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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.
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).
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
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.
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
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).
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
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.
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.
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.
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.
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,
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.
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.
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
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.
REFERENCES
1. Adlam. C.. Broughton, E. S., and Scott, M. T. Enhanced Resistance ofMice to Infection with Bacteria Following Pre-treatment with Coryne-bacterium parvum. Nature New Biol.. 235: 219 220, 1972.
2. Alexander, P.. and Evans. R. Endotoxin and Double Stranded RNARender Macrophages Cytotoxic. Nature New Biol. 232: 76-78, 1971.
4. Baldwin, R. W.. Bowen. J. G., and Price, M. R. Detection ofCirculating Hepatoma Antigen and Immune Complexes in TumorBearer Serum. Brit. J. Cancer, 28: 16-24, 1973.
5. Bartletl, G. L., Zbar, B., and Rapp. H. J. Suppression of Murine
Tumor Growth by Immune Reaction to the Bacillus-Calmeite-GuerinStrain of Mycobacterium bovis. J. Nati. Cancer Inst., 48: 245-257,1972.
6. Biozzi, G., Howard, J. G., Mouton, D., and Stiffel. C. Modificationsof Graft-vercui-Host Reaction Induced by Pretreatment of the Hostwith M. tuberculosis and C. parvum. Transplantation, 3: 170-177,1965.
7. Bluming. A. Z., Vogel, C. L., Zeigler, J. L., Mody, N., and Kamaya,C. Immunological Effect of BCG in Malignant Melanoma: TwoModes of Administration of the Compound. Ann. Intern. Med., 76:405-411, 1972.
8. Currie, G. A., and Bagshawe, K. D. Active Immunotherapy withCorynebacierium parvum and Chemotherapy in Murine Fibrosar-comas. Brit. Med. J., /: 541-544, 1970.
9. Deckers, P. J., Davis, R. C., Parker, G. A., and Mannick, J. A. TheEffect of Tumor Size on Concomitant Tumor Immunity. Cancer Res.,33: 33-39, 1973.
10. Fisher, J. C., Grace, W. R., and Mannick, J. A. The Effect ofNonspecific Immune Stimulation with Corynebacierium parvum onPatterns of Tumor Growth. Cancer, 26: 1379-1382, 1970.
11. Gillette, R. W., and Lance, E. M. Kinetic Studies on Macrophages.IV. Effect of Irradiation. J. Reticuloendothelial Soc., 14: 18-25, 1973.
12. Gutterman, J. U., McBride, C., Freireich, E. J, Mavligit, G., Frei, E.,Ill, and Hersh, E. M. Active Immunotherapy with B.C.G. forRecurrent Malignant Melanoma. Lancet, /: 1208 1212, 1973.
13. Halpern, B. N., Biozzi, G., Stiffel, C., and Mouton, D. Inhibition ofTumor Growth by Administration of Killed Corynebacierium parvum.Nature, 212: 853-854, 1966.
Corynebacierium parvum. J. Reticuloendothelial Soc., /: 77-96. 1964.15. Hellström,K. E., and Hellström,I. Immunological Enhancement as
Studied by Cell Culture Techniques. Ann. Rev. Microbio!., 24:373 398, 1970.
16. Hibbs, J. B., Jr., Lambert, L. H., Jr., and Remington, J. S. Control ofCarcinogenesis: A Possible Role for the Activated Macrophages.Science, /77: 998-1000, 1972.
18. Howard, J. G., Biozzi, G., Stiffel, C., Mouton, D., and Liacopoulos, P.An Analysis of the Inhibitory Effect of Corynebacierium parvum onGraft-viVJuj-Host Disease. Transplantation, 5: 1510-1524. 1967.
19. Israel, L., and Edelstein, R., Nonspecific Immunostimulation withCorynebacierium parvum in Human Cancer. In: ImmunologicalAspects of Neoplasia, M. D. Anderson Hospital and Tumor Instituteat Houston. Baltimore: The Williams & Wilkins Co., in press.
23. Mackaness. G. B. The Relationship of Delayed Hypersensitivity toAcquired Cellular Resistance. Brit. Med. Bull., 23: 52-54, 1967.
24. Mackaness, G. B. Delayed Hypersensitivity and Its Significance. In: E.C. Chamberleyne (ed.). Immunization in Tuberculosis, United StalesDepartment of Health, Education, and Welfare Publication No.
