Cory nebacterium granulosum-ináuced Protection against...

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[CANCER RESEARCH 34, 613-620, March 1974] Cory neb acterium granulosum-ináuced Protection against Artificial Pulmonary Métastasesof a Syngeneic Fibrosarcoma in Mice1 Luka Milas,2 Nancy Hunter, and H. Rodney Withers Section of Experimental Radiotherapy, The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77025 SUMMARY The treatment of mice with killed Corynebacterium granulosum bacteria before i.v. injection of viable syngeneic fibrosarcoma (FSA) cells greatly reduced the number and size of tumor nodules in the lung. It also reduced the number and size of tumor nodules and prolonged the survival of mice if injected after tumor cells. The antitumor effect was also apparent against FSA growing in the s.c. tissue, where C. granulosum induced permanent regression in more than 50% of cases. However, the strongest effect was against i.p.—transplanted cells; the number of tumor cells that killed normal mice within 15 days did not grow in C. granulosum-lreatea recipients. The antitumor activity of C. granulosum was expressed less against pulmonary de posits of a weakly antigenic mammary carcinoma than against those of FSA. Pretreatment with living Bacillus Calmette-Guérinbacteria also reduced the number of FSA métastases, but the effect was not as great as that obtained with C. granulosum. Thus, C. granulosum shows promise of being another efficient nonspecific immunostimulant for the treatment of solid tumors. INTRODUCTION Anaerobic corynebacteria are very potent stimulators of the RES.3 They increase phagocytic activity of their recipi ents (14, 34, 35), augment in them the production of antibodies (31, 34, 35) and the appearance of delayed hypersensitivity reactions (19, 31), and make them re sistant to graft-versus-host reaction (6, 18), to bacterial and protozoal infections (1, 32), and to tumors (3, 8, 10, 13, 19-21, 25, 26, 28, 39, 40, 53, 54). Studies on the induction of resistance against tumors by corynebacteria have so far been limited almost exclusively to Corynebacterium parvum. A number of experimental tumors would not grow in animals previously treated with the bacteria and, if they 'This investigation was supported by NIH Research Grants CA 6294 and CA 11138 from the National Cancer Institute, and by Institutional Research Grant RR 5511-11, Allotment IN-19. 2On leave of absence from Laboratory for Tumor and Transplantation Immunology, Department of Experimental Biology and Medicine, Insti tute Rudjer Boskovic, Zagreb, Croatia, Yugoslavia. 3The abbreviations used are: RES, reticuloendothelial system; FSA, fibrosarcoma; MCA, mammary carcinoma; BCG, Bacillus Calmetle- Guérin. Received September 28, 1973; accepted December 3, 1973. grew, their appearance was delayed and (or) their growth was retarded. As an adjunct to chemotherapy, C. parvum was very effective against s.c.-growing FSA in mice (8), and it greatly prolonged the survival of cancer patients (19, 20). We previously reported a remarkable reduction of experi mental pulmonary métastasesof syngeneic FSA by C. parvum given s.c. to mice before or after i.v. inoculation of tumor cells (28). We here report the effect of another Corynebacterium, C. granulosum, on artificial pulmonary métastases of a syngeneic methylcholanthrene-induced FSA in C3Hf/Bu mice. This Corynebacterium is a potent stimulator of the RES (35), but its antitumor activity has been insufficiently studied. Recently, Mathéet al. (26) reported that C. granulosum can, under certain conditions, induce in mice some degree of protection against semial- logeneic L1210 leukemia and Lewis and ICIGCIi solid tumors. MATERIALS AND METHODS Mice. C3Hf/Bu highly inbred mice of both sexes were 10 to 14 weeks old at the start of each experiment. They were bred in our own specific pathogen-free mouse colony and were maintained on a sterilized pellet diet and sterile water. (The mice carry only the following enteric bacteria: Clostridium sp., Peptostreptococcus sp., Bacillus sp., and Bacteroides sp.) During the experiments, 5 to 7 mice were kept to a cage. Experimental groups were comprised of 5 to 11 mice. Tumors. The FSA was induced in a young C3H/He female mouse by a single s.c. injection of 1 mg of methylcholanthrene suspended in peanut oil (44). The 1st through 4th generation isotransplants of this tumor had been kept in a liquid nitrogen refrigerator, and the experi ments were performed with tumors of the 5th generation. The tumor is strongly antigenic for its syngeneic hosts (43). The MCA of the 3rd isotransplant generation was used in 1 experiment. It arose spontaneously in a multiparous, mammary agent-positive C3H/He mouse (38). The 1st and 2nd generation isotransplants had been kept in a liquid nitrogen refrigerator. MCA is weakly antigenic for its syngeneic hosts (38). Tumor Cell Suspensions. FSA's were harvested from tumor-source mice when they were approximately 1to 2 cm in diameter. Nonnecrotic tissue was separated and finely minced with ophthalmic scissors. The mince was added to a beaker containing 0.025% trypsin in Solution A (8.0 g MARCH 1974 613 on March 23, 2019. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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[CANCER RESEARCH 34, 613-620, March 1974]

