(CANCER RESEARCH 38, 331-338, February 1978]

A Survey of the Growth Characteristics of and the Host Reactions toOne Hundred C3H/He Mammary Carcinomas1

Jan Vaage

Department ol Cancer Therapy Development, Pondville Hospital, Walpole, Massachusetts 02081

ABSTRACT

The ability of mouse mammary carcinomas to inducetransplantation resistance in syngeneic hosts has beentested by the surgery-challenge procedure. The characteristic has been determined for 100 different primarymammary carcinomas derived from 26 breeding femaleC3H/He mice and has been related to the time of appearance, to the s.c. growth rate, to the s.c. transplantabilityof all of the tumors, and to the i.v. transplantability(pulmonary growth) and pulmonary growth rate of 50 ofthe tumors.

Fifty-seven % of the tumors, when used for presensiti-zation, produced a degree of transplantation resistancein treated hosts, and the resistance was, where tested,individually specific. Twenty-one % of the tumors left littleor no effect, after surgical removal, on the growth ofsubsequent implants. Twenty-two % produced a degreeof promotion of the growth of challenge implants. Thegrowth promotion was, where tested, not individuallyspecific.

The tumor characteristics of immunogenicity, relativeneutrality, and growth promotion were found to be unrelated to the following: (a) the age of the original host attime of appearance of the primary tumor; (b) the growthrate of the tumor; and (c) the s.c. transplantability inunsensitized hosts. The ability to grow in the lungs afteri.v. implantation occurred most frequently among immu-nogenic tumors.

Most individual primary tumors were stable in repeatedtransplantations with respect to all characteristics tested.Three tumors lost immunosensitivity, and two tumorsgained immunogenicity and/or immunosensitivity in thecourse of repeated passages in untreated syngeneichosts. When these tumors were retested from primarytumors stored in liquid N2, the same changes recurred inthe same or nearly the same transplant generation.

INTRODUCTION

C3H mice, neonatally infected with MTV,2 have been

shown to be relatively immunogically tolerant to the MTV(1, 4, 5) and have been shown to develop transplantationresistance only against mammary tumor immunogens otherthan viral antigens or cell products controlled by the viralgenome (6, 13, 16). The clear difference between MTV-freeC3Hf mice and MTV-infected C3H mice in their immuneresponse to C3H mammary tumors (13, 17) has shown that

1 Supported by Grant CA-15960, awarded by the National Cancer Institute,

Department of Health. Education and Welfare. The work was started at theMassachusetts General Hospital and completed at the Pondville Hospital.

1 The abbreviation used is: MTV, mammary tumor virus.

Received August 15, 1977; accepted November 4, 1977.

immune tolerance to MTV-associated transplantation antigens is a characteristic of the C3H strain.

Earlier studies (13, 16, 17) produced evidence that about1 of 4 primary MTV-induced C3H mammary carcinomasinduced transplantation resistance in syngeneic mice andthat the immunogenic tumors most often did not sharecommon immunogens. A point of similarity, therefore,appeared to exist in the induction of neoantigens by chemical and viral oncogens (16). Studies by Old ef al. (7) andby Prehn (9, 10) on the immunogenicity of methylcholan-threne-induced mouse sarcomas provided evidence for thetheory that immunogenic tumors tend to be eliminated byimmune surveillance, unless they develop rapidly enoughto outpace host defenses. Later studies by Prehn (11, 12)suggested that the more immunogenic tumors were subjectto immunostimulation. According to either theory, immunogenic tumors would tend to appear before and growfaster than nonimmunogenic or weakly immunogenic tumors.

The purpose of this investigation has been to survey,with samples and tests numerous enough to present definitive evidence, transplantation immunogenicity in the C3Hmammary tumor system. The investigation is also an attempt to relate the transplantation immunogenicity of several primary mammary carcinomas to their time of appearance in the original host, their growth rate in syngeneichosts, and their transplantability s.c. and i.v., and is anattempt to test the constancy of some of these characteristics in serial in vivo passage.

