of growth.
In Vitro Cytotoxic Activity of Spleen Cells. Cytotoxic effect of
spleen cells of normal or tumor-bearing mice was assessed in a 4-hr
assay of 51Cr release as described previously (11 ). Some of the control
or tumor-bearing
mice were treated with 15 mg of silica (Santocel 58;
particle size 3 /un; Monsanto Co., St. Louis, Mo.) 3 days before the
Cytotoxic activity of their spleen cells was tested.
RESULTS
In the first series of experiments, the growth of a second
tumor graft was studied in tumor-bearing mice and in mice from
which primary tumors had been removed. In these studies,
there are certain difficulties concerning the survival of tumorbearing mice. Relatively small doses of reinoculated tumor cells
can have a long latent period, and tumor-bearing mice can die
before the tumors appear even in the control mice. High doses
of reinoculated tumor cells may overcome the antitumor resist
ance developed in tumor-bearing and tumor-excised mice. Our
previous experiments had shown that (C3H/eB x C57BL/6)
F, mice can tolerate the 3LL tumors at the size that usually
kills syngeneic C57BL/6 mice. Thus, FI mice were chosen for
these experiments. It was found previously that mice bearing
s.c. tumors larger than 1 cm in diameter had apparent resist
ance to the second tumor graft (15).
To facilitate the excision of the local tumor, 1 x 106 3LL
tumor cells were inoculated i.f.p. into (C3H/eB x C57BL/6)
FI mice. When tumors reached 10 to 12 mm, mice were divided
into 2 groups. In the first group, tumor-bearing legs were
amputated; in the second group, tumors were allowed to grow.
Tumor-bearing and tumor-excised mice were reinoculated in
the second leg with 1 x 106 3LL or T-10 tumor cells on the
day of surgery. Control mice were inoculated i.f.p. with the
same doses of tumor cells.
Most of the mice bearing the first tumor survived at least 8
days after tumor reinoculation, which allowed comparison of
JANUARY 1983
Tumor Immunity
the tumor growth of reimplanted tumor cells in the presence
and absence of the first tumor graft. Chart 1 shows the tumor
diameter in the reinoculated leg. The diameters of 3LL tumors
from reinoculated cells were similar in the tumor-excised and
control mice whereas in mice bearing the first 3LL tumor the
growth of the second 3LL tumor was substantially suppressed.
This inhibition was not tumor specific because growth of
reinoculated T-10 tumor cells was inhibited in the presence of
growing 3LL tumor. In contrast, there was no inhibition of T-10
tumor growth in mice that had had excision of 3LL tumor (Chart
1). Previous experiments (15) have demonstrated that growth
of the reinoculated tumor cells was effectively prevented in the
tumor-bearing T-cell-deficient animals (B-mice or nude mice).
Antitumor resistance of tumor-bearing mice with either intact
or deficient T-cell-mediated immunity could result from the
cytotoxic action of NK cells or macrophages, which have
cytotoxic action that is not tumor specific.
This assumption was analyzed using mice in which the func
tions of NK cells and macrophages were suppressed (C57BL/
6 mice treated with silica; beige mice). Beige mice are char
acterized by a low level of NK cell activity, and silica treatment
suppresses the functions of both NK cells and macrophages
(1, 5). C57BL/6 and beige mice were inoculated s.c. with 1
x 106 3LL tumor cells. When tumors reached 2 cm in diameter,
some of the tumor-bearing C57BL/6 mice and some of the
control C57BL/6 mice were inoculated i.p. with 15 mg of silica.
All mice were inoculated i.f.p. with 1 x 106 3LL tumor cells 24
hr later. Tumor growth i.f.p. was similar in silica-treated, control
C57BL/6 mice and in beige mice. However, in all groups of
mice bearing s.c. tumors, the growth of the second tumor graft
was strongly inhibited (Chart 2). Peritoneal macrophages of
normal or silica-treated tumor-bearing mice had no cytotoxic
activity against tumor cells assessed by in vitro technique (data
not shown).
The activity of NK cells in the tumor-bearing mice was tested
in vitro using a 4-hr assay of 51Cr release (Table 1). In a series
of experiments, the cytotoxic effect of spleen cells of intact
C57BL/6 mice at an effectortarget
ratio of 100:1 varied from
12 to 19%, whereas spleen cells of mice bearing 3LL tumor
>2 cm in diameter had lower cytotoxic activity which never
exceeded 6%. Cytotoxic activity in the silica-treated mice was
almost completely suppressed.
