[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

ABSTRACT

Antitumor resistance of mice bearing 3LL, M109, or Meth Atumors to a second tumor challenge was studied. The degreeof inhibition of a second tumor growth in the tumor-bearingmice was a function of both the size of the primary tumor massand the number of reinoculated tumor cells.

Antitumor resistance of tumor-bearing mice is tumor nonspecific and is observed only in the presence of the growing firsttumor. Following removal of 3LL tumor, the resistance to thesecond challenge with 3LL or T-10 tumor cells completelydisappeared.

Antitumor resistance of tumor-bearing mice does not appearto be mediated by T-cells, natural killer cells, or macrophages,since similar results were observed in immunologically competent and nude mice as well as in beige mice and in mice withfunctions of macrophages and natural killer cells suppressedby silica treatment. Growth inhibition of the reimplanted tumorcells does not appear to be a result of their elimination. Radio-labeled M109 tumor cells survived to the same degree at thelocal site of inoculation in tumor-bearing and non-tumor-bearing control mice. The number of surviving radiolabeled tumorcells inoculated into the footpad of ¡mmunocompetentandnude mice was similar and did not depend on the presence orabsence of growing first tumor. Despite the similarity in thesurvival of reinoculated tumor cells, their growth was inhibitedonly in the tumor-bearing mice. In mice bearing ¡mmunogenicMeth A tumors of different sizes (1 to 3 cm), the clearance ofthe reinoculated Meth A cells was significantly higher than innon-tumor-bearing mice. The number of surviving tumor cellswas similar in mice bearing small (<1 cm) or large (>3 cm)tumors. However, complete prevention of the second tumorgrowth was observed in mice bearing huge tumor mass (^3cm in diameter). These data may indicate that resistance ofmice bearing immunogenic and nonimmunogenic tumors ismediated by different mechanisms. The resistance to a secondtumor challenge in mice bearing nonimmunogenic tumor is duemainly to nonimmunological mechanisms. In contrast, the an-titumor resistance in mice bearing immunogenic tumor is afunction of antitumor immune reactions, although additionaleffects of nonimmunological mechanisms were also observed.

INTRODUCTION

The inhibition of growth of a second tumor graft in micebearing the original tumor was described by Ehrlich (6) in1906. This phenomenon was later attributed to the antitumorimmunological response and termed concomitant tumor immunity (2). Although the antitumor resistance of tumor-bearing

mice to a second challenge is rather strong, even at high dosesof tumor cell inocula, this experimental model did not attract

much attention. It required about 50 years to prove that thespecific immune response can be evoked during tumor growth,resulting in increased resistane - to the second tumor challenge(23). In this experimental model, the antitumor resistance ofthe host was analyzed after removal of the first tumor graft.The antitumor resistance in the animals following excision ofthe growing tumor became a favorite model for measuring theantitumor immune response, immunogenicity, and the antigenspecificity of the investigated tumors (18, 21, 23). In 1970,Fisher ef al. (7) proposed to distinguish concomitant immunity(resistance to a second tumor graft in the presence of the firstgrowing tumor) from sinecomitant immunity (resistance of tumor-excised animals to a second challenge). In many respects,

the resistance to a second tumor challenge shown by thetumor-excised host has different characteristics from the resistance observed in the tumor-bearing host.

In previous studies (15), we have found that the resistanceof mice to a second tumor graft in the presence of the growingtumor can be very strong and may completely prevent thegrowth of high doses of the reinoculated tumor cells. Thisresistance was a function of the volume of the primary tumormass and was not tumor specific. Indeed, mice bearing 3LLtumors were resistant to the reinoculation of 3LL, B16, or EL4tumors. Similarly, the growth of 3LL tumor cells was arrestedwhen they were reinoculated into mice bearing B16, EL4, or T-10 tumors. It appears that this resistance is not mediated via T-cell-mediated immunity because in T-cell-depleted animals (B-

mice or nude mice) bearing the first tumor graft, the inhibitoryeffect on the subsequently implanted tumor cells was as effective as in normal tumor-bearing mice (15).

