[CANCER RESEARCH 46, 6111-6115, December 1986]

Effects of Concomitant and Sinecomitant Immunity on Postsurgical Metastasis inMice1

Shinhachiro Nomi, Kazuyo Naito, Barry D. Kahan, and Neal R. PellisDepartment of Surgery, Division of Immunology and Organ Transplantation, The University of Texas Medical School at Houston, Houston, Texas 77030

ABSTRACT

The role of concomitant and sinecomitant ant ¡tumorresistance in theregulation of metastatic outgrowth was assessed using methylcholan-threne (MCA)-induced tumors in C3H/HeJ mice. Variants of neoplasmsMCA-F, MCA-D, and MCA-2A were selected for proclivity for spontaneous lung metastasis and expression of parental tumor-specific transplantation antigens. The incidence of spontaneous lung métastasesafterresection of a s.c. tumor of clone 9-4, a highly metastatic variant of theMCA-F tumor, was determined by both the size and the duration ofneoplastic disease. The coexistence of the primary local tumor retardedlung colonization both from spontaneous and after artificially inducedmétastases.Greater concomitant immunity leading to a reduced numberof artificial métastasesafter i.v. challenge with clone 9-4 cells was evidentin hosts bearing large (1.6 to 1.8 cm) compared to small (0.1 to 0.2 cm)burdens of the nonmetastatic MCA-F (/' < 0.005). Furthermore, i.v.

challenge of mice bearing antigenically different tumors revealed that theconcomitant inhibition was antigen specific with small tumor burdens,but nonspecific and possibly more efficacious with large tumor burdens.Therefore, concomitant antimetastatic immunity consists of both specific,immune-mediated resistance and nonimmunological mechanisms. Specific concomitant immunity decreases inversely with the progression ofthe primary, while nonimmunological inhibition of metastasis increasesduring late stages of primary growth. Abrogation of the strong nonspecificconcomitant inhibition by resection of the primary tumor may facilitatelung metastasis. On the other hand, significantly greater inhibition ofmétastasesoccurred after resection of 7- or 14-day neoplasms comparedto larger tumors (/' < 0.001 or 0.05). Sinecomitant inhibition is antigen

specific, probably representing an extension of specific concomitant immunity. These results suggest that adjunctive immunotherapeutic protocols for surgically treated hosts should augment existent specific immunity and promote nonspecific resistance, in order to minimize metastaticoutgrowth.

INTRODUCTION

This investigation determined the intensity and specificity oftwo forms of tumor resistance: concomitant immunity displayedby hosts bearing a progressive primary tumor growth against asecond tumor challenge; and sinecomitant resistance displayedto a second tumor challenge after excision of the primary tumor(1-5). The presence of a rapidly progressive primary neoplasminhibits distant tumor growth in many murine models of metastasis. Excision of a s.c. primary tumor accelerates metastatictumor growth in the lungs of mice (2, 6-8), while reinoculationof the primary tumor cells retards metastatic outgrowth (9).Concomitant antitumor immunity is indeed effective in thelungs (10). Since metastatic lesions are akin to second tumorgrafts, we investigated the role of concomitant and sinecomitantimmunity in the control of lung colonization and metastaticoutgrowth of spontaneous métastases.

Metastatic variants displaying a proclivity for the lung wereselected from three antigenically different MCA2-induced sar-

Received 1/2/85; revised 6/20/86, 9/4/86; accepted 9/5/86.1This work is supported by American Cancer Society Grant IM62J and

USPHS Grant CA29592.2The abbreviations used are: MCA, 3-methylcholanthrene; HBSS, Hanks'

balanced salt solution; LATA, local adoptive transfer assay; TSTA, tumor-specifictransplantation antigens.

comas which have antigens capable of provoking specific hostresistance (11-15). The effect of concomitant and sinecomitantimmunity, respectively, to abate métastaseswas assessed duringtumor progression and following resection, by enumeratinglung colonies following spontaneous or artificial (i.v. challenge)métastases.This study suggests that, in addition to ¡mimmo-logical resistance to metastatic outgrowth, there may be nonimmunological growth-controlling mechanisms which functiononly in hosts with large tumor burdens. Because of the antigenspecificity of sinecomitant tumor immunity, it is essential thatpostsurgical therapy augment both specific and nonspecificimmune mechanisms.