MATERIALS AND METHODS

Mice. The tumor donors were pedigree breeding femaleC3H/He mice, force-bred from the age of 6 weeks andremoved from breeding at the appearance of the firstmammary tumor, or at no later than 6 months of age. Theexperimental mice were 10- to 12-week-old line-bred femaleC3H/He animals. All the mice were from the defined-flora,pathogen-free breeding colony (mice carry only the entericbacteria Clostridium sp., Peptostreptococcus sp., Bacillussp., and Bacteroides sp.) maintained by the Department ofCancer Therapy Development at Pondville Hospital.

Tumors. The primary mammary carcinomas were ex-isledwith wide margins of normal mammary tissue and skin toprevent recurrences soon after they became palpable, usually at 3 to 5 mm. Most of the tumor donors were kept aftersurgery and observed for the development of further (notrecurrent tumors, which were also excised and tested.

A few of the donors were given injections of a cell suspension of their own tumor to test transplantation resistance inthe autochthonous as well as in the syngeneic hosts. Additional primary tumors that sometimes appeared in these

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J. Vaage

donors during the test were removed and discarded.Tumor Implantation. Tumors, nearly 10 mm, were re

moved from freshly killed or live anesthetized donor animals, and skin and necrotic tissue were removed beforethe tissue was placed in culture medium in a sterile Retridish kept on a bed of crushed ice. Implantation s.c. of 2pieces (1 cu mm) of living tumor tissue was used to initiateantitumor immunity. Tumors from treated animals werenever used. Removal of sensitizing tumors implanted s.c.was done under pentobarbital anesthesia. Unsensitizedcontrol animals were also anesthetized. A circular incisionwas made in the skin around the edge of the tumor, andthe tumor was removed by blunt dissection. The incisionwas closed with wound clips.

Disruption of tumor tissue to obtain suspensions of dispersed cells was accomplished with the use of 105 meshpolyester cloth (HC-1-105 screen cloth; TETKO, Inc., Elms-

ford, N. Y.) by a mechanical procedure described in previous publications (14, 15).

In all of the experiments reported here, s.c. sensitizationprocedures were done on the right side of the animals, ands.c. challenge implantations were done on the left side.The s.c. (10s cells) and i.v. (3 x 10" cells) challenge

implantations of presensitized (10/group) and control (10/group) mice were by injection of viable (trypan blue-nega

tive) tumor cells suspended in culture medium and consisting predominantly of single cells. The proportion of trypanblue-negative cells in the tumor cell suspensions was usu

ally about 25% or less. In each separate experiment, thechallenge of all the mice was completed within the briefestpossible time with cells from the same suspension.

Statistical Analyses. The effect of pretreatment is described as the s.c. growth value in treated mice divided bythe growth value in controls (growth index). The growthvalue was derived from the sum of the diameters of alltumors in a group divided by the number of implants. Theresults of repeated tests per tumor were averaged. Forcomparison of tumor growth, Student's f test was used.

Differences between groups were considered significantwhen the p value of comparison was 0.05 or smaller.

RESULTS

Transplantation Tests in Syngeneic Hosts. Table 1shows the accumulated results, and Tables 2 and 3 showthe summarized results of the tests in syngeneic C3H mice.Table 2 has been arbitrarily divided into 3 groups to showthe relationships of the growth index of the tumors to otherobservations. Table 3 shows the same relationships withthe 13 highest and the 15 lowest tumors on the growthindex scale.

C3H/He mammary carcinomas can, as previously reported (13, 16, 18), induce transplantation resistance insyngeneic hosts. This ability is presumed to depend onimmunogens which, as the data show (Chart 1; Table 1),may range from very strong to almost imperceptibly weak.Reciprocal cross-immunization tests between immuno-genic Tumor 9-15 versus Tumor 11-1, Tumor 9-15 versusTumor 6-1, and Tumor 6-1 versus Tumor 11-1 showed that

in these tumors the immunogens were individually specific

and not shared (data not shown). This is in agreement withpreviously reported results in the C3H mammary tumorsystem (13, 16, 17).