The tumor-nonspecific character of the resistance to a second tumor challenge and its independence from T-cell-medi-ated immunity suggest that either NK2 cells or macrophages

may be responsible for the inhibition of a second tumor growth.In the present study, the possible involvement of NK cells andmacrophages in the resistance of the tumor-bearing host to a

second tumor challenge was investigated. Inhibition of tumorgrowth can result from elimination of the tumor cells or prevention of their proliferation. To analyze the survival and growth oftumor cells reinoculated into normal or tumor-bearing mice, we

used a new radioisotope technique that makes it possible todetermine 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", andBALB/c-H-2a mice, 2 to 3 months old, were obtained from The JacksonLaboratory, Bar Harbor, Maine. (C3H/eB x C57BL/6) (H-2VH-2") F,

mice were supplied by the Animal Breeding Center of the WeizmannInstitute of Science, Rehovot, Israel.

Tumors. The following tumors were used: Lewis lung carcinoma1Present address: Surgery Branch, National Cancer Institute, NIH, Bethesda,

Md. 20205.Received March 24, 1982: accepted October 11,1982.

2The abbreviations used are: NK, natural killer; dUrd, deoxyuridine: i.f.p., intothe footpad.

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Mechanisms of Concomitant Tumor Immunity

(3LL) that arose spontaneously in C57BL/6 mice; Meth A fibrosarcomainduced by methylcholanthrene in BALB/c mice; Madison lung carcinoma (M109) spontaneously developed in BALB/c mice; YAC-1, atissue culture cell line of YAC, a Moloney virus-induced lymphoma of

A/J origin. These tumor cells were maintained in vitro in completeRoswell 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 byserial transfers in these mice.

Tumor Cell Survival in Vivo. Nonconfluent monolayers of cultivatedM109, Meth A cells, or nonsaturated cultures of YAC-1 lymphoma cellswere 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 incomplete Roswell Park Memorial Institute Medium 1640. Radiolabeledtumor 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 thenat various times following tumor cell inoculation. The level of radioactivity in the inoculated leg was measured with a low-energy y-scintillation counter (Ludlum model 2200; Ludlum Measurements Inc., Sweet-water, Texas) adjusted to detect y-rays between 10 and 40 keV, usinga detector with a 1-mm-thick, 1-inch-diameter Nal (T1 ) crystal with a 7mg per sq cm aluminum window. The detector was shielded with 2-mm-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 measuredduring 20 sec. Mice were marked, making it possible to follow the rateof clearance of radioactivity in individual mice. Vernier calipers wereused to measure the diameter of i.f.p. established tumors at variousperiods of growth.

In Vitro Cytotoxic Activity of Spleen Cells. Cytotoxic effect ofspleen cells of normal or tumor-bearing mice was assessed in a 4-hrassay 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 theCytotoxic activity of their spleen cells was tested.

RESULTS

In the first series of experiments, the growth of a secondtumor 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 tumor-

bearing mice. Relatively small doses of reinoculated tumor cellscan have a long latent period, and tumor-bearing mice can die

before the tumors appear even in the control mice. High dosesof reinoculated tumor cells may overcome the antitumor resistance 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 usuallykills syngeneic C57BL/6 mice. Thus, FI mice were chosen forthese experiments. It was found previously that mice bearings.c. tumors larger than 1 cm in diameter had apparent resistance 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 dividedinto 2 groups. In the first group, tumor-bearing legs wereamputated; in the second group, tumors were allowed to grow.Tumor-bearing and tumor-excised mice were reinoculated inthe 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 thesame doses of tumor cells.

Most of the mice bearing the first tumor survived at least 8days after tumor reinoculation, which allowed comparison of

the tumor growth of reimplanted tumor cells in the presenceand absence of the first tumor graft. Chart 1 shows the tumordiameter in the reinoculated leg. The diameters of 3LL tumorsfrom reinoculated cells were similar in the tumor-excised and

control mice whereas in mice bearing the first 3LL tumor thegrowth of the second 3LL tumor was substantially suppressed.

This inhibition was not tumor specific because growth ofreinoculated T-10 tumor cells was inhibited in the presence ofgrowing 3LL tumor. In contrast, there was no inhibition of T-10

tumor growth in mice that had had excision of 3LL tumor (Chart1). Previous experiments (15) have demonstrated that growthof the reinoculated tumor cells was effectively prevented in thetumor-bearing T-cell-deficient animals (B-mice or nude mice).Antitumor resistance of tumor-bearing mice with either intactor deficient T-cell-mediated immunity could result from thecytotoxic action of NK cells or macrophages, which havecytotoxic action that is not tumor specific.