Natural cell-mediated immunity also can be assessed in vivo
by the ability of mice to eliminate radiolabeled tumor cells
E
è
fi'
t
l
8.0-
•—•
Im*c1 Me* + 3LL
•-•3U.-EnM
t 31L
*—•3U. taring t 3LL
6.0
4.0
o
5
S
'S 3.0
2.0
246
DAYS OF TUMOR GROWTH
2.0
468
DAYS OF TUMOR GROWTH
Chart 1. Growth of reinoculated 3LL or T-10 tumor cells in tumor-bearing or
tumor-excised mice. F1 (C3H/eB x C57BL/6) mice bearing i.f.p. 3LL tumor (10
to 12 mm in diameter) were divided into 2 groups (10 mice/group): in Group 1.
local 3LL tumors were removed; in Group 2, 3LL tumors were allowed to grow.
All mice were challenged in second footpad with 1 x 10°of 3LL M ) or T-10 (B)
cells. Simultaneously, intact mice were inoculated i.f.p. with 1X10'
3U. or T-10
tumor cells. Bars, S.E.
139
E. Gorelik
9.0 r-
8.0
7.0
J
oc
i
6.0
!"
o
?4,
3.0
7
8
9
DAYS AFTER TUMOR
10
11
12
13
CELL REINOCULATION
Chart 2. Inhibition of growth of 3LL tumors in tumor-bearing C57BL/6-+/
+ , C57BL/6 beige, or C57BL/6-+/+
mice treated with silica. C57BL/6-+/
+
mice bearing s.c. 3LL tumors z2 cm in diameter were treated with silica (15 mg
i.p.). One day later, silica-treated (•)and nontreated tumor-bearing C57BL/6+ /
-I- mice (A) or tumor-bearing beige mice (•!were reinoculated i.f.p. with 1X10°
of 3LL cells. Control, non-tumor-bearing
C57BL/6-+/+
(A), silica-treated
C57BL/6-+/+
(O), or beige O mice were inoculated i.f.p. with 1 x 106 3LL
cells (10 mice/group).
in vivo were similar in mice with low and high NK cell activity.
Tumor growth was observed in syngeneic BALB/C+/+
and
BALB/c nude mice inoculated with radiolabeled M109 tumor
cells (11, 12).
The levels of radioactivity immediately after the inoculation
reflect the real number of injected tumor cells (Chart 4A).
Measurement of the radioactivity in the foot pads at different
times after tumor cell inoculation indicates the level of tumor
cell survival. One day after tumor cell inoculation, a decrease
in radioactivity was observed in all mice, but later the rate of
elimination of radioactivity from the footpad decreased. The
levels of radioactivity detected in normal and tumor-bearing
BALB/c mice were similar. In addition, no differences were
found in the clearance of radioactivity in BALB/c—1-/+ and
nude mice bearing s.c. tumors and in non-tumor-bearing mice.
The absolute levels of radioactivity in the footpads of nude
control mice were lower than in other mice, but this was a
result of the initial lower number of radiolabeled cells inoculated
i.f.p. in these mice (Chart 4/4). In general, the slope of the
elimination of radioactivity in nude mice was identical to the
slope for other BALB/c mice.
The similarity in the levels of radioactivity remaining in the
Table 1
NK activity of the spleen cells of intact or silica-treated
tumor-bearing
mice
C57BL/6 mice bearing s.c. 3LL tumor (»2 cm in diameter) received i.p. 15
mg of silica. Three days later, cytotoxic activity of spleen cells of treated and
nontreated mice was tested against YAC-1 tumor cells in a 4-hr '"Cr release
assay. A pool of 3 spleens was tested from each group.
% of cytotoxicity
Bars, S.E.
MiceIntact
inoculated i.v. from the lungs (24) or from the inoculation site
(11). The ability of normal or tumor-bearing C57BL/6 mice to
eliminate 125l-dUrd-labeled YAC-1 cells from the footpad was
studied; 0.5 x 106 radiolabeled YAC-1 cells were inoculated
i.f.p. in control C57BL/6 mice or mice bearing s.c. 3LL tumors
of different sizes (<1, >2, or >3 cm). The level of radioactivity
remaining in the leg was determined.
In C57BL/6 mice bearing 3LL tumors >3 cm, the elimination
of tumor cells from the inoculation site was substantially sup
pressed (Chart 3). This suppression was less in mice bearing
smaller 3LL tumors (2 to 3 cm in diameter). In mice bearing the
small s.c. 3LL tumors (<1 cm), the clearance of reinoculated
YAC-1 tumor cells was significantly greater than in other tumorbearing mice and was similar to the clearance in non-tumorbearing control mice. These experiments indicate that cytotoxic
activity of NK cells tested in vivo and in vitro against NKsusceptible tumor cells is impaired in tumor-bearing mice.
