[CANCER RESEARCH 30, 849-854, March 1970]

Effect of Oncolytic Anaerobic Spores on Animal Cell Cultures

P. Rousseau,1 A. Chagnon, and V. Predette

Institute of Microbiology and Hygiene of Montreal University [K. F., A. C.], and Department of Microbiology and Immunology of the MedicalSchool [P. R., V. F.], Montreal University, Montreal, Quebec, Canada

SUMMARY

Germinated spore suspensions of Clostridium M55 orClostridium C185 added to 11 animal cell cultures propagated in vitro exerted cytotoxic and inhibitory effects onlyon certain types of cells. Resistance of some cell culturesseemed to be related to the diploid character, while thesensitivity of others was related to the heteroploid character, which is a feature of most cancer cells. After only20 to 30 hr of contact, sensitivity of heteroploid cells togerminated clostridial spores was observed. Karyokineticactivity was arrested and cells, instead of elongating, remained spheroid; intracytoplasmic granules appeared andthe nucleus became pyknotic. Finally, some of the cellsbecame detached from the monolayer and eventuallyformed debris which could easily be seen after 3 to 5 dayscontact. Since at no time did the germinated clostridialspores penetrate inside the animal cells, it would appearthat they act at a distance, presumably through the agencyof a soluble substance.

INTRODUCTION

The diversity of metabolic products given off by microorganisms defies the calculations of the most fertile mind;this is sufficient reason for entertaining the hope that theymay harbor a truly specific antitumor agent. Several indications already point the way toward this possibility. In1925, Warburg (12) suggested that some types of cancercells in vitro use energy mechanisms similar to those ofcertain yeast cells. Several years later, Malmgren andFlanigan (5) demonstrated the strong affinity of Clostridium tetani spores for tumor tissue in the mouse. Finally,oncolysis of Ehrlich's solid tumor in the white mouse was

reported by Möseand Möse(6), who, after injecting sporesuspensions of Clostridium M55 into the tail vein of whitemice bearing 14-day-old tumors, saw massive colonizationof the germinating spores almost exclusively in the tumortissue. This elective nidation was translated in a few daysby a softening of the tumor, which eventually broke downto produce a purulent discharge. The animals usually diedfrom the extensive tissue loss. These preliminary experi-

1In partial fulfillment of the requirements for M.Sc. degree. Present

address: Laboratory of Molecular Biology, University of Wisconsin,Madison, Wis. 53706.

Received April 14, 1969; accepted August 25, 1969.

ments were confirmed with varying success in 1964 in dif-.ferent species of laboratory animals with spontaneous ortransplanted tumors (3, 4, 7, 9, 10), but no hint was givenas to the intimate mechanism of this effect. It was therefore felt that the addition of anaerobic spores to animalcell cultures propagated in vitro might throw some lighton this interesting phenomenon.

MATERIALS AND METHODS

Two anaerobic spore formers were used throughout thiswork: Clostridium M55, supplied by Dr. MösethroughDr. G. E. Boxer of Rahway, N. J., and Clostridium C185,obtained later from Dr. E. Thiele of the same laboratory.All these strains were stocked in brain medium sealedunder vacuum and kept in the dark at room temperature.When needed, they were rejuvenated by 2 successivetransfers in either Prévot'sVF broth (8) or Trypticase

Soy Broth (Baltimore Biological Laboratory, Baltimore,Md.) contained in Hall tubes; all media used for rejuvenation, as well as for production, were freshly regeneratedbefore seeding by placing them in boiling water or in theunpressurized autoclave for 20 min, and then cooling themat 45°.Spore suspensions were prepared by transferring

the actively growing cultures into cellophane tubes according to the method of Vinet and Predette (11), usingTrypticase Soy Broth as the production medium; thespent medium was replaced daily for 3 days, at whichtime the cultures were left to sporulate for 1 week. Thismethod has the advantage of providing heavy growths ofrefractile spores without having to resort to special methods of concentration. Harvesting was done by centrifuga-tion in 600-ml bottles at 1370 X g for 60 min in the cold.The sediment was washed twice with chilled 0.9% NaClsolution and three times with chilled phosphate-bufferedsaline: final dilution was made to 50% T at 600 m^. Suspensions were kept frozen at —20°until needed.