Some tumors could, while growing in a host, provideconditions that promoted the growth of subsequent implants of the same tumor. This ability, which was expressedstrongly or weakly in a given tumor, is presumed to dependon tumor factors unrelated to transplantation antigensbecause reciprocal cross-reactivity tests with Tumor 14-2versus Tumors 6-1, 9-6, 9-15, and 11-1 showed that exposure to a growth-promoting tumor would enhance thegrowth of other C3H mammary tumors used to challenge.The growth indexes in these reciprocal cross-reactivity

tests were as follows (with sensitizing tumor versus challenge tumor):

Sensitizingtumor14-214-214-214-26-19-69-1511-1Challengetumor6-19-69-1511-114-214-214-214-2Growthindex1.211.141.061.190.931.180.960.98

There was no significant difference between immuno-genic and nonimmunogenic groups of tumors in theiraverage time of appearance or in their average initial growthrate (Chart 1; Tables 2 and 3).

There were no significant differences between immuno-

genic and nonimmunogenic groups of tumors in changinggrowth rates or in changing s.c. transplantability withrepeated passages (Tables 2 and 3). Changes in transplant-

ability were determined by the number of living cells neededto produce s.c. growth in at least 8 of 10 unsensitizedcontrol mice. Past and present experience has shown that,for C3H mammary carcinomas, 10s cells are most often

adequate for initial in vivo passages.The proportion of tumors that would grow (in the lungs)

after i.v. implantation was higher among tumors with agrowth index less than 0.95 than among tumors with agrowth index near 1 or higher (Table 2). This relationshipwas not apparent in the comparison of tumors with agrowth index less than 0.50 and tumors with a growthindex greater than 1.10 (Table 3).

The rate of growth in the lungs (measured in normalmice as days from i.v. injection to the time of necropsy,which was determined by signs of dyspnea or poor health)was invariably slower than the rate of growth of the sametumor implanted s.c. (measured in normal mice as daysrequired by the sensitizing tumor implant to reach anaverage size of 3 mm) (Table 1).

The following tests were done to test the homogeneity ofprimary tumors with respect to individual characteristics.Each of the primary tumors appearing in Donor Animals 31and 32 was split by discarding the center portion of eachtumor and by implanting and studying the opposite polesseparately. With only 1 exception (Tumor 31-5A.B), thetumors were uniform with respect to the ratio of growth insensitized and normal hosts, s.c. and i.v. transplantability,

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Comparison of Mouse Mammary Tumors

Table 1The accumulated results of transplantation studies with mammary carcinomas in syngeneic C3H mice

Numbers missing in a sequence of tumors from individual donors are tumors that failed to grow s.c. in syngeneic hosts: Tumor 9-14

was not a mammary carcinoma by histological examination. Test animals were sensitized by s.c. implants of a tumor in the right flank andwere challenged by 2 s.c. implantations of suspended cells at the left shoulder and hip. The rate of growth s.c. was measured as daysrequired by the sensitizing tumor implant to reach an average size of 3 mm. The rate of growth in the lungs was measured as days fromi.v. injection to the time of necropsy after signs of dyspnea or poor health.

Growth rates.c.Mouse568910111214162021Tumor12345134512123456789101112131516171819123121341234567111234Age

(days)370387387425425372372384384246400286298355369369381386386399399411437464437471488488488441441510426426262332525323348380409409442442340443360367395462Generation14860463239485021141723302442262042302822443328312829492823402022222236281420192315272619342918482117Succeedinggenerations35(2)a38(2)28(2)31

(2)13(9)17(6)28(10)15(2)14(2)16(5)15(6)13(4)18(2)20(4)14(6)18(2)32(5)14(2)20(2)22

(3)20(8)16(2)10(3)18(8)41

(6)18(6)73(2)13(4)15(5)27(6)35

(10)17(2)15(5)21

(2)29(2)26(6)15(5)10(6)17(6)20(5)15(2)15(5)11

(6)14(2)13(4)25(5)20

(3)17(3)16(2)20(3)Transplantability

s.c.NC6NCNCNCNCNCNCNCNCNCDecreasedNCNCNCIncreasedNCNCNCNCNCDecreasedNCNCIncreasedNCNCDecreasedNCNCNCNCNCNCNCNCDecreasedDecreasedNCNCNCNCNCIncreasedNCNCNCNCNCNCNCNo.