This assumption was analyzed using mice in which the functions of NK cells and macrophages were suppressed (C57BL/6 mice treated with silica; beige mice). Beige mice are characterized by a low level of NK cell activity, and silica treatmentsuppresses the functions of both NK cells and macrophages(1, 5). C57BL/6 and beige mice were inoculated s.c. with 1x 106 3LL tumor cells. When tumors reached 2 cm in diameter,

some of the tumor-bearing C57BL/6 mice and some of thecontrol 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 ofmice bearing s.c. tumors, the growth of the second tumor graftwas strongly inhibited (Chart 2). Peritoneal macrophages ofnormal or silica-treated tumor-bearing mice had no cytotoxic

activity against tumor cells assessed by in vitro technique (datanot shown).

The activity of NK cells in the tumor-bearing mice was testedin vitro using a 4-hr assay of 51Cr release (Table 1). In a series

of experiments, the cytotoxic effect of spleen cells of intactC57BL/6 mice at an effectortarget ratio of 100:1 varied from12 to 19%, whereas spleen cells of mice bearing 3LL tumor>2 cm in diameter had lower cytotoxic activity which neverexceeded 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

Eè 8.0-fi'

tl 6.0

o5

2.0

•—•Im*c1 Me* + 3LL•-•3U.-EnM t 31L*—•3U. taring t 3LL

4.0

246

DAYS OF TUMOR GROWTH

S'S 3.0

2.0468

DAYS OF TUMOR GROWTH

Chart 1. Growth of reinoculated 3LL or T-10 tumor cells in tumor-bearing ortumor-excised mice. F1 (C3H/eB x C57BL/6) mice bearing i.f.p. 3LL tumor (10to 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.

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9.0 r-

8.0

7.0

Joc

6.0

i!"o

?4,

3.0

7 8 9 10 11 12

DAYS AFTER TUMOR CELL REINOCULATION

13

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 mgi.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-treatedC57BL/6-+/+ (O), or beige O mice were inoculated i.f.p. with 1 x 106 3LL

cells (10 mice/group). Bars, S.E.

inoculated i.v. from the lungs (24) or from the inoculation site(11). The ability of normal or tumor-bearing C57BL/6 mice toeliminate 125l-dUrd-labeled YAC-1 cells from the footpad wasstudied; 0.5 x 106 radiolabeled YAC-1 cells were inoculated

i.f.p. in control C57BL/6 mice or mice bearing s.c. 3LL tumorsof different sizes (<1, >2, or >3 cm). The level of radioactivityremaining in the leg was determined.

In C57BL/6 mice bearing 3LL tumors >3 cm, the eliminationof tumor cells from the inoculation site was substantially suppressed (Chart 3). This suppression was less in mice bearingsmaller 3LL tumors (2 to 3 cm in diameter). In mice bearing thesmall s.c. 3LL tumors (<1 cm), the clearance of reinoculatedYAC-1 tumor cells was significantly greater than in other tumor-bearing mice and was similar to the clearance in non-tumor-

bearing control mice. These experiments indicate that cytotoxicactivity of NK cells tested in vivo and in vitro against NK-susceptible tumor cells is impaired in tumor-bearing mice.

Survival and growth of reinoculated radiolabeled tumor cellscan be a useful method for understanding the mechanism ofthe tumor growth suppression in tumor-bearing mice. It is still

unclear whether the growth inhibition of the second tumor graftresults from the elimination of the reinoculated tumor cells orfrom the suppression of their proliferation. To make this determination, BALB/c mice and BALB/c nude mice were inoculated 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 2x 10s 12Sl-dUrd-labeled M109 tumor cells. Control BALB/c

mice, BALB/c nude mice, and allogeneic C57BL/6 and CBA/J mice received the same number of radiolabeled M109 tumorcells. Previously, we have shown that M109 tumor cells resistant to the cytotoxic action of NK cells in vitro and their survival

in vivo were similar in mice with low and high NK cell activity.Tumor growth was observed in syngeneic BALB/C+/+ andBALB/c nude mice inoculated with radiolabeled M109 tumorcells (11, 12).