MATERIALS AND METHODS

Animals and Tumors. Three non-cross-reactive sarcomas (MCA-F,MCA-D, and MCA-2A) were induced in female inbred C3H/HeJ mice(Jackson Laboratories, Bar Harbor, ME) by s.c. injection of 3-methylcholanthrene and maintained by serial s.c. propagation in 5- to 7-wk-old female C3H/HeJ mice, as previously described (12).

Selection of Sublines. Sublines were obtained from a sixth in vivopassaged generation of MCA-F, MCA-2A, and MCA-D by a modification of the method of MacPherson (16). For example, after six invivo passages, the MCA-F tumor was cultured to confluency anddissociated by treatment with 0.05% trypsin solution. One-tenth ml ofa cell suspension (10 cell/ml) was dispensed into each well in a 96-well,flat-bottomed microtest tray (Falcon No. 3040), and single cell isolateswere established as sublines. Subline 9, which displayed a high proclivityto form lung colonies after i.v. challenge, was chosen for furtherselection of metastatic variants using the method of Fidler (17). Asingle cell suspension of subline 9 prepared from the confluent mono-layers by 10-min treatment with 0.05% trypsin in versene solution wasinoculated i.v. at a dose of 10s viable cells in 0.2 ml of HBSS into C3H/

HeJ mice. On Day 21 the hosts were sacrificed by ether inhalation, andpulmonary colonies were aseptically dissected from normal tissue andcultured in vitro, when the cells had grown to confluency, 10' viable

cells were injected i.v. into naive C3H/HeJ mice. After this cycle wasrepeated 4 times, a daughter line was designated cIone-9-4 for MCA-F, and for the other tumors by similar methods, MCA-2A-L and MCA-D-L. All sublines shared TSTA with the parental tumor by cross-immunoprotection tests.

Spontaneous and Experimental Metastatic Model. The number ofspontaneous lung colonies was enumerated 14 or 21 days after resectionof neoplasms generated by injection of 10 to IO6(usually IO6)clone-9-

4 cells into the left hind footpad or left flank. For the artificialmetastasis model, tumors resected from 3 to 28 days after footpad orflank injection of 2 x 10s nonmetastatic MCA-F tumor cells wereimmediately challenged by tail vein inoculation of 5 x IO4 clone-9-4,MCA-2A-L, or MCA-D-L cells. Pulmonary colonies were microscopically enumerated in mice sacrificed at 14 days or 21 days after tumorexcision by India black ink insufflation by the method of Wexler (18).The significance of differences in the number of metastatic coloniesbetween groups was determined using the Kruskal-Wallis test; probability values were estimated from the ßstatistics calculated for eachgroup.

Assessment of Immunity. LATA assessed the kinetics of cellularimmunity in tumor-bearing mice. Spleen cells (2 x IO6), harvested atserial stages from tumor-bearing mice and admixed with IO4 MCA-F

tumor cells (effectontarget ratio, 200:1) in 0.2 ml of HBSS, were

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immediately injected s.c. into the flanks of 10 naive mice. Tumor sizemeasured serially with calipers was expressed as the mean of twoperpendicular diameters.

RESULTS

Spontaneous Lung Métastases of Clone-9-4 Initiated withDifferent Numbers of Cells. The number of clone-9-4 tumorcells in the initial neoplastic inoculum determined the extentof spontaneous lung colonies at 21 days following left hind limbamputation. Ten cells failed to produce any colonies; IO2or IO3cells, 0 to 7 colonies; and IO4, 10s, or IO6cells, 10 (range, 2 to

27), 9 (1 to 33), or 19 (12 to 62) colonies, respectively. The lastthree groups displayed a significantly greater number of colonies than the hosts given injections of IO3 tumor cells (Table1). The group receiving IO6 cells had a significantly higherincidence of lung colonies than those hosts receiving 10" or 10s

cells (P< 0.001). Thus there appeared to be a threshold size ofthe primary tumor in order to achieve métastases.

Effects of Tumor-bearing Time upon Lung Colonization. Varying numbers of lung métastasesappeared when the tumor wasamputated 3, 5, 7, 14, or 21 days after inoculation of 10*clone-

9-4 cells (Table 2). While hosts resected at 3, 5, or 7 daysshowed few lung métastases,there was an appreciable incidenceof lung metastasis after 14 (median, 7; range, 1 to 35) or 21(28; 5 to 75) days. Therefore, the extent of lung métastaseswasinfluenced by primary tumor size and/or the duration of tumor-bearing period before amputation.