oftimes

tested222254522324234232228228462254152422436432432932223Growth

ratio(presensitized/

control)0.941.070.431.100.860.380.591.130.790.930.260.641.021.090.921.001.171.100.961.020.640.861.200.520.680.711.651.110.761.100.500.900.490.531.000.400.970.571.290.481.060.630.990.970.410.911.231.290.870.69Implants

i.v.Growth

Pulmonary rategrowthgeneration-+

107(5)+88(5)+

107(2)+

68(5)+

127(3)-+

76(2)+98(4)+

110(4)+111(5)__-1-

110(5)+

92(6)+

64 (4)

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J. Vaage

Table 1-Continued

Growth rates.c.Mouse263031323334353637383940Tumor5678234511A1B2A2B3A3B4A4B5ASB6A6B7A7B1A1B2A2B3A3B11111123456789111234561235Age(days)477477556566317390403403447343343362362386386386386396396450450450450343343377377404404380489261489366390390390433455477477517517533170310345375375385255348373400Gen

eration1232231362325252523201830233029202329223343232731274750222429554235422414233014211815182914242334202429342928Succeedinggenerations21

(2)18(5)20(4)26(4)13(7)25(7)21

(2)20(3)11

(4)18(7)18(5)17(6)18(6)28(4)26(4)18(6)19(6)41

(5)18(5)13(6)20(6)18(5)18(4)18(7)15(7)46(4)49(4)13(8)15(8)28(3)51

(2)13(8)27(7)27(2)12(6)7(6)18(7)12(7)5(6)12(6)18(5)18(4)12(3)18(3)19(5)15(6)18(3)20(4)12(6)19(8)12(10)14(3)11

(5)28(2)Transplantability

s.c.NCNCNCDecreasedIncreasedIncreasedNCNCIncreasedNCNCNCNCNCNCNCNCDecreasedDecreasedNCNCNCNCDecreasedDecreasedDecreasedDecreasedIncreasedIncreasedNCNCNCNCNCIncreasedNCNCNCNCNCNCDecreasedNCNCNCNCNCNCDecreasedNCIncreasedNCNCNCNo.

oftimes

tested242345223444433336633333333443244233454433223423357232Growth

ratio(presensitized/

control)1.360.500.380.570.641.020.970.621.080.55

1 ._,0.67f°'610.671

-,6Q0.53)'1'23h

131.03Õ1'130.66

) n-„0.70(U'bo0.551.510.72l

0__0.72)'0.94)1.08Õ1'010.38)0.58)

U0°'0!H0080.07

)u°'9(H0840.78)1.580.980.590.961.110.930.560.700.210.971.040.891.060.940.980.920.680.960.960.400.950.780.880.541.03Implants

i.v.Growth

Pulmonary rategrowthgeneration+

108(2)+86(4)-+

127(4)+

79(5)_—+

98(3)+102(3)+

124(5)+112(5)+

90(7)+82(7)--+

161(3)+157(3)_-+

108(6)+"112(6)--+

112(2)+121(3)+

58(7)-+

48(5)-+

102(6)+63(6)+

111(2)-+

74(6)+109(4)-——+

134 (5)

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Comparison of Mouse Mammary Tumors

Table 1 —¿�Continued

Growth rates.c.Mouse414243Tumor6125671Age

(days)400205355333333333185Generation123322520302720Succeedinggenerations20

(8)19(5)11

(3)18(2)18(2)19(2)20

(2)Transplantability

s.c.DecreasedNCNCNCNCNCNCNo.

oftimes

tested5322222Growth

ratio(presensitized/

control)1.010.650.790.741.060.351.35Implants

i.v.GrowthPulmonary

rategrowthgeneration+

175(7)+

111(4)+102(3)—+

40 (2)

°Numbers in parentheses, generation number.b NC, no change.

Table 2

Summarized results of transplantation studies

The 3 groups were divided arbitrarily to distinguish amonggrowth characteristics of immunogenic (<0.95), neutral (0.95 to1.05), and growth-promoting (>1.05) tumors.