Survival and growth of reinoculated radiolabeled tumor cells
can be a useful method for understanding the mechanism of
the tumor growth suppression in tumor-bearing mice. It is still
unclear whether the growth inhibition of the second tumor graft
results from the elimination of the reinoculated tumor cells or
from the suppression of their proliferation. To make this deter
mination, BALB/c mice and BALB/c nude mice were inocu
lated s.c. with 1 x 106 M109 tumor cells. When s.c. tumors
reached >2 cm in diameter, mice were inoculated i.f.p. with 2
x 10s 12Sl-dUrd-labeled M109 tumor cells. Control BALB/c
mice, BALB/c nude mice,
J mice received the same
cells. Previously, we have
ant to the cytotoxic action
140
and allogeneic C57BL/6 and CBA/
number of radiolabeled M109 tumor
shown that M109 tumor cells resist
of NK cells in vitro and their survival
±0.8E'C
±0.5
±0.2
Silica-treated non-tumor3.4
0.16.2
±
3.0 ±
0.15.4
2.9 ±
0.13.1
bearing
Tumor-bearing
±0.2
±0.1
±0.3
Silica-treated tumor-bearing100:1"15.73.9 ±0.133:110.3 3.1 ±0.111:16.4
2.9 ±0.1
Effectortarget ratio.
6 Mean ±S.E.
c Significantly different (p < 0.01) from other groups.
100,000
10,000
1000
100
16
24
40
48
HOURS AFTER YAC-1 CELL INOCULATION
Chart 3. Elimination of radiolabeled YAC-1 cells from footpad of control mice
or mice bearing s.c. 3LL tumor of different sizes. Control C57BL/6 mice or mice
bearing s.c. 3LL tumors («1, »2. or »3 cm in diameter) were inoculated i.f.p.
with 0.5 x 10e [125l)dUrd-labeled YAC-1 cells. Level of remaining radioactivity
i.f.p. was measured at various periods after tumor cell inoculation
group). Bars. S.E.
CANCER
RESEARCH
(5 mice/
VOL.
43
»Vi-v
Mechanisms of Concomitant Tumor Immunity
10,000 r
100,000
10.000
1000 -
~
24
6
8
10
12
14
DAYS AFTER INOCULATION RADIOLABELED
TUMOR CELLS
1000
2
4
6
8
10
12
DAYS AFTER TUMOR CELL INOCULATION
Chart 4. Elimination of radiolabeled M109 tumor cells from footpad of control and tumor-bearing mice. In A. control syngeneic BALB/C-+/+
O, BALB/c nude
(A), allogeneic C57BL/6 (•),and CBA/J (O) mice were inoculated i.f.p. with 2 x 105['25l]dUrd-labeled
M109 tumor cells. Radiolabeled M109 tumor cells (2 X 10s)
were also reinoculated i.f.p. in BALB/C-+/+
(•)or BALB/c nude mice (A) bearing s.c. M109 tumor a2 cm in diameter. At various periods after inoculation of
radiolabeled cells, the level of radioactivity remaining i.f.p. was measured. In ß,control CBA/J mice (O), BALB/C-+/+
mice (O), or BALB/C-+/ + mice bearing s.c.
M109 tumor a2 cm (•)were inoculated i.f.p. with 1 x 106 radiolabeled M109 tumor cells (10 mice/group).
foot pads of BALB/C-+/ + and nude mice bearing the first
tumor graft and of non-tumor-bearing mice may indicate: (a)
that cytotoxic T-cells did not develop in BALB/C-+/+
mice;
or (o) that NK cells, macrophages, or other potential cytotoxic
agents were not active in all mice investigated or that they were
equally efficient in tumor-bearing and non-tumor-bearing mice
and in immunocompetent and T-cell-depleted nude mice. The
potential efficiency of immune reaction in the elimination of
inoculated radiolabeled M1 09 tumor cells was indicated by the
elimination of these cells in allogeneic C57BL/6 and CBA/J
mice. Seven days after inoculation of radiolabeled cells, the
slope of radioactivity in allogeneic and syngeneic mice was
identical (Chart 4A). Later, however, the rate of clearance of
tumor cells rapidly increased in the allogeneic mice and, 13
days after tumor cells were injected, all radioactivity in the
allogeneic mice had completely disappeared (Chart 4A). The
rate of clearance of M109 tumor cells was slightly higher in
C57BL/6 mice than in CBA/J mice. Although the levels of
radioactivity were similar in the footpads of BALB/C-+/+
and
nude mice bearing s.c. tumors and in non-tumor-bearing mice,
the rates of tumor cell growth in these mice were different
(Chart 5/0. In intact BALB/C-+/ + and BALB/c nude mice,
i.f.p. tumors grew progressively, but the growth of reinoculated
M109 tumor cells was strongly suppressed in mice bearing s.c.