Influence of Primary Tumor upon the Growth of SpontaneousLung Métastases. The effect of the presence of a primaryneoplasm upon the number of lung métastaseswas determined

Table 1 Influence of the size of the original clone-9-4 tumor inoculum on theincidence of spontaneous lung métastases

Six groups of mice were given injections in each group of 10 to 10" clone-9-4cells into the left hind footpad on Day -14. Tumor-bearing legs were amputatedon Day 0. All mice were sacrificed, and lung colonies were enumerated on Day21.

No. ofinoculated

cells*1010'

10'IO410'10"Tumor

wt at Lungresection colony

(g)*incidence0

000.10.10.60/10

6/107/10

10/1010/1010/10Lung

colonizationMedian0

22

109

19Range0

0-70-7

2-271-33

12-62PeNS*

NS<0.005

<0.001<0.001

•Footpad inoculation of clone-9-4 cells in 0.05 ml of HBSS.b Mean tumor weight with each tumor weight calculated by the difference

between the weight of the tumor-bearing leg minus the weight of control normalleg from normal mice.

c /"-values determined by the Kruskal-Wallis test comparing the incidenceof lung colonies in each group to the 10' group.

" NS, not significant.

Table 2 Effect of tumor-bearing time upon the extent of lung colonizationAll groups of mice were given injections intrafootpad of 1 x 10*clone-9-4 cells

on Day 0. On Day 3, 5, 7, 14, or 21 tumor-bearing legs were amputated. Micewere sacrificed 21 days after their tumor resection.

Resected tumor Lung coloniesResection Lung colony

day" Wt (g)* Size (cm) incidence Median Range F

357142100.030.131.003.761.432.055/106/106/1010/1010/100117280-80-130-181-355-75NS1*NS<0.005<0.001

" Day of tumor-bearing leg amputation.'' Mean tumor weight calculated as described in Table 1.

the Kruskal-Wallis test; incidence

Table 3 Influence of the primary tumor upon the growth of spontaneous lungmétastases

Five groups of mice were given injections intrafootpad of 10' clone-9-4 cellson Day 0. Mice of Groups 1 to 3 were sacrificed on Days 14,21, or 35, respectively.Mice of Group 3 were sham operated on Day 14. Tumor-bearing limbs wereamputated on Day 14 or 21 in Groups 4 and 5. All three groups were sacrificedon Day 35 to enumerate lung colonies.

Group12

345Primary

resectionSham

Day 14Day 14Day 21Day

ofsacrifice14

21353535No.

oflungcoloniesn12

10121615Median0

01

1430Range0

0-10-8

0-1305-122fNS*

NS<0.001

«cO.OOl

'Statistical differences determined by

of lung colonies in the Day 7 resection group was compared with other groups.* NS, not significant.

" Statisticaldifferencedeterminedbythe Kruskal-Wallistest; incidenceof lungcoloniesin Group 3 wascomparedwith other groups.

*NS, not significant.

by amputation at varying times after footpad inoculation of IO6clone-9-4. While nonamputees displayed few lung colonies 14,21, or 35 days after implantation of clone-9-4, primary tumorresection on day-14 or 21 induced significant lung colonizationat Day 35, namely 14 (0 to 130) or 30 (5 to 122) colonies,respectively (Table 3). Thus resection of the primary neoplasmfacilitates lung colonization, and the presence of the primaryneoplasm inhibits lung colonization, even in mice bearing tumors at the late stage of 5 wk.

Specificity of Inhibition of Lung Métastasesin Tumor-bearingMice. The immunological specificity of the inhibition of lungcolonization by primary tumor was investigated using an artificial i.v. metastasis model. Mice already bearing large (> I cm)or small (<0.5 cm) s.c. tumors in the flank and mice inoculateds.c. with IO6 MCA-F, antigenically distinct IO6 MCA-D, or 5x 10s MCA-2A cells were challenged i.v. with 5 x IO4 MCA-Fclone-9-4 or MCA-2A-L cells (Table 4). Hosts displaying 1.3-cm primary tumors (Group 2) showed decreased numbers ofnot only homotypic MCA-F clone-9-4 [Table 4; 1 (0 to 4); P <0.001], but also antigenically distinct MCA-2A-L: [Table 4; 29(12 to 57); P < 0.001] pulmonary colonies. Hosts bearing smallprimary tumors (Groups 3 and 4) showed specific inhibition ofMCA clone-9-4 lung colonies (4 or 14; P < 0.001), but not ofMCA-2A-L colonies (120 or 138; not significant). Mice giveninjections of IO6MCA-D cells (Table 4) or MCA-F cells (Table