GrowthindexNo.oftumors"AgeRangeMedianMeanGrowth

rates.c.,generation1RangeMedianMeanGrowth

rate withs.c.transfers*IncreasedDecreasedNo

changeTransplantability

withs.c.transfers'*IncreasedDecreasedNo

changeTransplantability

i.v.+-Immunogenic

tumors0.9558170-566386387.114-502627.744

(76)c6(10)8(14)6(10)8(14)44

(76)25

(43)6(10)Neutral

tumors0.95-1.0521298-533403424.514-552526.215(71)06(29)2(10)2(10)17

(80)4(19)5(24)Growth-

promotingtumors>1.0522348-517386.5411.515-6029.530.313

(59)2(9)7(32)1

(4)3(14)18

(82)4(18)6(27)

0 The numbers total 101 because Tu mor 13-5A.B was considered

as 2 tumors.6 The s.c. growth rate of a tumor was considered to be un

changed if the time for the tumor to reach an average size of 3 mmdiffered by 10% or less between early and late transplant generations.

c Numbers in parentheses, percentage of tumors in the group.'' Changes in transplantability with repeated s.c. transfers were

determined by the number of cells needed to produce s.c. growthin at least 8 of 10 unsensitized control mice.

s.c. and i.v. growth rate, and the constancy of thesecharacteristics in successive transplantation generations(Table 1). The opposite poles of Tumor 31-5 were found to

Table 3

Summarized selected results of transplantation studies

The 2 groups were selected to compare the growth characteristics of the most immunogenic (<0.50) and the most growth-promoting (>1.10) tumors."' *

GrowthindexNo.oftumorsAgeRangeMedianMeanGrowth

rate s.c., generation1RangeMedianMeanGrowth

rate with s.c.transfers"IncreasedDecreasedNo

changeTransplantability

with s.c.transfers6IncreasedDecreasedNo

changeTransplantability

i.v.+-Most

immunogenictumors<0.5015332-556380396.322-482930.811

(73)c04(27)1

(7)5(33)9(60)6(40)3(20)Most

growth-

promotingtumors>1.1013348-488384404.518-492830.68

(62)05(38)02(15)11

(85)4(31)2(15)

a, 6 See Table 2, Footnotes b and d.Numbers in parentheses, percentage of tumors in the group.

differ in 3 respects. Part A showed a decreasing growthrate with s.c. propagation and was strongly immunogenic,while Part B showed a slightly increasing growth rate andwas strongly growth promoting (Table 1). Part A appearedto be toxic, making the hosts cachectic without evidenceof métastasesbefore the s.c. implant reached 1 cm. Part Breached a size of 2 cm or larger without apparent ill effectson the host. For confirmation of the data, Parts A and Bwere tested again from generation 2 (from liquid N2) togeneration 4 with similar results.

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01

02

03

•¿�.4

i"i 06

ÃœS i.T

Chart 1. The relationship between the time of develop-ment of mammary carcinomas (age of donor in days) in theoriginal C3H host and the response of syngeneic hosts tosensitizing tumor implants (growth ratio). PRESENS., presen-sitized.

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560

ARE OF HOST (DAYS)

The following experiments were done to test the constancy of the growth characteristics of a tumor in repeatedtransplantations.

Tumors 9-13, 9-16, and 9-19 had all changed from agrowth ratio of less than 0.6 to a growth ratio close to 1 intheir 7th, 5th, and 4th respective transplant generations,indicating a loss of immunogenicity or a loss of immuno-sensitivity. For identification of the cause, cross-sensitiza-tion tests were done between the second transplant generation of Tumor 9-13 (from liquid N2), which had alreadybeen shown to be immunogemc, and the tumor in thegeneration succeeding the change. The results of thesetests showed that the tumor had lost immunosensitivitywhile retaining immunogenicity. Each of the generationswas still capable of inducing transplantation resistance,but pretreatment with neither the early nor the late generations significantly influenced the growth of challenge implants of a late transplant generation. The average growthindexes in once-repeated cross-sensitization tests were asfollows (in which G is generation): sensitizing tumor 9-13 G2 versus G 2 challenge, 0.61; sensitizing tumor 9-13 G 2versus G 8 challenge, 0.89; sensitizing tumor 9-13 G 8versus G 8 challenge, 0.94; sensitizing tumor 9-13 G 8versus G 2 challenge, 0.61. With continued passage andtesting, Tumor 9-13 lost immunogenicity in generation 11and remains (now in generation 13) negative for bothimmunosensitivity and immunogenicity. The growth indexes in 1 cross-sensitization test were as follows: sensitizing tumor 9-13 G 2 versus G 12 challenge, 1.03; sensitizingtumor 9-13 G 12 versus G 12 challenge, 1.09; sensitizingtumor 9-13 G 12 versus G 2 challenge, 0.97.