M109 tumor. In the presence of the first tumor, this suppression
was even more profound in nude mice than in BALB/C-+/ +
mice (Chart 5A ). Nude mice bearing s.c. M109 tumor appeared
to be very ill. They lost weight and were pale and less active
than nude mice that had no s.c. tumors. Similar results were
obtained when, in parallel, BALB/c mice bearing s.c. M109
tumors (>2 cm in diameter) were reinoculated i.f.p. with a
higher dose of radiolabeled M109 tumor cells (1 x 106). The
slope of radioactivity in the inoculated legs of tumor-bearing
and control BALB/c mice was similar (Chart 48). Fourteen
days after reinoculation of tumor cells, a high level of radioac
JANUARY 1983
tivity remained at the site of transplantation, although a slight
decrease in radioactivity was found in BALB/c mice not bearing
s.c. tumors. At this period, these mice developed i.f.p. tumors
that were >0.5 cm in diameter, which made it difficult to
measure the level of radioactivity in the leg. In allogeneic CBA/
J mice inoculated with radiolabeled M9 tumor cells, the tumor
cells were completely eliminated (Chart 46). In contrast to the
similarity in the survival of reinoculated M109 cells in tumorbearing and control BALB/c mice, there was a striking differ
ence in the growth of reinoculated tumor cells (Chart 5B). In
the presence of s.c. M109 tumor, the proliferation of the
reinoculated M109 tumor was strongly suppressed.
All of these experiments were performed using weakly ¡mmunogenic spontaneous 3LL and M109 tumors. To assess the
contribution of the immunological mechanisms in the elimina
tion of reinoculated tumor cells and in the suppression of their
growth in tumor-bearing mice, the immunogenic methylcholanthrene-induced Meth A tumor was used. The antigenic and
immunogenic properties of Meth A tumor have been reported
in numerous investigations (3, 21, 25). BALB/c mice were
inoculated s.c. with 1 x 106 Meth A tumor cells. Mice bearing
s.c. Meth A tumors that were >3, >2, or >1 cm in diameter
were inoculated i.f.p. with 0.8 x 106 125l-dUrd-labeled Meth A
tumor cells. The level of radioactivity at the inoculation site
progressively decreased in all mice. However, mice bearing
s.c. Meth A tumors had more efficient clearance of radioactive
tumor cells than did normal non-tumor-bearing BALB/c mice
inoculated i.f.p. with radioactive Meth A tumor cells (Chart 6).
Although the levels of radioactivity remaining in the footpads of
all tumor-bearing mice were similar, large differences were
observed in the growth rates of reinoculated tumor cells (Chart
7). In mice bearing tumors >3 cm, growth of i.f.p. reinoculated
tumor cells was completely suppressed. There was substantial
inhibition in the development of second-footpad tumors in mice
bearing first s.c. tumors >2 cm in diameter. Fourteen days
141
E. Gorelik
11.0
10.0
9.0
8.0
O 6.0
g
¡,0
4.0 3.0 -
B
6
10
14
18
22
26
30
DAYS AFTER TUMOR CELL INOCULATION
DAYS AFTER i.f.p. INOCULATION OF
TUMOR CELLS
Chart 5. Resistance of tumor-bearing mice to a second tumor challenge. A, growth of radiolabeled M109 tumor cells (2 x 105) inoculated i.f.p. in control BALB/
C-+/+ (O) or BALB/c nude mice (A) or in tumor-bearing BALB/C-+/+
(•)or BALB/c nude mice (A). B. growth of M109 tumor cells (1 x 106) inoculated i.f.p. in
control BALB/c
mice (LTDor in (•)BALB/c
mice bearing s.c. M109 tumor a2 cm (10 mice/group).
Bars, S.E.
100.000
10,000
1000
3.0 -
8
11
14
17
20
23
DAYS AFTER TUMOR CELL INOCULATION
2
4
6
8
10
12
DAYS AFTER INOCULATION OF TUMOR
CELLS
Chart 7. Resistance of tumor-bearing mice to second tumor challenge. Con
trol BALB/c mice (O) or BALB/c mice bearing s.c. Meth A tumors 21 (•),22
(A), or >3 cm (•)were inoculated with 0.8 x 10s radiolabeled Meth A tumor
Chart 6. Elimination of radiolabeled Meth A tumor cells from footpad of control
or tumor-bearing mice. Control BALB/c mice (O) or BALB/c mice bearing s.c.