Cory neb acterium granulosum-ináuced Protection against ArtificialPulmonary Métastasesof a Syngeneic Fibrosarcoma in Mice1

Luka Milas,2 Nancy Hunter, and H. Rodney Withers

Section of Experimental Radiotherapy, The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas 77025

SUMMARY

The treatment of mice with killed Corynebacteriumgranulosum bacteria before i.v. injection of viable syngeneicfibrosarcoma (FSA) cells greatly reduced the number andsize of tumor nodules in the lung. It also reduced thenumber and size of tumor nodules and prolonged thesurvival of mice if injected after tumor cells. The antitumoreffect was also apparent against FSA growing in the s.c.tissue, where C. granulosum induced permanent regressionin more than 50% of cases. However, the strongest effectwas against i.p.—transplanted cells; the number of tumorcells that killed normal mice within 15 days did not grow inC. granulosum-lreatea recipients. The antitumor activity ofC. granulosum was expressed less against pulmonary deposits of a weakly antigenic mammary carcinoma thanagainst those of FSA. Pretreatment with living BacillusCalmette-Guérinbacteria also reduced the number of FSAmétastases,but the effect was not as great as that obtainedwith C. granulosum. Thus, C. granulosum shows promise ofbeing another efficient nonspecific immunostimulant for thetreatment of solid tumors.

INTRODUCTION

Anaerobic corynebacteria are very potent stimulators ofthe RES.3 They increase phagocytic activity of their recipi

ents (14, 34, 35), augment in them the production ofantibodies (31, 34, 35) and the appearance of delayedhypersensitivity reactions (19, 31), and make them resistant to graft-versus-host reaction (6, 18), to bacterialand protozoal infections (1, 32), and to tumors (3, 8, 10, 13,19-21, 25, 26, 28, 39, 40, 53, 54). Studies on the induction ofresistance against tumors by corynebacteria have so farbeen limited almost exclusively to Corynebacteriumparvum. A number of experimental tumors would not growin animals previously treated with the bacteria and, if they

'This investigation was supported by NIH Research Grants CA 6294and CA 11138 from the National Cancer Institute, and by InstitutionalResearch Grant RR 5511-11, Allotment IN-19.

2On leave of absence from Laboratory for Tumor and Transplantation

Immunology, Department of Experimental Biology and Medicine, Institute Rudjer Boskovic, Zagreb, Croatia, Yugoslavia.

3The abbreviations used are: RES, reticuloendothelial system; FSA,fibrosarcoma; MCA, mammary carcinoma; BCG, Bacillus Calmetle-Guérin.

Received September 28, 1973; accepted December 3, 1973.

grew, their appearance was delayed and (or) their growthwas retarded. As an adjunct to chemotherapy, C. parvumwas very effective against s.c.-growing FSA in mice (8), andit greatly prolonged the survival of cancer patients (19, 20).

We previously reported a remarkable reduction of experimental pulmonary métastasesof syngeneic FSA by C.parvum given s.c. to mice before or after i.v. inoculation oftumor cells (28). We here report the effect of anotherCorynebacterium, C. granulosum, on artificial pulmonarymétastasesof a syngeneic methylcholanthrene-induced FSAin C3Hf/Bu mice. This Corynebacterium is a potentstimulator of the RES (35), but its antitumor activity hasbeen insufficiently studied. Recently, Mathéet al. (26)reported that C. granulosum can, under certain conditions,induce in mice some degree of protection against semial-logeneic L1210 leukemia and Lewis and ICIGCIi solidtumors.