Germination of the spores was obtained by placingthe thawed suspensions of spores into dialysis tubing immersed in Morgan's Medium 199 buffered at pH 7.4 withEarle's balanced salt solution: the process was followed

by staining, as well as by a decrease in absorbance at 600nifi. After centrifugation at 13,000 X g for 15 min, thegerminated spores were kept in an ice bath until theywere added to the animal cell cultures.

Eleven cell cultures (Table 1) were examined after addition of the germinated spore suspensions. Six of these

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P. Rousseau, A. Chagnon, and V. Predette

Table 1Animal cells examined

Label Origin TypeC'.iromosome No.

Culture

LeL929EhrlichME

(Swiss)ME(C3H/Hej)RK-13RKLLC-MK-12ÃœMKHeLaWl-38MouseMouseMouseMouseMouseRabbitRabbitMonkeyMonkeyHumanHumanFibroblasticFibroblasticFibroblasticFibroblasticFibroblasticEpithelioidMixedEpithelioidMixedEpithelioidFibroblasticHeteroploidHeteroploidHeteroploidDiploidDiploidHeteroploidDiploidHeteroploidDiploidHeteroploidDiploidEstablishedEstablishedEstablishedPrimaryPrimaryEstablishedPrimaryEstablishedPrimaryEstablishedNonestablished

were heteroploid and 5 were diploid. Five were of mouseorigin, 2 were of human origin, and the remainder camefrom either the rabbit or the monkey. Finally, only 3 cellcultures were prepared from embryonic material; all theothers originated from immature and adult animals. Only2 of the cell lines were malignant for adult mice: the Lestrain, kindly supplied by Dr. S. Fedoroff of the University of Saskatchewan, and the Ehrlich strain, graciouslyfurnished by Dr. J. F. Morgan, also of Saskatchewan. Allthe other cell lines, including material from tumor tissuesuch as the HeLa strain, were nonmalignant. Strains ofpermanent cell lines (Le, L929, Ehrlich, HeLa, RK-13,and LLC-Mk2) have been maintained with the help ofthe usual methods. RK and GMK cell cultures, as wellas human diploid cells (WI-38), were prepared accordingto the technique of Chagnon and Pavilanis (1, 2). Mousediploid cell cultures were obtained from 16- to 18-day-oldembryos from either Swiss or C3H/HeJ mice. It was firstestablished that Clostridium M55 spores do not producevegetative cells in any of the media used for propagatingor suspending the animal cells, while Bacillus subtilis andBacillus cereus spores were able to do so under identicalconditions. In no case have we been able to isolate afterward any viable vegetative cells from the cell cultures towhich had been added the germinated clostridial sporesuspensions.

For our tests, germinated spore suspensions containing0.7 mg/ml, dry weight, were diluted decimally in phosphate-buffered saline from 10~' to 10~', andaliquots rang

ing from 0.1 to 0.5 ml of each dilution were added to 5 to6 tubes of each type of 20-hr-old animal cells. In otherwords, germinated spores ranging in number from 20 to20 X IO6 were used for about 100,000 to 150,000 cells.

Cells were grown in roller tubes, Leighton tubes, andsmall Petri dishes. Since cell production occurred moreor less rapidly in a closed system, according to the type ofcell system, the pH of each culture was consistently adjusted to from 6.8 to 6.9 by adding sodium bicarbonatesolution. Incubation of Petri dish cell cultures in a 5%COa-95% air incubator consistently gave a uniform pHin all cultures at the end of the experiments.

RESULTS

Evidence of oncolytic activity in our strain of Clostridium M55 is shown in Fig. 1, where it can be seen that a

gaping hole in the Ehrlich solid type of tumor has resultedfrom the i.v. injection of 20 to 40 X IO6spores in C3H/

HeJ mice. Identical results were obtained on solid tumorsinduced by injection of Le cells. Fig. 2 shows a microscopic section of an Le tumor 3 days after injection of thespores; it may be seen that part of the tumor is still unaltered. Seven days after such an injection, however, complete lysis of the central portion of the tumor has occurredand only sporangia and vegetative cells can be seenamong the debris of the cancer cells (fig. 3). M55 and C185spores were equally effective under these conditions inwhite mice as well as in C3H/HeJ mice bearing eitherspontaneous tumors or tumors produced by injection ofLe cells propagated in vitro.