Meth A tumors a! (•),>:2 (A), or z3 cm O were inoculated i.f.p. with 8 x 106
cells (8 to 10 mice/group),
radiolabeled Meth A tumor cells. At various periods after tumor cell inoculation,
the level of radioactivity remaining i.f.p. was measured (8 to 10 mice/group).
Bars, S.E.
the foot. Tumors developed in the 3 surviving mice, although
tumor size was smaller than in control mice (Chart 7). In mice
that had s.c. tumors >1 cm at the time of i.f.p. reinoculation
with Meth A cells, the appearance and growth of tumors were
similar to those in control mice. On Day 12 after reinoculation
of tumor cells, the first s.c. tumors increased in size and after
Day 12 growth retardation of i.f.p. tumors was observed. On
Day 19 after reinoculation, substantial differences in the size
of i.f.p. tumors were found in these and in control mice. In
general, suppression of growth of reinoculated tumor cells in
mice bearing small s.c. tumors was less profound than in mice
bearing large s.c. tumors (Chart 7).
after reinoculation of Meth A cells i.f.p., visible tumors devel
oped in 3 of 9 surviving mice. All mice had symptoms of
cachexia. However, tumor-bearing mice in which growth of the
second tumor graft i.f.p. was completely prevented had more
profound symptoms of cachexia and loss of activity than mice
in which some growth of i.f.p. tumor was observed; 15 to 19
days after i.f.p. reinoculation of radiolabeled Meth A cells, 6 of
the 9 mice were dead. None of them had established tumors in
142
ears, S.E.
CANCER
RESEARCH
VOL. 43
Mechanisms of Concomitant
Resistance of tumor-excised
Table 2
and tumor-bearing
mice to the second tumor
challenge
CharacteristicsAbility
thegrowth
to inhibit
secondtumor
of a
graftImmunogenicity
ofreinoculated
tumorcellsAntigen
oftumorspecificity
cellsHostTumor-excised
miceWeakOnly
miceStrongImmunogenic
andnonimmunogenicCan
immunogenicSpecificImmunocompetentTumor-bearing
nonspecificCan
be
immunosuppressedor
be
immunodeficient
DISCUSSION
Resistance of mice to a second tumor graft in the presence
of the growing primary tumor or after its excision is thought to
be mediated by immunological mechanisms, concomitant and
sinecomitant immunity, respectively (7). However, these types
of immunity have different characteristics (Table 2). In general,
sinecomitant immunity was observed when the removed tumors
were relatively small and the number of cells used for the
second challenge was very small. Larger numbers of reinocu
lated tumor cells can overcome the antitumor resistance of
these animals. Antitumor resistance was observed when tumor
reinoculation was performed 7 to 10 days after removal of the
first tumor. This period was probably required for the restora
tion of immune functions suppressed by the growing tumor
(17-19, 22). In the present study as well as in the previous
study (15), we have found that tumor-bearing mice had a high
level of resistance to the second tumor graft, even when
relatively high doses of tumor cells were reinoculated. This
resistance can be eliminated by simple excision of the first
tumor; 1 x 106 3LL tumor cells inoculated into tumor-excised
mice grew at the same rate as in the control mice, whereas
their growth was inhibited in the presence of the first tumor.
Similar results were obtained by DeWys (4) and Kerney and
Nelson (17).
Antitumor resistance may disappear after tumor excision
because the continuing presence of antigenic material is nec
essary to maintain antitumor immunity (8). However, excision
of tumor does not mean that all antigenic material was removed,
because 3LL tumor is a metastasizing tumor and at the time of
excision metastatic cells have spread and settled in the lungs
(13,14). In addition, 1 x 1063LL tumor cells were reinoculated
i.f.p. These data may support the assumption that the inhibition
of growth of the second graft was mostly mediated by the first
tumor mass, with little or no involvement of the host immune
system. Furthermore, antitumor sinecomitant immunity has
been observed in immunologically competent hosts after exci
sion of immunogenic tumors (21, 23). Resistance to a second
tumor challenge in tumor-bearing mice was observed against
both immunogenic and nonimmunogenic tumors. Thus, strong
inhibition was found when weakly immunogenic 3LL, B16, or
M109 tumor cells were reinoculated into tumor-bearing mice.
In addition, this antitumor resistance of tumor-bearing mice is
not tumor specific. Mice bearing a 3LL tumor had inhibited
growth of reinoculated antigenic non-cross-reactive
B16 or
EL4 tumors, and mice bearing B16, EL4, or T-10 tumors had
arrested growth of reinoculated 3LL tumor cells (15). The
nonspecific character of the inhibition of rechallenge tumor
cells has been observed by several investigators (17, 20).