MATERIALS AND METHODS

Mice. C3Hf/Bu highly inbred mice of both sexes were 10to 14 weeks old at the start of each experiment. They werebred in our own specific pathogen-free mouse colony andwere maintained on a sterilized pellet diet and sterile water.(The mice carry only the following enteric bacteria:Clostridium sp., Peptostreptococcus sp., Bacillus sp., andBacteroides sp.) During the experiments, 5 to 7 mice werekept to a cage. Experimental groups were comprised of 5 to11 mice.

Tumors. The FSA was induced in a young C3H/Hefemale mouse by a single s.c. injection of 1 mg ofmethylcholanthrene suspended in peanut oil (44). The 1stthrough 4th generation isotransplants of this tumor hadbeen kept in a liquid nitrogen refrigerator, and the experiments were performed with tumors of the 5th generation.The tumor is strongly antigenic for its syngeneic hosts (43).

The MCA of the 3rd isotransplant generation was used in1 experiment. It arose spontaneously in a multiparous,mammary agent-positive C3H/He mouse (38). The 1st and2nd generation isotransplants had been kept in a liquidnitrogen refrigerator. MCA is weakly antigenic for itssyngeneic hosts (38).

Tumor Cell Suspensions. FSA's were harvested fromtumor-source mice when they were approximately 1 to 2 cmin diameter. Nonnecrotic tissue was separated and finelyminced with ophthalmic scissors. The mince was added to abeaker containing 0.025% trypsin in Solution A (8.0 g

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Luka Milas, Nancy Hunter, and H. Rodney Withers

NaCI, 0.4gKCl, 1.0 g glucose, and 0.35 g NaHCO3 in 1000ml of water) and stirred on a magnetic stirrer for 20 to 30min at room temperature. Approximately l g of tumortissue was mixed with 20 ml of the solution containingtrypsin. At the beginning of stirring, DNAase was added tothe mixture to achieve a final concentration of 0.1 mg/ml.After the mixture was stirred, the undigested tissue wasallowed to settle in a beaker. The upper two-thirds of thesuspension was removed, passed through a stainless steelmesh (200 wires/inch), and washed 2 to 3 times bycentrifugation for 5 min at 1200 rpm and resuspension infresh Medium 199 containing 5% of syngeneic normalmouse serum. Standard hemocytometer counts were made.Viability of the cells was determined by phase microscopyand by observation of their impermeability to 0.25% trypanblue in Medium 199. It was found to be regularly more than95%. All suspensions were morphologically homogeneous,with slight variation in size of cells, and they consistedexclusively of single cells. Occasionally, a few nonmalignantblood cells could be identified in the suspension.

A suspension of cells from a MCA was prepared by i.mechanical method. Two s.c.-growing tumors of about 2 cmin diameter were harvested from tumor-source mice, andnonnecrotic tissue was separated and finely minced. Themince was added to Solution A and, after being thoroughlymixed, was transferred to centrifuge tubes positioned vertically in crushed ice. After about 15 min of settling, theupper two-thirds of the suspension was removed, passedthrough a stainless steel mesh (200 wires/inch), and cen-trifuged at 1200 rpm for 5 min. Supernatant was discarded,and the pellet was resuspended in Medium 199 and cen-trifuged again. The new pellet was resuspended in freshMedium 199, and the viability of tumor cells was determined as described for FSA cells. The viability was 30%.About 80% of the cells were single cells; the remainder werein clumps of 2 to 5 cells. The trypsin digestion method wasnot used with this MCA because it does not improve thequality of the suspension.

Tumor Métastases(Colonies, Nodules) in Lung. To obtainFSA colonies in the lung of normal of C. granulosum-treated mice, usually IO5viable tumor cells, suspended in

0.5 ml of Medium 199, were injected i.v. In 1 experiment,however, the number of injected cells varied from IO4to 6.4x IO5cells. In whole body-irradiated mice, only 2.5 x IO4FSA cells were injected i.v. The recipients were sacrificed 14to 16 days after the injection of FSA cells, and their lungswere removed and fixed in Bouin's solution. Colonies of

tumor cells were seen as white, round nodules on the surfaceof the yellowish lung and were counted with the naked eyeor, in doubtful cases, by use of a dissecting microscope thatmagnified them 6 times. The colonies were counted on all 5lung lobes. Size of the colonies was measured with adissecting microscope fitted with an ocular micrometer.More than 50 colonies were measured per experimentalgroup, usually within the same 2 lobes of each mouse.