Transplant generation 2 of Tumors 9-10, 10-2, 14-1, 16-1,31-1A, and 31-1B, and (as described above) Tumors 31-5Aand 31-5B were removed from liquid N2 and retested forimmunogenicity in transplant generations 3 and 4. Theresults were similar to those of the earlier tests and have

been included in Table 1.Tumors 9-13, 9-16, and 9-19 had lost immunosensitivity

in their seventh, fifth, and fourth respective transplantgenerations. Tumors 10-2 and 40-1 had gained immunogenicity and/or immunosensitivity in their fifth and seventhrespective transplant generations. The shifts in the growthindexes were as follows:

Tumor9-139-169-1910-240-1Generation1-61-41-32-41-6Growthindex0.450.580.551.090.99Generation7-85-64-55-107-10Growthindex1.061.160.940.310.37Av.0.520.710.760.500.78

For determination of whether these changes would recur ifthe tumors were tested again, growths were started fromgeneration 2 kept in liquid N2. Each of the tumors demonstrated the same characteristics observed in its earliertests, and each tumor recapitulated the shift in immunogenicity. Tumors 9-13, 9-16, and 9-19 again lost immunogenicity, this time in their sixth, sixth, and fourth respectivegenerations. Tumors 10-2 and 40-1 became immunogenicin their third and sixth respective generations.

Tumors 21-4, 21-6, 31-2A and 31-2B, 38-2, and 40-1 wereselected because they were immunogenic in the s.c. transplant studies and also grew in the lungs after i.v. injection(Table 1). Nodules of pulmonary growth were removed andimplanted s.c. to presensitize mice against s.c. and i.v.challenges. Cells from these implants were used to challenge. The results of the s.c. challenges are presented asthe growth index, and the results of the i.v. challenges arepresented as the incidence of pulmonary growth in sensitized mice versus controls as follows:

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Comparison of Mouse Mammary Tumors

Tumor21-421-631-2A31-2B38-240-1s.c.0.710.600.640.510.700.88i.v0/10

vs.0/10vs.3/10vs.0/10vs.0/10vs.1/10

vs.10/108/1010/104/1010/1010/10

The results showed that each tumor had remained immu-

nogenic after i.v. injection and growth in the lungs.Transplantation Tests in the Autochthonous Hosts. For

determination of the transplantability of tumors in theautochthonous versus syngeneic hosts, the following donors and tumors were used: 8-2, 11-2, 16-1, 20-1, 30-1, 33-1, 34-1, 35-1, 36-1, and 37-1. To reduce the risk of compro

mising immune resistance factors in the aging primaryhosts, I removed their tumors at a size of 5 mm or less,with the mice under short-acting inhalation anesthesia

(Penthrane; Abbott Laboratories, North Chicago, III.). Themice were given s.c. injections of a suspension of 105

viable cells prepared from their own primary tumor directlyafter its excision. At the same time pieces of the tumorwere implanted into normal syngeneic mice to start thetesting process.

Of the 10 tumors implanted into both autochthonous andsyngeneic hosts, 9 were rejected and only Tumor 36-1grew as an autografi, while only 4 (8-2, 11-2, 16-1, and 35-

1) showed immunogenicity in syngeneic hosts (Table 1).

DISCUSSION

The objective of this study was to determine the frequency with which transplantation immunogens occuramong MTV-induced mammary carcinomas arising sponta

neously in breeding female C3H/He mice and to relate thistumor characteristic to as many other growth characteristics as could conveniently be studied in syngeneic hosts.

The donor mice developed their first mammary carcinomaat an average age of 343 days. This was considerably laterthan the average of 280 days reported in a previous studyof MTV oncogenesis (17). The discrepancy is related to theearlier age (6 weeks versus 9 weeks) at which the micewere started in breeding in the present study. Other studies,in C3H and C3Hf mice, which will be reported separately,have found that the age at the first primary mammarycarcinomas is, as in the human, inversely related to theage at first pregnancy.