4) at the time of i.v. challenge did not inhibit lung colonizationof either clone-9-4 or MCA-2A-L. On the other hand, micebearing large MCA-D tumors did inhibit lung colonization bythe antigenically distinct MCA-F clone-9-4 cells (P < 0.001).Thus hosts bearing a small tumor burden display specific concomitant immunity, while animals carrying a large tumor burden display nonspecific resistance to metastasis.

Specificity of Inhibition of Lung Métastasesafter Tumor Resection (Sinecomitant Immunity). Groups of mice either bearingor resected of tumors were challenged i.v. with 5 x IO4 clone-9-4 homotypic, MCA-2A-L, or MCA-D-L heterotypic cells.Mice bearing large 14-day MCA-F tumors nonspecifically retarded lung colonization by not only MCA-F clone-9-4, butalso MCA-2A-L or MCA-D-L (P < 0.001 to 0.005). Hostsresected of primary MCA-F neoplasms showed specific inhibition of clone-9-4, but not MCA-2A-L or MCA-D-L lung métastases (Table 5). Thus tumor excision abrogates nonspecificresistance, thereby enabling measurement of specific antitumorimmunity.

Influence of Tumor Size upon Artificial Lung MétastasesinTumor-bearing Mice. Since inhibition of metastatic growth bythe primary tumor may be due to factors which affect tumorcells after they enter the vascular compartment, the influence

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Table 4 Immunological specificity of lung colony inhibition after i.V.tumor challenge in mice bearing small or large tumorsGroups of mice bearing large (>1 cm) or small (<0.5 cm) s.c. tumors, 1 x IO6MCA-F, 1 x 10' MCA-D, or 5 x 10* MCA-2A cells, were challenged i.v. with 5 x

IO4clone-9-4 or MCA-2A-L on Day 0. All mice were sacrificed on Day 14, and lung colonies were enumerated.

Tumor size(cm)"GroupChallenge

with clone-9-4ofMCA-F1234567Challenge

withMCA-2A-L1234567Primary

tumorinjectionNoneMCA-F,

2 x IO5,-14dayMCA-F,1 x IO3,-14dayMCA-F,1 x 10«,OdayMCA-D,2 x IO5, -14dayMCA-D,1 x IO3, -14dayMCA-D,1 x 10«, 0dayNoneMCA-F,

2 x 10', -14dayMCA-F,1 x IO3, -14dayMCA-F,1 x 10«, 0dayMCA-2A,

1 x IO5, -14dayMCA-2A,1 x IO3,-14dayMCA-2A,5 x IO5, O dayDayO1.30.31.50.21.30.31.40.2Day

142.81.81.62.71.51.42.81.61.42.91.21.3n*10691010101067910101010Lung

coloniesMedian32141412263316829120138114928Range17-920-40-157-271-218-1012-85120-19112-5767-14164-1830-984-1390-54P<0.001<0.001<0.001<0.001NSCNS<0.001NSNS<0.001<0.025<0.001*<0.001<0.025<0.001<0.005<0.001<0.001<0.001<0.001<0.001NS<0.01NS

" Mean tumor size of the mice at the time of i.v. injection (Day 0) or sacrifice (Day 14).* Statistical difference was determined by the Kruskal-Wallis test; significance of differences in lung colonies versus normal control or versus large MCA-F-bearing

group.c NS, not significant.

Table 5 Immunological specificity of resistance toward pulmonary métastasesafter tumor resection

Mice were given injections s.c. of 2 x IO5MCA-F cells into the flank on Day-14; on Day +9 the mice were randomized into six groups. Three groups wereallowed to have continued tumor growth; the other three groups were resectedcuratively. Another three groups of non-tumor-bearing mice were sham operatedon Day 0. Each set of three groups was i.v. challenged with 5 x IO4 of eitherclone-9-4, MCA-2A-L, or MCA-D-L cells. All mice were sacrificed on Day 14 toenumerate lung colonies.