To obtain colonies of MCA in lung, the mice were giveni.v. injections of 3 or 6 x IO5viable cells. The animals were

sacrificed 28 days later; lungs were removed and fixed inBouin's solution. Counting of colonies is the same as for

FSA.

C. granulosum. Formole-killed C. granulosum bacteriawere generously supplied by Professor Raynaud, InstitutePasteur, Garches, France. They were supplied in 2-mlampuls with a bacterial concentration of 10 mg/ml. Thebatch number was 5196 with a relative phagocytic activityK ¡Ko= 4.00. The activity of this batch oîC. granulosum isvery stable (35). C. granulosum was diluted with Solution Aso that each mouse received a desired concentration ofbacteria in 0.4 ml. With the exception of the 1st experiment,the dose of C. granulosum was 0.5 mg and was given to micei.p. For s.c. treatment, equal volumes of C. granulosum (0.1ml) were injected into both axillary and inguinal regions.

BCG. Living BCG bacteria from Pasteur Institute, Paris,France, were generously provided through Dr. Gutterman(Department of Developmental Therapeutics, M. D. Anderson Hospital, Houston Texas). Mice were given injections(s.c., i.p., or i.v.) of 1 mg of BCG suspended in 0.4 ml ofSolution A.

Whole-Body Irradiation. The mice were confined, 5 to around Lucite chamber, and exposed to 200, 400, 600, or 800rads to the whole body from a single 137Cssource. Thesource-to-skin distance was 28 cm and the dose rate was 285rads/min, as measured by calibrated ionization chambersand lithium fluoride thermoluminescent dosimeters.

Analysis of Results. The results were statistically evaluated by Student's t test. Differences between groups were

considered significant if the p value of comparison was 0.05or smaller.

RESULTS

Effect of Dose and Route of Injection of C. granulosum.Mice were given s.c., i.p., or i.v. injections of 1.0, 0.5, or 0.1mg of C. granulosum. Two of 7 mice that received 1 mg ofthe bacteria i.v. died within 1 day. No mortality, however,was observed in other groups. Seven days after the treatment with C. granulosum, mice were inoculated i.v. with IO5

viable FSA cells and, 16 days thereafter, were sacrificed todetermine the incidence of pulmonary métastases(Table 1).The number of lung colonies was greatly reduced in micetreated with C. granulosum, from 38.1 ±5.2 (control value)to 0.7 to 3.1, depending on dose and route of injection of theimmunostimulant.

Size of FSA Colonies in C. granulosum-trezted Mice. Thesize of all pulmonary colonies (total, 74) found in micetreated with the above-described doses and routes ofinjection of C. granulosum (Table 1) was compared with thesize of 63 colonies counted on always the same 2 lobes ofeach lung of control mice. C. granulosum significantlyreduced the size of colonies: 0.6 ±0.04 mm compared with0.9 ±0.06 mm in control mice (p < 0.0005).

Effect against Different Numbers of Tumor Cells. Themice were treated with 0.5 mg of C. granulosum i.p. and, 7days later, were given injections of FSA cells, ranging innumber from IO4to 6.4 x IO5.The number of métastases

was determined 14 days later (Table 2). C. granulosummarkedly inhibited the lung métastasesin mice of allgroups. Even 6.4 x IO5tumor cells, a sufficient number to

cause the lungs of normal recipients to be totally replaced by

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tumor tissue, produced only about 8 colonies in lungs ofmice treated with C. granulosum.

Effect of C. granulosum against Tumor Cells Injected i.V.,i.p., and s.c. The degree of specific immune resistance totumors may depend on the anatomical location in which thereaction takes place (37, 47, 50). To determine whether theeffect of C. granulosum varies against tumor cells injectedby different routes, IO6, 5 x IO5, or IO5viable FSA cells

were injected i.V., ¡.p.,or s.c. into groups of 6 to 8 mice,either normal or treated with the immunostimulant 7 daysearlier. Mice that received s.c. tumor cells were checkedtwice a week for the presence and size of tumors. Tumordiameter was measured by Vernier calipers. Mice giventumor cells i.v. or i.p. were autopsied shortly after they diedand checked for the presence of tumors in lung or abdominal cavity.