From the distribution of tumors by the host responseshown in Chart 1, it appeared that C3H mammary carcinomas possessed in various degrees the ability to inducetransplantation resistance against, or the promotion of,their own growth. By cross-sensitization tests it was determined that growth-promoting factors, unlike transplanta

tion immunogens, were not individually specific. Whiletumor immunogenicity is a major area of emphasis incancer research, the "negative" phenomenon of self-pro

motion of growth has received less attention. From thedistribution of the tumors by their time of appearance andby the host response, it appeared that the 2 characteristicsof immunogenicity and self-promotion were unrelated to

the time of appearance of the tumors and were also not

related to the growth rate of the tumors (Chart 1; Tables 2and 3). This would suggest that mammary tumorigenesis inC3H mice is not noticeably influenced by the immunesurveillance mechanism proposed by Burnet (2). Furthermore, changes in growth rate and in s.c. transplantabilitywith repeated passages in syngeneic hosts were also notrelated to growth resistance or growth promotion (Tables 2and 3). These results differ from the observations of Old efal. (7) and Prehn (9, 10) that mouse sarcomas, induced bymethylcholanthrene, were more often immunogenic whenthey appeared soon after the s.c. implantation of the carcinogen. Their experimental conditions were different,however, from those reported here in that methylcholanthrene is a recognized immunosuppressant, producingmaximum suppression at the time when most of the anti-

genie sarcomas appeared.The discrepancy between the number of tumors that

were susceptible to rejection by sensitized syngeneic hostsand the greater number of the same tumors that wererejected after s.c. implantation into the autochthonoushosts cannot be explained yet, but it may suggest thatsyngeneic mice are not entirely adequate as tumor hostsand that the effectiveness of anticancer resistance in thehuman host may be greater than what may be assumedfrom most animal experiments.

The ability of mammary carcinoma cells, implanted viathe tail vein, to grow in the lungs of unsensitized hosts wasproportionately more frequent among tumors with a growthindex less than 0.95 than among tumors considered neutral(growth index, 0.95 to 1.05) or growth promoting [growthindex greater than 1.05 (Table 2)]. Pulmonary growth wasnot significantly more frequent, however, among the mostimmunogenic tumors than among the most growth-promot

ing tumors (Table 3). This suggests that the growth ofdisseminated malignant cells may be assisted by weak butnot by strong immune factors. The data are compatiblewith the theory of Prehn (11) that a mild immune reactionmay stimulate the replication of neoplastic cells and withthe theory of Fidler (3) that activated peripheral lymphocytes may promote metastasis formation by aggregatingcirculating cancer cells. The rate of growth of tumorsdeveloping in the lungs after i.v. injection was invariablyslower, and often very much slower, than the growth of thesame tumor implanted s.c. (Table 1). The rate of growth inthe lungs versus the rate of growth s.c. was not related toimmunogenicity.

From the results of the tests that demonstrated thehomogeneity of primary tumors, it seems that most individual MTV-induced C3H mammary tumors may be the prog

eny of a single transformed cell. The identifying characteristics that were the same in the opposite poles of 9 individual primary tumors (Table 1), at a size of about 5 mm, werealso persistent through several transplant generations andwere repeatable in tests started over again from earlytransplant generations kept in liquid N2. The 1 exception,Tumor 31-5A.B, among the 10 tested, displayed so many

clear differences in its 2 poles that the possibility must beconsidered that 2 neighboring clones may have mergedinto a single tumor.

Tumor growth characteristics were stable in repeateds.c. as well as pulmonary passages and were reproducible

FEBRUARY 1978 337

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J. Vaage

in repeated passages of tumors started from samples storedin liquid N,. Also, occasional tumors lost or gained responsiveness in immunogenicity tests during the course ofrepeated s.c. passages. The same shifts in immunogenicityand/or immunosensitivity recurred when these tumors wereagain tested in sequential s.c. passages of tumors startedfrom generation 2 samples kept in liquid N2. The changesrecurred in the same transplant generation or in one closelybefore or after. The latter observation opens the opportunityto speculate that these repeatable changes, which presumably were scheduled by genetic regulation long before theyappeared, may have been an expression of the same mechanism that programs the aging process inherent in allnormal metazoan cells. Also, by further speculation, if somebiological timers can still function in some cancers, couldnot the timer that limits life span also function and explainsome spontaneous regressions? A similar phenomenonmay have been observed by Outzen and Custer (8), when afrog sarcoma differentiated to a benign form in both theprimary tumor and in an autotransplant at approximatelythe same time. The authors attributed the phenomenon toepigenetic influences.