The levels of radioactivity immediately after the inoculationreflect the real number of injected tumor cells (Chart 4A).Measurement of the radioactivity in the foot pads at differenttimes after tumor cell inoculation indicates the level of tumorcell survival. One day after tumor cell inoculation, a decreasein radioactivity was observed in all mice, but later the rate ofelimination of radioactivity from the footpad decreased. Thelevels of radioactivity detected in normal and tumor-bearing

BALB/c mice were similar. In addition, no differences werefound in the clearance of radioactivity in BALB/c—1-/+ andnude mice bearing s.c. tumors and in non-tumor-bearing mice.

The absolute levels of radioactivity in the footpads of nudecontrol mice were lower than in other mice, but this was aresult of the initial lower number of radiolabeled cells inoculatedi.f.p. in these mice (Chart 4/4). In general, the slope of theelimination of radioactivity in nude mice was identical to theslope for other BALB/c mice.

The similarity in the levels of radioactivity remaining in the

Table 1NK 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. 15mg of silica. Three days later, cytotoxic activity of spleen cells of treated andnontreated 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

MiceIntact

Silica-treated non-tumor-bearing

Tumor-bearingSilica-treated tumor-bearing100:1"15.7

±0.8E'C

3.4 ±0.16.2

±0.33.9 ±0.133:110.3

±0.53.0 ±0.15.4

±0.23.1 ±0.111:16.4

±0.22.9 ±0.13.1

±0.12.9 ±0.1

Effectortarget ratio.6 Mean ±S.E.c Significantly different (p < 0.01) from other groups.

100,000

10,000

1000

10016 24 40 48

HOURS AFTER YAC-1 CELL INOCULATION

Chart 3. Elimination of radiolabeled YAC-1 cells from footpad of control miceor mice bearing s.c. 3LL tumor of different sizes. Control C57BL/6 mice or micebearing s.c. 3LL tumors («1, »2. or »3cm 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 (5 mice/group). Bars. S.E.

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»Vi-v

Mechanisms of Concomitant Tumor Immunity

10,000r

1000 -

100,000

10.000

~ 1000

24 6 8 10 12 14DAYS AFTER INOCULATION RADIOLABELED

TUMOR CELLS

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 ofradiolabeled 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 firsttumor 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 cytotoxicagents were not active in all mice investigated or that they wereequally efficient in tumor-bearing and non-tumor-bearing miceand in immunocompetent and T-cell-depleted nude mice. The

potential efficiency of immune reaction in the elimination ofinoculated radiolabeled M1 09 tumor cells was indicated by theelimination of these cells in allogeneic C57BL/6 and CBA/Jmice. Seven days after inoculation of radiolabeled cells, theslope of radioactivity in allogeneic and syngeneic mice wasidentical (Chart 4A). Later, however, the rate of clearance oftumor cells rapidly increased in the allogeneic mice and, 13days after tumor cells were injected, all radioactivity in theallogeneic mice had completely disappeared (Chart 4A). Therate of clearance of M109 tumor cells was slightly higher inC57BL/6 mice than in CBA/J mice. Although the levels ofradioactivity were similar in the footpads of BALB/C-+/+ andnude 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 reinoculatedM109 tumor cells was strongly suppressed in mice bearing s.c.M109 tumor. In the presence of the first tumor, this suppressionwas even more profound in nude mice than in BALB/C-+/ +

mice (Chart 5A ). Nude mice bearing s.c. M109 tumor appearedto be very ill. They lost weight and were pale and less activethan nude mice that had no s.c. tumors. Similar results wereobtained when, in parallel, BALB/c mice bearing s.c. M109tumors (>2 cm in diameter) were reinoculated i.f.p. with ahigher 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). Fourteendays after reinoculation of tumor cells, a high level of radioac

tivity remained at the site of transplantation, although a slightdecrease in radioactivity was found in BALB/c mice not bearings.c. tumors. At this period, these mice developed i.f.p. tumorsthat were >0.5 cm in diameter, which made it difficult tomeasure the level of radioactivity in the leg. In allogeneic CBA/J mice inoculated with radiolabeled M9 tumor cells, the tumorcells were completely eliminated (Chart 46). In contrast to thesimilarity in the survival of reinoculated M109 cells in tumor-bearing and control BALB/c mice, there was a striking difference in the growth of reinoculated tumor cells (Chart 5B). Inthe presence of s.c. M109 tumor, the proliferation of thereinoculated M109 tumor was strongly suppressed.