Tumor take and survival of mice are shown in Table 3.The i.v. injection of tumor cells produced tumors in lungs ofall control mice, and their survival depended upon thenumber of injected cells: 16.2 ±0.7 days for IO6, 19.1 ±2.2

Table 1Effect of dose and route of injection of C. granulosum on formation of

pulmonary métastasesgenerated by I0b FSA cells inoculated i.v.

Treatment withC. granulosum Métastasesin lung

No. ofmice with

métastases/Route of total no. of

Dose (mg) injection mice No. /lung Range

No10.50.1treatments.c.i.p.i.v.s.c.i.p.i.v.s.c.i.p.i.v.7/76/76/73/56/72/63/76/74/77/738.1±5.2"2.0

±0.81.9±0.51.4±0.71.6±0.40.7±0.41.0±0.43.1±0.80.9±0.32.0±0.320-610-60-40-30-30-20-2060-21-3

' Mean ±S.E.

C. granulosum and Fibrosarcoma Métastases

days for 5 x 10s, and 28.3 ±2.4 days for IO6tumor cells. In

mice treated with C. granulosum, no tumors were formed byIO5cells; 5 x IO5tumor cells generated tumors in 2 of 7mice, and IO6tumor cells generated tumore in 5 of 7 mice.

Treated mice developing tumors survived longer thancontrol mice.

Tumor take and survival of control mice given i.p.injections of FSA cells were similar to those of micereceiving tumor cells i.v. Tumor cells given i.p. to C.granulosum-lre-dled mice produced no tumors, however.

Compared with i.v. and i.p. injection, the s.c. injection oftumor cells led to a lower take of tumors in control mice.Thus, only 3 of 7 mice given injections of IO5 FSA cells

developed tumors. In mice treated with C. granulosum, avery peculiar pattern of growth of s.c. tumors was observed.Tumor take was not reduced, but more than one-half ofestablished tumors regressed. In the group receiving IO5

cells, however, all 7 mice developed tumors (more than incontrol group), but 4 of them regressed. Therefore, of a totalof 19 tumors developing in these mice, 11 regressed. Thetumors began to regress when they measured between 6 and14 mm in diameter. Tumors that did not regress grew moreslowly than those in control animals. Chart 1 shows thegrowth pattern of tumors in normal and C. granulosum-treated mice after the injection of IO6tumor cells.

Effect of C. granulosum Given after Tumor Cells. Micewere given i.v. injections of IO5FSA cells and, 2 or 7 dayslater, were treated with C. granulosum. One-half of treatedmice were sacrificed 14 days after tumor cell inoculationto check for the number of métastasesand the other halfwas left for a determination of the effect of the immunostimulant on the survival of the recipients. The results(Table 4) show that the number of lung colonies was significantly reduced if the mice were treated 2 days, but notif they were treated 7 days, after receiving tumor cells. Also,the colonies were smaller in mice that received C.granulosum 2 days after tumor cells; the diameter of 71colonies measured on 2 lung lobes of each mouse was 0.4 ±0.04, compared with 0.9 ±0.08 mm for 66 colonies on thesame 2 lung lobes of control mice.

Mice that received only tumor cells lived 22.7 ±0.8 days

Table 2The number of pulmonary métastasesinduced by different numbers of FSA cells injected i.v. into

normal or C. granulosum-treated C3Hf/Bu mice

Métastasesin lungs of

No. ofFSAcellsinjected(x

10')1248163264Mice

withmétastases/total

no.ofmice5/77/77/78/87/77/77/7Normal

miceNo.

/lung2.0

±0.8"8.0

±1.711.4±2.132.5±6.4112

±8.1ConfluentConfluentRange0-63

164-1817-5683-145Mice

treatedMice

withmétastases/total

no.ofmice1/72/74/70/72/74/77/7with

C.granulosumNo.