28. Milas, L., and Mujagic, H. Protection by Corynebacierium parvumagainst Tumor Cells Injected Intravenously. European J. Clin. Biol.Res., 17: 498-500, 1972.
29. Mitchell, M. S., Kirkpatrick, D., Mokyr, M. B., and Gery, I. On theMode of Action of BCG. Nature New Biol., 243: 216 218, 1973.
30. Morton, D., Eilber, F. R., Malmgren, R. A., and Wood, W. C.Immunological Factors which Influence Response to Immunotherapyin Malignant Melanoma. Surgery, 68: 158-164, 1970.
34. O'Neill, G. J., Henderson, D. C., and White, R. G. The Role of
Anaerobic Coryneforms on Specific and Nonspecific ImmunologicalReactions. I. Effect on Particle Clearance and Humoral and Cell-Mediated Immunological Responses. Immunology. 24: 977 995, 1973.
35. Raynaud, M., Kouznetzova, B., Bizzini, B.. and Chermann. J. C.Etude de 1' Effect Immunostimulant de Diverses Espèces de
38. Silobrcic, V., and Suit, H. D. Tumor-specific Antigen(s) in aSpontaneous Mammary Carcinoma of C3H Mice. I. Quantitative CellTransplants into Mammary-Tumor-Agent-Positive and -Free Mice. J.Nati. Cancer Inst.. 39: 1113 1119, 1967.
39. Smith, L. H.. and Woodruff, M. F. A. Comparative Effect of TwoStrains of C. parvum on Phagocytic Activity and Tumour Growth.Nature, 219: 197 198, 1968.
40. Smith, S. E., and Scott, M. T. Biological Effects of Corynebacleriumparvum. HI. Amplification of Resistance and Impairment of ActiveImmunity to Murine Tumours. Brit. J. Cancer, 26: 361 367, 1972.
41. Stjernswärd.J. Immunological Changes after Radiotherapy for Mammary Carcinoma. Ann. Inst. Pasteur, 122: 883 894, 1972.
42. Stjernswärd,J.. Jondal, M., Vanky, F., Wigzell. H., and Sealy, R.Lymphopenia and Change in Distribution of Human B and TLymphocytes in Peripheral Blood Induced by Irradiation for Mammary Carcinoma. Lancet, /.' 1352 1356, 1972.
43. Suit, H. D., and Kastelan, A. Immunologie Status of Host andResponse of a Methylcholanthrene-Induced Sarcoma to Local X-Irradiation. Cancer, 26: 232 238, 1970.
44. Suit, H. D., and Suchato, D. Hyperbaric Oxygen and Radiotherapy of
48. Weiss, D. Current Aspects of Tumor Immunology. Israel J. Med. Sci.,9: 205-216, 1973.
49. Weiss, D. W., Bonhag, R. S., and DeOme, K. B. Protection Activity ofFractions of Tubercle Bacilli against Isologous Tumors in Mice.Nature, 190: 889-891, 1961.
50. Wexler, H., Chretien, P. B.. and Ketcham. A. S. The Fate ofCirculating Methylcholanthrene Tumor Cells in Mice with Tumor-Specific Immunity. Cancer, 28: 641-646, 1971.
53. Woodruff, M. F. A., and Boak, J. L. Inhibitory Effect of Injections ofCorynebacterium parvum on the Growth of Tumour Transplants inIsogeneic Hosts. Brit. J. Cancer, 20: 345-355, 1966.
54. Woodruff, M. F. A., and Inchley, M. P. Synergistic Inhibition ofMammary Carcinoma Transplants in A-Strain Mice by AntitumorGlobulin and C. parvum. Brit. J. Cancer, 25: 584-593, 1971.
55. Zarling, J. M., and Tevethia, S. S. Transplantation Immunity toSimian Virus 40-Transformed Cells in Tumor-Bearing Mice. I.Development of Cellular Immunity to Simian Virus 40-SpecificTransplantation Antigens during Tumorigenesis by TransplantedCells. J. Nati. Cancer Inst., 50: 137-147, 1973.
57. Zbar, B., Bernstein, I. D., and Rapp, H. J. Suppression of TumorGrowth at the Site of Injection with Living Bacillus-Calmette-Guerin.}. Nati. Cancer Inst., 46: 831-839, 1971.
58. Zbar, B.. Wepsic, H. T., Borsos, T., and Rapp, H. J. Tumor-GraftRejection in Syngeneic Guinea Pigs: Evidence for a Two-StepMechanism. J. Nati. Cancer Inst., 44: 473 481, 1970.