All of these experiments were performed using weakly ¡m-

munogenic spontaneous 3LL and M109 tumors. To assess thecontribution of the immunological mechanisms in the elimination of reinoculated tumor cells and in the suppression of theirgrowth in tumor-bearing mice, the immunogenic methylchol-anthrene-induced Meth A tumor was used. The antigenic andimmunogenic properties of Meth A tumor have been reportedin numerous investigations (3, 21, 25). BALB/c mice wereinoculated 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 diameterwere inoculated i.f.p. with 0.8 x 106 125l-dUrd-labeled Meth A

tumor cells. The level of radioactivity at the inoculation siteprogressively decreased in all mice. However, mice bearings.c. Meth A tumors had more efficient clearance of radioactivetumor 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 ofall tumor-bearing mice were similar, large differences were

observed in the growth rates of reinoculated tumor cells (Chart7). In mice bearing tumors >3 cm, growth of i.f.p. reinoculatedtumor cells was completely suppressed. There was substantialinhibition in the development of second-footpad tumors in micebearing first s.c. tumors >2 cm in diameter. Fourteen days

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11.0

10.0

9.0

8.0

O 6.0

g¡,0

4.0 -

3.0 -

B6 10 14 18 22 26 30

DAYS AFTER i.f.p. INOCULATION OF DAYS AFTER TUMOR CELL INOCULATION

TUMOR CELLSChart 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

2 4 6 8 10 12

DAYS AFTER INOCULATION OF TUMORCELLS

Chart 6. Elimination of radiolabeled Meth A tumor cells from footpad of controlor 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

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.

after reinoculation of Meth A cells i.f.p., visible tumors developed in 3 of 9 surviving mice. All mice had symptoms ofcachexia. However, tumor-bearing mice in which growth of the

second tumor graft i.f.p. was completely prevented had moreprofound symptoms of cachexia and loss of activity than micein which some growth of i.f.p. tumor was observed; 15 to 19days after i.f.p. reinoculation of radiolabeled Meth A cells, 6 ofthe 9 mice were dead. None of them had established tumors in

3.0 -

8 11 14 17 20 23

DAYS AFTER TUMOR CELL INOCULATION

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

cells (8 to 10 mice/group), ears, S.E.

the foot. Tumors developed in the 3 surviving mice, althoughtumor size was smaller than in control mice (Chart 7). In micethat had s.c. tumors >1 cm at the time of i.f.p. reinoculationwith Meth A cells, the appearance and growth of tumors weresimilar to those in control mice. On Day 12 after reinoculationof tumor cells, the first s.c. tumors increased in size and afterDay 12 growth retardation of i.f.p. tumors was observed. OnDay 19 after reinoculation, substantial differences in the sizeof i.f.p. tumors were found in these and in control mice. Ingeneral, suppression of growth of reinoculated tumor cells inmice bearing small s.c. tumors was less profound than in micebearing large s.c. tumors (Chart 7).

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Table 2Resistance of tumor-excised and tumor-bearing mice to the second tumor

challenge

CharacteristicsAbility

to inhibitthegrowthof asecondtumor

graftImmunogenicityofreinoculated

tumorcellsAntigen

specificityoftumorcellsHostTumor-excised

miceWeakOnly

immunogenicSpecificImmunocompetentTumor-bearing

miceStrongImmunogenic

andnonimmunogenicCan

benonspecificCan

beimmunosuppressedorimmunodeficient

DISCUSSION

Resistance of mice to a second tumor graft in the presenceof the growing primary tumor or after its excision is thought tobe mediated by immunological mechanisms, concomitant andsinecomitant immunity, respectively (7). However, these typesof immunity have different characteristics (Table 2). In general,sinecomitant immunity was observed when the removed tumorswere relatively small and the number of cells used for thesecond challenge was very small. Larger numbers of reinoculated tumor cells can overcome the antitumor resistance ofthese animals. Antitumor resistance was observed when tumorreinoculation was performed 7 to 10 days after removal of thefirst tumor. This period was probably required for the restoration of immune functions suppressed by the growing tumor(17-19, 22). In the present study as well as in the previousstudy (15), we have found that tumor-bearing mice had a high

level of resistance to the second tumor graft, even whenrelatively high doses of tumor cells were reinoculated. Thisresistance can be eliminated by simple excision of the firsttumor; 1 x 106 3LL tumor cells inoculated into tumor-excised

mice grew at the same rate as in the control mice, whereastheir growth was inhibited in the presence of the first tumor.Similar results were obtained by DeWys (4) and Kerney andNelson (17).