/lungRange<1

0-1<102<1

01<1

0-2<10-28.4

1 45

' Mean ±S.E.

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Luka Milas, Nancy Hunter, and H. Rodney Withers

Table 3Tumor take and survival of CiHf/ Bu mice treated with C. granulosum and a week later given

i.V., i.p., ors.c. injections of syngeneic FSA cells

No. of mice withtumor/total no.

Tumor cell injection of mice" Survival time (days)

Routei.v.i.p.s.c.No.105

x10105

x10105

x10«10't'10't'10*BControl6/67/77/77/78/86/76/67/73/7C.

granulosum5/72/70/70/70/70/73/72/73/7Control16.19.28.21315.117.33.51.54.59.32016±±±±±±=fc±±0.2.2.115.37117°24,4.775,3.5C.granulosum40.2

±8.324,82°61,

67,>100C78,>100153,>IOO, >100°

1Tumor take was scored up to 100 days.'Mean ±S.E.

Individual values.

32

28

24

1 20

I '•

£ 12

rf/Vn// *#?A0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Days After Fibrosarcoma Inoculation

Chart I. Growth of FSA in C3Hf/Bu mice, normal ( ) or treatedwith C. granulosum (- - - -). Vertical bars on control curve, S.E. The curvesfor treated mice trace the growth pattern of individual tumors.

(Table 4). However, 3 of 7 and 2 of 7 mice receiving C.granulosum 2 and 7 days, respectively, following tumor cellinoculation survived permanently. The other mice that diedlived longer than controls: 38.8 ±7.4 days for mice thatreceived C. granulosum 2 days after tumor cells and 30.6 ±3.6 days for those treated at 7 days. Therefore, in somecases, C. granulosum was able to induce complete regression of already established métastases,while in others itprolonged survival by about 35 to 70%.

Protection against Irradiation-induced Enhancement ofPulmonary métastases.Irradiation of mice before injectionof tumor cells increases the incidence of pulmonary colonies(27, 52). To determine whether C. granulosum can modifythe enhancing effect of irradiation on métastases,the micewere given i.p. injections of 0.5 mg C. granulosum and, 6days afterwards, were irradiated with 200, 400, 600, or 800

rads to the whole body. One day later the mice received i.v.injections of 2.5 x IO4FSA cells. Controls consisted of miceirradiated but not treated with C. granulosum, and unir-radiated mice, both untreated and treated with C.granulosum. The mice were sacrificed 14 days after tumorcell inoculation, with the exception of those irradiated with800 rads that were sacrificed 3 days earlier because ofirradiation sickness. Table 5 presents results showing thatirradiation of normal mice markedly increased the numberof lung métastases.In contrast to this, irradiation wastotally ineffective in increasing the yield of pulmonarynodules from the injection of 2.5 x IO4 cells into mice

previously treated with C. granulosum.Effect of C. granulosum on MCA. To determine the effect

of C. granulosum on the growth of a weakly antigenictumor, 3 or 6 x IO5 viable cells from spontaneous MCA

were injected i.v. into C3Hf/Bu mice, normal or treatedwith the immunostimulant (0.5 mg i.p.) 7 days earlier.Twenty-eight days later, approximately one-half of therecipients were sacrificed to determine the number ofcolonies in lung and the other one-half was kept to measuretheir survival time. Both the number of lung colonies andsurvival of recipients are presented in Table 6. C.granulosum significantly reduced the number of colonies inmice that received 6 x IO5but not in those that received 3 x10s viable cells. Also, C. granulosum prolonged the survival

of tumor cell recipients. In contrast to the effect on thenumber of lung colonies, the effect upon survival was moreevident when 3 x IO5tumor cells were injected.