JANUARY 1983
Tumor Immunity
Inhibition of the growth of the second tumor graft was ob
served in the immunologically deficient mice. The growth of
reinoculated tumor cells was equally inhibited in tumor-bearing
B-mice and nude mice as well as immunologically competent
mice (15). These results may indicate that the observed antitumor resistance of tumor-bearing mice is not mediated by Tcells. As an alternative, NK cells and macrophages could be
considered as possible effector cells that participate in the
rejection of the second tumor graft. This seems unlikely, how
ever, because it has been shown in many experiments that, at
the latest stages of tumor growth, the functions of the immune
system, including those of NK cells and macrophages, are
suppressed (9, 16, 19, 22). The present study supports the
findings that NK cell function, which was tested in vitro and in
vivo, was strongly suppressed in mice bearing 3LL tumors 2 to
3 cm in diameter. The growth of reinoculated 3LL tumor cells
was strongly suppressed in silica-treated or in NK-deficient
beige mice bearing s.c. 3LL tumors, indicating that NK cells
and macrophages cannot be considered to be effector cells
responsible for the rejection of tumor cells reinoculated into
tumor-bearing mice.
Radioisotope technique showed that radiolabeled, NK-resistant M109 tumor cells reinoculated into tumor-bearing mice
were not eliminated but survived at the inoculation site to the
same degree as in non-tumor-bearing mice. The same results
were obtained when radiolabeled cells were inoculated into
control and tumor-bearing nude mice. It is remarkable that
survival of reinoculated, nonimmunogenic M109 tumor cells
was similar in BALB/c mice and in BALB/c nude mice. When
CBA/J and C57BL/6 mice were inoculated with allogeneic
M109 tumor cells, these cells were quickly eliminated. Al
though reinoculated M109 tumor cells survived in syngeneic
mice, their proliferation was strongly suppressed in BALB/c
mice and in nude mice in the presence of large M109 tumors.
These results also indicate that data on survival of tumor cells
are not sufficient to predict the development and growth rate
of subsequent tumors. Cytostatic mechanisms may suppress
tumor cell proliferation and keep these cells in the dormant
state or inhibit their growth. By inoculation of radiolabeled
immunogenic Meth A tumor cells into tumor-bearing mice, it
was found that their elimination was greater than in control
mice. These differences in the elimination of reinoculated Meth
A tumor cells can be explained by the participation of immune
reaction developed by the host during growth of the first Meth
A tumor. This reaction was not sufficient to destroy all rein
oculated cells as it was observed in allogeneic CBA/J mice.
The levels of survival of reinoculated Meth A tumor cells were
similar in mice bearing small and large tumors. However, com
plete prevention of tumor growth was observed only in mice
bearing large tumors. In mice bearing small tumors, growth of
reinoculated Meth A cells was partially suppressed.
These data give the experimental basis for the conclusion
that inhibition of the growth of the second tumor graft in tumorbearing mice is mostly mediated by nonimmunological mech
anisms. However, immune response developed during the
growth of immunogenic tumor cells may have some additional
effect on the elimination of reinoculated tumor cells and the
suppression of their growth.
In experiments performed by North and Kirstein (20), mice
bearing Sal tumor had greater resistance to the reinoculation
of identical Sal tumor than to non-cross-reactive BP3 or MC5
143
E. Gorelik
tumor cells. Lymphocytes of mice bearing Sal tumor were
specifically cytotoxic against Sal tumor cells (20). In thymectomized, lethally irradiated, and bone marrow-reconstituted
Bmice bearing Meth A or Sal tumor, resistance to a second
tumor challenge substantially decreased. However, some antitumor resistance remained; hence, the growth of a second
graft in B-mice was significantly inhibited (3, 20).
These data may support the hypothesis that immunologically
specific and nonspecific factors can be involved in the inhibition
of the second tumor graft growth. Specific inhibition became
apparent in the presence of specific immune response evoked
against immunogenic tumor cells. The hypothesis that concom
itant tumor immunity is responsible for the resistance of the
tumor-bearing host to a second tumor challenge was based on
the data obtained by Bashford ef al. (2) using outbred mice
and allogeneic tumors. This assumption was supported when
allogeneic lymphoma in hamsters and syngeneic, strongly im
munogenic tumors in mice were used (3, 7, 8, 17, 20). These
tumors induced strong immune response but grew progres
sively. However, reinoculated tumor cells can be eliminated by
developed immune reactions.