ACKNOWLEDGMENTS

I appreciate the valuable assistance and expert surgical technique ofJanet Bury. Robin McDonald, Thaya DuBois. and Robert Kenrick. I thankDr. Gloria Heppner, Dr. Richmond T. Prehn, and Dr. George Klein for theirconstructive evaluations of the experimental results.

REFERENCES

1. Blair, P. B. Immunological Aspects of the Relationship between Hostand Oncogenic Virus in the Mouse Mammary Tumor System. Israel J.Med. Sci., 7: 161-186, 1971.

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Burnet, F. M. Cancer: A Biological Approach. I The Process of Control.Brit. Med. J.. 1: 779-786, 1957.Fidler, I. J. Immune Stimulation-Inhibition of Experimental Cancer Metastasis. Cancer Res., 34: 491-498, 1974.Lavrin, D. H., Blair, P. B., and Weiss, D. W. Immunology of SpontaneousMammary Carcinomas in Mice. IV. Association of the Mammary TumorVirus with the Immunogenicity of C3H Nodule and Tumors. CancerRes., 26. 929-934, 1966.Morton, D. L. Acquired Immunological Tolerance and Carcinogenesisby the Mammary Tumor Virus. I. Influence of Neonatal Infection withthe Mammary Tumor Virus on the Growth of Spontaneous MammaryAdenocarcinomas. J. Nati. Cancer Inst., 42. 311-320. 1969.Morton, D. L., Miller, G. F., and Wood, D. A. Demonstration of Tumor-specific Immunity against Antigens Unrelated to the Mammary TumorVirus in Spontaneous Mammary Adenocarcinomas. J. Nati CancerInst., 42: 289-301, 1969.Old, L. J., Boyse, E. A., Clarke, D. A., and Carswell, E. AntigenicProperties of Chemically Induced Tumors. Ann. N. Y. Acad. Sci., 707.80-106, 1962.Outzen, H. C., and Custer, R. P. Differentiation of a Methyl-cholan-threne-induced Sarcoma to a Benign Plexiform Fibroneural Tumor inan Adult Frog (Rana pipiens). Am. J. Pathol., 85. 183-194, 1976.Prehn, R. T. Specific Isoantigenicities among Chemically Induced Tumors. Ann. N. Y. Acad. Sci., 707. 107-113, 1962.Prehn, R. T. The Relationship of Immunology of Carcinogenesis. Ann.N. Y. Acad. Sci.. 764. 449-457. 1969.Prehn, R. T. The Immune Reaction as a Stimulator of Tumor Growth.Science, 776. 170-171, 1972.Prehn, R. T. Immunostimulation of the Lymphodependent Phase ofNeoplastic Growth. J. Nati. Cancer Inst., 59. 1043-1049, 1977.Vaage, J. Nonvirus-associated Antigens in Virus-induced Mouse Mammary Tumors. Cancer Res., 28. 2477-2483, 1968.Vaage. J. Influence of Tumor Antigen on Maintenance versus Depressionof Tumor-specific Immunity. Cancer Res., 33. 493-503. 1973.Vaage, J. Transplantation Procedures in Tumor Immunology. MethodsCancer Res. 8. 33-58, 1973.Vaage, J., Kalinowsky, T., and Olson, R. Antigenic Differences amongVirus-induced Mouse Mammary Tumors Arising Spontaneously in theSame C3H/Crgl Host. Cancer Res., 29. 1452-1456. 1969.Vaage, J., and Medina, D. Virus Oncogenesis and Tumor Immunogenicity in the Mouse Mammary Tumor System. Cancer Res., 34: 1319-1324,1974.Weiss, D. W.. Faulkin, L. J., and DeOme. K. B. Acquisition of HeightenedResistance and Susceptibility to Spontaneous Mouse Mammary Carcinomas in the Original Host. Cancer Res., 24: 732-741, 1964.

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