Effect of Viable BCG on FSA Métastases.Groups of 9 to10 mice were given s.c., i.p., or i.v. injections of 1 mg of liveBCG and 7 days later they, in addition to 9 normal mice,were inoculated i.v. with IO5viable FSA cells. The number

of lung colonies was determined 16 days after the inoculation of tumor cells. By itself, the i.v. injection of BCGinduced nodular changes in lungs that made scoring of lungnodules uncertain. In mice given s.c. injections of BCG, thenumber of lung colonies was 32.4 ±3.6, 7.4 ±2.4 in thosegiven i.p. injections, and 64.4 ±8.1 in control mice. Thus,

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C. granulosum and Fibrosarcoma Métastases

Table 4The effect of C. granulosum given after FSA cells on the number of lung colonies and survival ofC3Hf/Bu

Métastasesin lung Survival of recipients

Days C. granulosumwasgivenaftertumor

cellsNo

treatment27"

Mean ±S.E."p<0.005.'

p < 0.01.Mice

withmétastases/total

no.ofmice7/77/77/7No.ofmétastases"99.3

±15.039.7±7.2"82.0

±8.0Range37-14216-6044105Mice

withtumors/totalmice11/114/75/7,Survival"(days)22.7

±0.838.8±7.4"30.6±3.6'Range21-2724-5824-44

Table 5The effect of whole-body irradiation on FSA métastasesin lungs of C3Hf/Bu mice, normal or

treated with C. granulosum"Irradiation was delivered I day before the i.v. injection of 2.5 x 10*FSA cells. C. granulosum

was given to mice 6 days before irradiation and 7 days before tumor cells injections.

Lung métastasesin

Normal mice Mice treated with C. granulosum

Irradiationdose(rads)None200400600800Mice

withmétastases/total

no.ofmice7/76/67/76/610/10No.ofmétastases11.4

±2.0°34.3

±5.931.3±6.075.0±6.387.5±7.0Range3

1718-5812-5560-9751-111Mice

withmétastases/total

no. of No.ofmicemétastases3/7

<12/71.7±0.82/7

<11/7<12/10

<1Range0-20-60-10-10-1

" Mean ±S.E.

Table 6Effect of C. granulosum on the number of lung colonies and survival of C3Hf/Bu mice given injections of

MCAcellsTreatment

ofrecipientsNoneC.

granulosum"

Mean ±S.E."p<0.005.cp< 0.025Wrt

rtfINO.OlMCAcellsinjected(xlOs)3636Métastases

inlungMice

withmétastases/total

no.ofmice6/77/75/75/6No.ofmétastases"3.3

±0.96.3±1.42.0±0.82.3±0.5rRange072-130-503Survival

ofrecipientsMice

withtumors/total

ofofmice7/77/77/75/5Survival"(days)55.9

±2.853.1±4.676.9±5.6'64.4

±3.7Range47-6539-72599856-73

viable BCG reduced the incidence of pulmonary colonies,but it appears that the effect is not as strong as that obtainedwith killed C. granulosum (Tables 1 and 2).

DISCUSSION

Nonspecific stimulators of the RES recently have been

used with success in the immunological therapy of a varietyof tumors of experimental animals (3, 5, 8, 10, 13, 21, 22,26, 28, 33, 39, 40, 49, 51, 53, 54, 56-58) and of humans (7,12, 19, 20, 25, 30). Other than BCG (5, 7, 12, 22, 30, 33, 49,56 58), C. parvum (3, 8, 10, 13, 19-21, 25, 26, 28, 39, 40,53, 54) has most frequently been used for that purpose. Ourpresent study shows that another Corynebacterium, C.

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Luka Muas, Nancy Hunter, and H. Rodney Withers

granulosum. can elicit strong antitumor activity and mightalso be used for immunotherapy of solid tumors. Thenumber and size of pulmonary métastasesgenerated byi.v.-injected FSA cells was greatly reduced in mice previously treated with C. granulosum. Even the number oftumor cells that, within 14 days from injection, completelyreplace the lung tissue of normal mice with tumor tissueproduced only a few colonies in mice treated with thisimmunostimulant. However, treated mice were more resistant to FSA cells injected i.p. and less resistant to thoseinjected s.c. (Table 3). This confirmed previous observationsby one of us (37) and by others (47, 50) that immunologicalresistance is not equally expressed against tumors growingat different anatomical locations. Given to mice 2 days aftertumor cells, C. granulosum was also effective. Administered7 days following tumor cell inoculation, it did not affect thenumber of lung colonies as checked 7 days later, but itprolonged the survival of mice. Some mice survived permanently, implying that C. granulosum induced completeregressions of established tumors. The peculiar observationwas that FSA cells injected s.c. into C. granulosum-trealedmice generated tumors as in normal mice, but when tumorsin C. granulosum-lrenled mice grew to a certain size, morethan 50% of them regressed completely. This is the 1streport, to the authors' knowledge, that syngeneic solid

tumors first appeared and then regressed totally in animalspretreated with nonspecific immunostimulants.