The mechanisms involved in the nonimmunological suppres
sion of growth of a second tumor in the host bearing a nonimmunogenic tumor are mostly unknown. Ehrlich (6) explained
the antitumor resistance of tumor-bearing mice by developing
the hypothesis of athreptic immunity. According to this hypoth
esis, the resistance to the second tumor graft is a result of the
depletion in the tumor-bearing host of essential nutritional
substances that are required for the growth of the second
tumor. In mice bearing large tumors, cachexia and other symp
toms of malignant disease were more profound, and inhibition
of the growth of the second tumor graft was greater. Weight
loss, which is common for a tumor-bearing organism, can be
modulated in normal mice by food restriction. Nutrient restric
tion may be accompanied by strong inhibition of the develop
ment of spontaneous or induced tumors as well as transplanted
tumors (10, 28). Since cachexia and other symptoms of malig
nant disease and resistance to the challenge of the second
nonimmunogenic tumor usually disappeared after removal of
the tumor, it is possible that the growing tumor is responsible
for these phenomena.
Numerous data indicate that increase in tumor mass in the
tumor-bearing organism takes place at the expense of the host
tissues and is accompanied by severe metabolic disorders,
impairment of enzyme function in the host tissues, and distur
bance of endocrine regulation. No tissue functions normally in
the tumor-bearing organism (26), and it appears that these
conditions are less favorable to the proliferation of reinoculated
tumor cells than are those in control animals.
It was also suggested that the tumor produces some factors
with antiproliferative activity (4, 27, 29). The nature of these
antimitotic factors is mostly unknown. Chalones, polyamines,
or other nonspecific antiproliferative factors could be consid
ered as the candidates for this role (27, 29). The concentration
of these antimitotic factors probably increases with the size of
the primary tumor mass. Thus, in the late stage of tumor
growth, there is greater suppression of the proliferation of
reinoculated tumor cells. The antiproliferative factors produced
or induced by the primary tumor may inhibit the growth of
metastatic cells that can be considered to be a spontaneous
second tumor graft; hence, the accelerated growth of metas
144
tasis is manifested following removal of the primary tumor (13,
14, 27). It is possible that production of antiproliferative sub
stances and the development of metabolic and hormone dis
orders in tumor-bearing animals could be responsible for sup
pression of the growth of reinoculated tumor cells or sponta
neous métastases.
Therefore, the resistance of tumor-bearing and tumor-ex
cised mice to the challenge of the second tumor is mediated
by different mechanisms. There is increasing evidence that the
resistance of a tumor-bearing host to the second tumor graft is
mostly a nonimmunological phenomenon. However, antitumor
immune response evoked against immunogenic tumor may
contribute to the protection of the growth of the second tumor
graft in the tumor-bearing host. Since the resistance to the
second tumor challenge in tumor-bearing mice may be due to
nonimmunological mechanisms, this experimental model can
not be used for the assessment of the antitumor immune
reactions of tumor-bearing mice.
ACKNOWLEDGMENTS
I thank Anne McCarthy for extensive technical editing assistance.
REFERENCES
1. Allison, A., Harrington, J., and Birbeck M. An examination of the cytotoxic
effect of silica on macrophages. J. Exp. Med., 124: 141-147, 1966.
2. Bashford, E., Murray, J., Haaland, M., and Bowen, W. General results of
propagation of malignant new growths. In: E. Bashford (ed.). Third Scientific
Report on the Investigations of the. Imperial Cancer Research Fund, Vol. 3,
pp. 262-268. London: Taylor and Francis, 1908.
3. Berendt, M., North, R., Kirstein, D. The ¡mmunological bases of endotoxininduced tumor regression. Requirement for a pre-existing state of concom
itant anti-tumor immunity. J. Exp. Med., 148: 1560-1569,
1978.
4. DeWys, W. Studies correlating the growth rate of a tumor and its métastases
and providing evidence for tumor-related systemic growth-retarding factors.
Cancer Res., 32: 374-379, 1972.
5. Djeu, J., Heinbaugh, J., Viera W., Holden, H., and Herberman, R. The effect
of immunopharmacological
agents on mouse natural cell-mediated cytotoxicity and on its augmentation by Poly I:C. Immunopharmacology,
Õ.231244, 1979.
6. Ehrlich, P. Experimentelle Karzinom-Studien on Mäusen. Arch. Inst. Exp.
Ther Frankfurt, 1: 65, 1906.
7. Fisher, B., Safer. E.. and Fisher, E. Comparison of concomitant and sinecomitant tumor immunity. Proc. Soc. Exp. Biol., J35: 68-71, 1970.