Table 6 Influence of tumor size upon the incidence of artificial lung métastasesin tumor-bearing mice

Groups of mice were given injections s.c. into flank of 2 x IO5 MCA-F cellson Day 0 or 14. On Day 21 the mice were challenged i.v. with 5x10* clone-9-4cells; on Day 28 primary tumors were curatively excised. Non-tumor-bearingmice were also challenged i.v. with 5 x IO4clone-9-4 cells on Day 21 and sham

operated on Day 28. All mice were sacrificed on day 42 to enumerate lungcolonies.

LungcoloniesTumor

resection_+No

tumor,sham-operated_+No

tumor,sham-operated_+No

tumor, sham-operatedChallenge

i.v.Clone-9-4Clone-9-4Clone-9-4MCA-2A-LMCA-2A-LMCA-2A-LMCA-D-LMCA-D-LMCA-D-Ln151515131414121212Median48384413115841927Range0-140-337-6413-9337-17848-1960-127-4010-47f<0.001<0.001<0.005NS"<0.001NS

Day ofinoculationExperiment

1014NoneExperiment

2014NoneTumor

size(cm)at

i.v. challenge1.80.11.60.2atresection2.51.12.21.0n799101010Lung

coloniesMedian12284042638Range8-325-6216-920-303-9812-96P°<0.005NS*<0.005NS

" Statistical differences determined by the Kruskal-Wallis test; incidence oflung colonies in sham-operated group was compared with other groups.

NS, not significant.

of primary tumor size upon lung colonization was tested in anartificial metastasis model in which mice bearing 0.1- to 0.2-cm or 1.6- to 1.8-cm nonmetastatic MCA-F tumors were giveninjections i.v. of 5 x IO4 clone-9-4 cells. To avert death fromlocal tumor burden, the primary, nonmetastatic MCA-F neoplasm was excised 7 days after i.v. injection of the metastaticvariant, at which time the tumors were 1.0 to 1.1 or 2.2 to 2.5cm in diameter. Mice were sacrificed 2 wk after excision forenumeration of lung colonies. Mice with large tumor burdenshad fewer lung colonies than those with small tumor burdens(Table 6; 12 versus 28 or 4 versus 26; P < 0.005). Since therewas no difference between the noninoculated and the groupwith 1 wk of primary tumor growth, it was considered aninsufficient interval to retard lung colonization.

Influence of Tumor Size upon Artificial Métastasesin Tumor-excised Mice. The relationship of tumor progression to resistance toward artificial lung métastaseswas determined by allowing an inoculum of 2 x 10s MCA-F tumor cells to grow prior

to curative excision (Table 7). The number of lung colonies inhosts after removal of a 3-day tumor (median, 23) was almost

" Statistical differences among incidences of lung colonies determined by theKruskal-Wallis test with comparison of the incidence of lung colonies in thenoninoculated control group.

* NS, not significant.

Table 7 Influence of tumor size upon the number of artificial métastasesintumor-excised mice

Groups of mice were given injections s.c. into right flank of 2 x 10* MCA-Fcells on Day -28, -14, -7, or -3 prior to curative excision on Day 0 withsimultaneous i.v. injection of 5 x IO4clone-9-4 cells. Another 10 sham-operated

mice were given injections i.v. on Day 0. All mice were sacrificed on Day 21 forenumeration of lung colonies.

Lung coloniesDay of tumor Tumor size (cm)

injection at resection" n Median Range

None-3-7-14-280.10.92.4101099924232103011-635-440-113-555-114NSC<0.001<0.05NS

°Mean tumor size at resection in cm.* Statistical significance of differences was assessed by the Kruskal-Wallis test

based upon comparison with the incidence of lung colonies in the noninoculatedcontrol group.

' NS, not significant.

the same as normal controls (median, 24). However, there wassignificant retardation of lung colonization at 7 days [2 (0 toll);P< 0.001] or 14 days [10 (3 to 55); P < 0.05]. However,resection of 28-day tumors failed to induce inhibition. Spleencells from mice bearing MCA-F tumors for 3, 7, 14, or 28 days

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were tested for tumor neutralization in local adoptive transferassays (Table 8). Spleen cells from hosts bearing neoplasms for7 or 14 but not 28 days inhibited MCA-F tumor outgrowthsignificantly (P < 0.05). Therefore inhibition of experimentallung métastasesat different stages of tumor development afterexcision paralleled the level of cellular immunity assessed inlocal adoptive transfer assays.