The mechanisms whereby C. granulosum exerted anantitumor effect were not sought in this study but possiblythey are similar to those of other nonspecific immuno-stimulators. Probably, by inducing proliferation of thelymphoreticular tissues, C. granulosum potentiated theresponse to tumor-specific antigens. Consistent with this isthe stronger effect achieved against FSA than againstweakly antigenic MCA. Additional evidence would be thats.c.-growing tumors first appeared and then regressed in C.granulos um-lK'died mice. It is possible, however, that thecells from the lymphoreticular tissues of nonspecificallystimulated animals can destroy tumor cells in a nonspecificway. Macrophages are particularly effective in this way (2,16), although recently it has been reported that T-cells fromBCG-treated mice can also destroy tumor cells (29). Othermechanisms, recently discussed by Weiss (48), might alsohave been involved in the antitumor activity of C.granulosum.

It is important to mention that C. granulosum did induceresistance even against MCA. which contains very feebletumor antigens (38). The effect was expressed both in thereduction of lung métastasesand in the prolongation ofsurvival of mice. However, reduction of métastaseswasobserved with the inoculation of a higher number of tumorcells, a finding for which we have no satisfactory explanation at present. The resistance to MCA was less than thatobserved against antigenic FSA.C. granulosum-'mduced resistance to pulmonary métas

tases cannot be abolished by irradiating the animals with200 to 800 rads to the whole body 1 day before injection oftumor cells. Irradiation is known to markedly inhibithumoral and cellular response (45), the function of B- and

T-cells, respectively. The reasons for the lack of suppressiveactivity of irradiation in C. granulosum-lre'dled mice are notclear. It might be that, under the influence of C. granulosum. all types of lymphoid cells proliferated to such anextent that enough of them remained undamaged afterirradiation to respond efficiently against tumor antigens. Orthe macrophages, which are not depleted by irradiation (11,36), may be sufficient to deal alone, by nonspecific means,with the injected tumor cells. The observation that C.granulosum-ire'dled mice are most resistant to tumor cellsinjected i.p. (Table 3) suggests that macrophages mightindeed play an important role in this resistance. Whateverthe mechanism(s) might be, the observation is important forcancer therapy; there are reports that radiotherapy of alocal tumor in patients might be immunosuppressive andthus facilitate the formation of métastases(41, 42). If suchan immunosuppressive effect occurs, nonspecific immuno-stimulators given before local tumor irradiation could notonly counteract this effect of irradiation, but also result indestruction of tumor cells, apart from irradiation. Houc-hens et al. (17) recently reported similar protection againsttumor growth in cyclophosphamide-immunosuppressedmice that had previously received BCG and that weresubsequently inoculated with Maloney sarcoma virus.

The use of live bacteria in immunotherapy of solid tumorshas been advocated (5, 12, 56-58) because they stimulatepredominantly cellular immune response (23, 24), and cellsof solid tumors are generally considered to be destroyed byimmune cells. However, factors present in the serum oftumor hosts can interfere with the action of lymphocytes (4,15), although there are recent studies, performed both invivo and in vitro, suggesting that the role of enhancingserum factors in the relentless growth of solid tumors mayhave been overemphasized (9, 27, 46, 55). Killed bacteriaare generally thought to stimulate humoral immunity, butkilled C. parvum. like viable BCG, has manifested a strongadjuvant effect against a variety of solid tumors (8, 10, 19,20, 28, 39, 40, 53, 54). In the present study, viable BCGshowed no advantage over killed C. granulosum in reducingthe number of FSA colonies. In fact, BCG was lesseffective, particularly if applied s.c. Furthermore, s.c.-grow-ing tumors regressed in mice pretreated with C. granulosum. a phenomenon not known, to the authors' knowledge,

to be applicable for BCG treatment.

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1974;34:613-620. Cancer Res   Luka Milas, Nancy Hunter and H. Rodney Withers  Mice

inArtificial Pulmonary Metastases of a Syngeneic Fibrosarcoma -induced Protection againstCorynebacterium granulosum

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