8. Gershon, R. Regulation of concomitant immunity. Activation of suppressor
cells by tumor excision. Isr. J. Med. Sci., TO. 1012-1023,
1974.
9. Gershon, J. Systemic and in situ natural killer activity in tumor-bearing mice
and patients with cancer. In: R. Herberman (ed.), Natural Cell-Mediated
Immunity against Tumors, pp. 1047-1062. New York: Academic Press, Inc..
1980.
10. Giovanella. P., Shepard, R., Stehlin, J., Venditti, J., and Abbott, B. Calorie
restriction: effect on growth of human tumors heterotransplanted
in nude
mice. J. Nati. Cancer Inst.. 68. 249-257. 1982.
11. Gorelik, E., and Herberman, R. Radioisotope assay for evaluation of in vivo
natural cell-mediated resistance of mice to local transplantation of tumor
cells. Int. J. Cancer, 27: 709-720. 1981.
12. Gorelik. E., Kedar, E., Sredni, B., and Herberman, R. In vivo antitumor
effects of local adoptive transfer of mouse and human cultured lymphoid
cells. Int. J. Cancer, 28. 157-164, 1981.
13. Gorelik, E., Segal. S.. and Feldman, M. Growth of a local tumor exerts a
specific inhibitory effect on progression of lung métastases. Int. J. Cancer,
21: 61 7-625, 1978.
14. Gorelik, E., Segal, S.. and Feldman. M. Control of lung metastasis progres
sion in mice: role of growth kinetics of 3LL Lewis lung carcinoma and host
immune reactivity. J. Nati. Cancer Inst.. 65. 1257-1264.
1980.
15. Gorelik, E., Segal, S., and Feldman, M. On the mechanism of tumor
"concomitant immunity." Int. J. Cancer, 27. 847-856. 1981.
16. Kano, J., and Friedman, M. Immunosuppression and the role of suppressive
factors in cancer. Adv. Cancer Res., 25. 271-322, 1977.
17. Kearney, R., and Nelson. D. Concomitant immunity to syngeneic methylcholanthrene-induced
tumors in mice. Occurrence and specificity of con
comitant immunity. Aust. J. Exp. Biol. Med. Sci.. 51: 723-735. 1973.
18. Klein. G., Sjögren, H., Klein, E., and Hellström, K. Demonstration of resist-
CANCER
RESEARCH
VOL.
43
Mechanisms of Concomitant
19.
20.
21.
22.
23.
ance against methylcholanthrene-induced
sarcomas in the primary autoch
thonous host. Cancer Res.. 20. 1561-1572,
1960.
Nelson, D., and Kearney. R. Macrophages and lymphoid tissues in mice with
tumor immunity. Br. J. Cancer. 34: 221-226, 1976.
North, R.. and Kirstein, D. T-cell-mediated concomitant immunity to syngeneic tumors. I. Activated macrophages as the expressors of nonspecific
immunity to unrelated tumors and bacterial parasites. J. Exp. Med.. 145:
275-292. 1977.
Old, L., and Boyse, E. Immunology of experimental tumors. Annu. Rev.
Med., Õ5.167-186, 1964.
Otu, A., Russell. R., Wilkinson, P., and White, R. Alteration of mononuclear
phagocyte function induced by Lewis lung carcinoma in C57BL/6 mice. Br.
J. Cancer, 36: 330-340, 1977.
Prehn. R.. and Main, J. Immunity to methylcholanthrene-induced
sarcomas.
J. Nati. Cancer Inst., 78. 769-778, 1957.
JANUARY
1983
Tumor Immunity
24. Riccardi, C., Santon, A.. Barlozzari. T., Puccetti, P., and Herberman. R. In
vivo natural reactivity of mice against tumor cells. Int. J. Cancer, 25. 475486, 1980.
25. Rogers, M., and Law, L. Some immunogeneic and biochemical properties of
tumor-associated transplantation antigens (TATA) obtained in soluble form
or solubilized from two methylcholanthrene-induced
sarcomas, Meth A and
CI-4. Int. J. Cancer, 27. 789-796, 1981.
26. Shapot V. On the multiform relationships between tumor and the host. Adv.
Cancer Res., 30. 89-150, 1979.
27. Sugarbaker, E., Thornthwaite, J., and Ketcham, A. Inhibitory effect of a
primary tumor on metastasis. Prog. Cancer Res. Ther.. 5. 227-240, 1977.
28. Tannenbaum, A., and Silverstone. H. Nutrition in relation to cancer. Adv.
Cancer Res., 1: 451-501. 1952.
29. Urushizaki, K. Humoral regulation in cell-mediated immunity of cancer pa
tients. Tumor Res.. 11: 1-18, 1976.
145