DISCUSSION

In the methylcholanthrene-induced murine fibrosarcomamodel, the presence of a primary neoplasm retarded, but itsresection facilitated, lung colonization. The incidence of post-surgical spontaneous métastasesdepends upon the stage of thetumor progression, namely both tumor size (Table 1) andduration of growth (Table 2). Presumably these factors affectboth the number of cells which intravasate during tumor growthand the induction of antitumor immunity in tumor-bearing ortumor-resected hosts. Although seeding of tumor cells is thelungs probably occurs prior to resection of the primary tumor,outgrowth is inhibited by concomitant immunity until the primary tumor is resected. Analysis of both concomitant andsinecomitant immunity revealed that the former may be specificor nonspecific, while the latter is TSTA specific. Furthermore,suppression of metastatic outgrowth by large s.c. growing primaries may be, in part, due to nonimmunological mechanisms.Nonimmunological inhibition of metastatic seedings may bedue to single or to combination effects of growth factors andtheir respective receptors. As reviewed by Goustin et al. (19)growth factors may function between and among cell populations in various arrays, e.g., autocrine, paracrine. It may be thatprimary tumor growth regulates distal seeding by altering (a)tissue growth factor concentration, (b) presentation of growthfactor receptors, or (c) production of biologically inactive competitors. At present we have no direct evidence to support anysingle hypothesis. Furthermore, the production of a singlemetastasis inhibition factor by the primary tumor remains aplausible, yet undocumented, mechanism.

To observe the effects of the presence or the removal of aprimary tumor on métastases,the artificial model was studied.Using the artificial metastasis mode, the number of lung colonies following i.v. injection was greater in mice bearing smalltumors than in animals with large tumor burdens (Table 6).Similar results were obtained in concomitant inhibition of s.c.inoculated Meth A in BALB/c mice (3), 3LL in BALB/c xDBA/2 F, mice (3), or EL4 lymphoma in C57BL/6J mice (10),and in pulmonary métastasesfollowing i.v. inoculation of methylcholanthrene-induced syngeneic murine sarcoma in C3Hf/Bumice (20). Thus, concomitant inhibition is stronger in hosts

Table 8 Kinetics of cellular immunity in MCA-F-bearing miceSpleen cells (2 x IO6) from hosts bearing MCA-F tumors for 3, 7, 14, or 28

days or from normal mice (none) were admixed with 1 x IO4MCA-F tumor cells

(200:1) in 0.2 ml of HBSS, and immediately injected s.c. into the right flank ofsecondary virgin syngeneic ( .111 IId mice. Tumor size was serially measuredwith calipers; tumor sizes were compared at 21 days after inoculation.

Tumor-bearingdayNone

37

1428n10

109

1010Tumor

size(mm)11.0±0.27*

12.8 ±0.768.0 ±1.338.3 ±1.10

11.6± 1.49P"<0.05

<0.05<0.05NSC

" Statistical differences were determined by Student's t test by comparison with

reactions containing normal spleen cells.* Mean ±SE.' NS, not significant.

bearing large tumor burdens than small tumor ones. Investigation of the components of the immune response was performedusing isolated spleen cells. This population was chosen becauseit is well characterized and conveniently obtained as a representative population. However, the degree of inhibition of métastases is not necessarily reflected in the tumor-neutralizingactivity of spleen cells from tumor-bearing donors and may bebetter reflected in the lymphoid populations of the lung. Theinhibition of lung colonization in whole-body irradiated miceby spleen cells transferred from tumor-bearing mice was optimal 12 days after tumor inoculation (19). Further, the comparison of concomitant suppression of metastasis with kinetics ofcellular immunity (LATA) during tumor progression in mice(Table 8) showed that, when the primary tumor was largeenough to nonspecifically suppress metastasis, the specific cellular immunity as determined by LATA was weak. In contrast,inhibition of lung colonization after excision of a relativelysmall tumor (7- or 14-day tumor) was much stronger than afterexcision of a large tumor (Table 7). North and Bursuker (4)reported that the concomitant immunity was generated 6 daysafter IO6 Meth A challenge, peaked on Day 9, and then rapidly

decayed. They also showed that the postexcision immunity afterthe removal of primary tumor on Day 16 was down-regulatedby the activity of T-suppressor populations in tumor-bearinghosts (5). The inhibition of lung colonization presented hereinparalleled the cellular immunity measured by LATA at the timeof tumor excision (Table 8). Further, the inhibition after tumorresection was antigen specific (Table 5). Therefore after resection of a small tumor resection, sinecomitant immunity doesnot inhibit colonization by an antigenically different variant.

