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