Antitumor resistance may disappear after tumor excisionbecause the continuing presence of antigenic material is necessary to maintain antitumor immunity (8). However, excisionof tumor does not mean that all antigenic material was removed,because 3LL tumor is a metastasizing tumor and at the time ofexcision 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 inhibitionof growth of the second graft was mostly mediated by the firsttumor mass, with little or no involvement of the host immunesystem. Furthermore, antitumor sinecomitant immunity hasbeen observed in immunologically competent hosts after excision of immunogenic tumors (21, 23). Resistance to a secondtumor challenge in tumor-bearing mice was observed against

both immunogenic and nonimmunogenic tumors. Thus, stronginhibition was found when weakly immunogenic 3LL, B16, orM109 tumor cells were reinoculated into tumor-bearing mice.In addition, this antitumor resistance of tumor-bearing mice isnot tumor specific. Mice bearing a 3LL tumor had inhibitedgrowth of reinoculated antigenic non-cross-reactive B16 orEL4 tumors, and mice bearing B16, EL4, or T-10 tumors had

arrested growth of reinoculated 3LL tumor cells (15). Thenonspecific character of the inhibition of rechallenge tumorcells has been observed by several investigators (17, 20).

Mechanisms of Concomitant Tumor Immunity

Inhibition of the growth of the second tumor graft was observed in the immunologically deficient mice. The growth ofreinoculated tumor cells was equally inhibited in tumor-bearingB-mice and nude mice as well as immunologically competentmice (15). These results may indicate that the observed anti-tumor resistance of tumor-bearing mice is not mediated by T-

cells. As an alternative, NK cells and macrophages could beconsidered as possible effector cells that participate in therejection of the second tumor graft. This seems unlikely, however, because it has been shown in many experiments that, atthe latest stages of tumor growth, the functions of the immunesystem, including those of NK cells and macrophages, aresuppressed (9, 16, 19, 22). The present study supports thefindings that NK cell function, which was tested in vitro and invivo, was strongly suppressed in mice bearing 3LL tumors 2 to3 cm in diameter. The growth of reinoculated 3LL tumor cellswas strongly suppressed in silica-treated or in NK-deficient

beige mice bearing s.c. 3LL tumors, indicating that NK cellsand macrophages cannot be considered to be effector cellsresponsible for the rejection of tumor cells reinoculated intotumor-bearing mice.

Radioisotope technique showed that radiolabeled, NK-re-sistant M109 tumor cells reinoculated into tumor-bearing mice

were not eliminated but survived at the inoculation site to thesame degree as in non-tumor-bearing mice. The same results

were obtained when radiolabeled cells were inoculated intocontrol and tumor-bearing nude mice. It is remarkable that

survival of reinoculated, nonimmunogenic M109 tumor cellswas similar in BALB/c mice and in BALB/c nude mice. WhenCBA/J and C57BL/6 mice were inoculated with allogeneicM109 tumor cells, these cells were quickly eliminated. Although reinoculated M109 tumor cells survived in syngeneicmice, their proliferation was strongly suppressed in BALB/cmice and in nude mice in the presence of large M109 tumors.These results also indicate that data on survival of tumor cellsare not sufficient to predict the development and growth rateof subsequent tumors. Cytostatic mechanisms may suppresstumor cell proliferation and keep these cells in the dormantstate or inhibit their growth. By inoculation of radiolabeledimmunogenic Meth A tumor cells into tumor-bearing mice, itwas found that their elimination was greater than in controlmice. These differences in the elimination of reinoculated MethA tumor cells can be explained by the participation of immunereaction developed by the host during growth of the first MethA tumor. This reaction was not sufficient to destroy all reinoculated cells as it was observed in allogeneic CBA/J mice.The levels of survival of reinoculated Meth A tumor cells weresimilar in mice bearing small and large tumors. However, complete prevention of tumor growth was observed only in micebearing large tumors. In mice bearing small tumors, growth ofreinoculated Meth A cells was partially suppressed.

These data give the experimental basis for the conclusionthat inhibition of the growth of the second tumor graft in tumor-bearing mice is mostly mediated by nonimmunological mechanisms. However, immune response developed during thegrowth of immunogenic tumor cells may have some additionaleffect on the elimination of reinoculated tumor cells and thesuppression of their growth.