Antigenic differences between the parental tumor and itsmétastaseshave been reported in several systems (21-23). Possibly the exquisite specificity of sinecomitant immunity is oneof the reasons that metastatic facilitation may occur even afterremoval of a relatively small tumor. In mice bearing largetumors, concomitant inhibition of lung colonization was nonspecific (Table 4). Gorelik (3) also showed that suppression ofreinoculated radiolabeled M109 tumor cells (2 x IO5) into

BALB/c or BALb/c nude mice bearing a tumor greater than 2cm was the same in both strains and survived to the same extentat the local site of inoculation in tumor-bearing and non-tumor-bearing control mice. The growth of reinoculated 3LL, B16,EL4, or T-10 cells was equally inhibited in thymectomized,irradiated, and bone marrow reconstituted C57BL mice andnude mice as well as immunologically competent mice bearingtumors more than 1.5 cm (2). Therefore all primary tumorswhen large enough inhibit not only immunogenic, but also lowor nonimmunogenic tumors whether in immunocompetent orimmunodeficient hosts. These results suggest that, in additionto immunity, other nonimmunological host factors may regulate the rate of tumor cell proliferation at specific tissue sitesin hosts bearing large tumor burdens. The induction of nonspecific concomitant tumor inhibition has not been well understood. Niederkorn and Streilein (24) indicated that immunologically privileged, intracamerally inoculated P815 mastocytomacells induced specific concomitant immunity which was T-celldependent and radiosensitive, but because the intraocular tumoris small, the mice display specific concomitant immunity.

Serum factors in mice bearing methycholanthrene-inducedsarcoma did not influence the number of pulmonary métastases(20, 25). Unfortunately, estimation of the serum content ofTSTA is not feasible because of the lack of specific antibodies.Gorelik (3) showed that some concomitant suppression of metastasis was not due to T-cells, natural killer cells, or macro-

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phages, but probably to nonimmunological mechanisms in micebearing nonimmunogenic tumors and to antitumor immunereactions with additional nonimmunological mechanisms inmice bearing immunogenic tumors. The experiments usingthe methylcholanthrene-induced sarcoma described herein suggest that there are two types of concomitant inhibition. Theone is specific cellular immunity, which is dominant in smalltumor-bearing mice and decreases with tumor growth. Theother is nonspecific inhibition, possibly nonimmunologicalmechanisms, which prevails in animals with large tumor burdens and increases with tumor growth. The nonspecific inhibition is strong, but disappears with tumor excision. This may bea contributory factor to the metastatic facilitation that followsresection of large tumors. The sinecomitant immunity, whichis presumably the continuation of the specific cellular concomitant immunity, depends upon the tumor stage at resection, andit does not afford resistance to antigenically different metastaticfoci (Table 5). Since postsurgical therapy with immunospecificreagents probably augments specific immunity, control of post-surgical metastasis may require additional activation of nonspecific immune and nonimmunological factors to control proliferation of antigenically distinct metastatic foci.

ACKNOWLEDGMENTS

The authors thank Elena Vess for excellent secretarial assistance andScott Smith for technical assistance.

REFERENCES

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3. Gorelik, E. Resistance of tumor-bearing mice in a second tumor challenge.Cancer Res., 43:138-145, 1983.

4. North, R. J., and Bur-inker. I. Generation and decay of the immune responseto a progressive fibrosarcoma. I. Ly 12' suppressor T cells down-regulatethe generation of Ly 1~2* effector T cells. J. Exp. Med., 159: 1295-1311,

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1986;46:6111-6115. Cancer Res   Shinhachiro Nomi, Kazuyo Naito, Barry D. Kahan, et al.   Postsurgical Metastasis in MiceEffects of Concomitant and Sinecomitant Immunity on

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