In experiments performed by North and Kirstein (20), micebearing Sal tumor had greater resistance to the reinoculationof identical Sal tumor than to non-cross-reactive BP3 or MC5

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E. Gorelik

tumor cells. Lymphocytes of mice bearing Sal tumor werespecifically cytotoxic against Sal tumor cells (20). In thymec-tomized, lethally irradiated, and bone marrow-reconstituted B-

mice bearing Meth A or Sal tumor, resistance to a secondtumor challenge substantially decreased. However, some an-titumor resistance remained; hence, the growth of a secondgraft in B-mice was significantly inhibited (3, 20).

These data may support the hypothesis that immunologicallyspecific and nonspecific factors can be involved in the inhibitionof the second tumor graft growth. Specific inhibition becameapparent in the presence of specific immune response evokedagainst immunogenic tumor cells. The hypothesis that concomitant tumor immunity is responsible for the resistance of thetumor-bearing host to a second tumor challenge was based on

the data obtained by Bashford ef al. (2) using outbred miceand allogeneic tumors. This assumption was supported whenallogeneic lymphoma in hamsters and syngeneic, strongly immunogenic tumors in mice were used (3, 7, 8, 17, 20). Thesetumors induced strong immune response but grew progressively. However, reinoculated tumor cells can be eliminated bydeveloped immune reactions.

The mechanisms involved in the nonimmunological suppression of growth of a second tumor in the host bearing a nonim-

munogenic tumor are mostly unknown. Ehrlich (6) explainedthe antitumor resistance of tumor-bearing mice by developing

the hypothesis of athreptic immunity. According to this hypothesis, the resistance to the second tumor graft is a result of thedepletion in the tumor-bearing host of essential nutritionalsubstances that are required for the growth of the secondtumor. In mice bearing large tumors, cachexia and other symptoms of malignant disease were more profound, and inhibitionof the growth of the second tumor graft was greater. Weightloss, which is common for a tumor-bearing organism, can be

modulated in normal mice by food restriction. Nutrient restriction may be accompanied by strong inhibition of the development of spontaneous or induced tumors as well as transplantedtumors (10, 28). Since cachexia and other symptoms of malignant disease and resistance to the challenge of the secondnonimmunogenic tumor usually disappeared after removal ofthe tumor, it is possible that the growing tumor is responsiblefor these phenomena.

Numerous data indicate that increase in tumor mass in thetumor-bearing organism takes place at the expense of the hosttissues and is accompanied by severe metabolic disorders,impairment of enzyme function in the host tissues, and disturbance of endocrine regulation. No tissue functions normally inthe tumor-bearing organism (26), and it appears that theseconditions are less favorable to the proliferation of reinoculatedtumor cells than are those in control animals.

It was also suggested that the tumor produces some factorswith antiproliferative activity (4, 27, 29). The nature of theseantimitotic factors is mostly unknown. Chalones, polyamines,or other nonspecific antiproliferative factors could be considered as the candidates for this role (27, 29). The concentrationof these antimitotic factors probably increases with the size ofthe primary tumor mass. Thus, in the late stage of tumorgrowth, there is greater suppression of the proliferation ofreinoculated tumor cells. The antiproliferative factors producedor induced by the primary tumor may inhibit the growth ofmetastatic cells that can be considered to be a spontaneoussecond tumor graft; hence, the accelerated growth of metas

tasis is manifested following removal of the primary tumor (13,14, 27). It is possible that production of antiproliferative substances and the development of metabolic and hormone disorders in tumor-bearing animals could be responsible for sup

pression of the growth of reinoculated tumor cells or spontaneous métastases.

Therefore, the resistance of tumor-bearing and tumor-excised mice to the challenge of the second tumor is mediatedby different mechanisms. There is increasing evidence that theresistance of a tumor-bearing host to the second tumor graft is

mostly a nonimmunological phenomenon. However, antitumorimmune response evoked against immunogenic tumor maycontribute to the protection of the growth of the second tumorgraft in the tumor-bearing host. Since the resistance to thesecond tumor challenge in tumor-bearing mice may be due tononimmunological mechanisms, this experimental model cannot be used for the assessment of the antitumor immunereactions of tumor-bearing mice.

ACKNOWLEDGMENTS

I thank Anne McCarthy for extensive technical editing assistance.

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1983;43:138-145. Cancer Res   Elieser Gorelik  ChallengeResistance of Tumor-bearing Mice to a Second Tumor

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