Concerning the in vitro experiments, control culturesof Le cells are shown in Fig. 4 (35-hr-old) and Fig. 5 (120hr old); it is evident that during this interval of time thespheroid cells have elongated and formed a continuoussheet or monolayer.

Effect on Le Cells. If 0.5 ml of a 1 to 1000 dilution ofgerminated M55 spore suspension is added to a 18-hr-old culture of malignant Le cells, it will be noticed after30 hr of contact that the density of the population is diminished; moreover, the remaining cells do not elongate(Fig. 6). Some cells may even swell and show intracyto-plasmic granules. Finally, the cells tend to form aggregates rather than a monolayer; after 48 hr of incubation,some cells may be seen floating in the culture medium,and after 5 to 6 days of contact, some cells undergo pyk-nosis and lysis. A similar state of affairs was obtained withC185 spores on Le cells after 120 hr (Fig. 7). If, after 50hr of contact, the spores are removed by washing andfresh medium is added, it will be found that some cellsare still capable of elongating and forming a monolayer,thereby suggesting a reversible effect of enzymatic nature. At no time did the spores penetrate inside the animal cell, an indication that those effects occurred throughthe agency of a soluble substance. Since Clostridium M55spores do not produce toxin of any kind (as determined bymouse inoculation of cellophane tube cultures), the observed cytotoxic and inhibitory effects must be attributedto some other agent.

Effect of Ehrlich Cells. Germinated spores of Clostridium M55 added to malignant Ehrlich cells propagated invitro produced cytotoxic effects similar to those describedfor Le cells. Once again, it was noted that the spores donot penetrate the animal cells before destroying them.

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Oncolytic Anaerobic Spores

Effect on L929 Cells. Growth of L929 cells was significantly altered by germinated M55 spores. Here also, theoncolytic spores seem to act at a distance, probablythrough the agency of a soluble substance.

Effect of HeLa Cells. Both M55 and C185 germinatedspore suspensions were effective within 3 days in producing detachment of HeLa cells from the glass and therebyreducing the density of the cell population. Although originating from tumor tissue, these cells are nonmalignantafter propagation in vitro. Although of epithelioid nature,they are altered as well as fibroblastic cells. Once more,it appears that oncolysis occurs through a soluble agent.

Effect on RK 13 Cells. Although nonmalignant and epithelioid, these cells were altered by 20 hr of contact witheither M55 or C185 germinated spore suspensions. After3 days, gaps in the monolayer are easily visible, as are cellaggregates and floating cells. Two days later, the cellsstill attached show pyknosis. Penetration of the sporesinside the animal cells again was not seen.

Effect on LLC-MK-2 Cells. Slightly less sensitive thanthe preceding cell line, these epithelioid and nonmalignant cells showed after only 3 days the same alterationsdescribed for RK-13 cells. Oncolysis takes place withoutpenetration of the spores inside the animal cells.

Effect on WI-38 Cells. Originating in the human lung,these cells are fibroblastic and nonmalignant. No alteration could be detected in this diploid cell strain even after6 days of contact with germinated spore suspensions ofeither M55 or C185.

Rabbit Kidney Cells. Primary cultures of rabbit kidney(containing both fibroblastic and epithelioid cells) showedafter only 3 days a very slight decrease in confluenceby contact with either M55 or C185 germinated spores,and was most difficult to evaluate. Incubation for 3 moredays did not increase this barely visible effect.

Monkey Kidney Cells. Primary cultures of monkey kidney showed a greater number of detached cells than thepreceding cell culture, but no other visible cytotoxic orinhibitory effect could be seen, even after 6 days of incubation in the presence of C185 or M55 germinatedspores.

Effect on Embryonic Cells (Swiss). In this case, no difference could be found between the control cultures andthose cultures which had been incubated with germinatedM55 spores, even when 2 different lots of spore materialwere used (Fig. 8).

Effect on Embryonic Cells (C3H/HeJ). Here again, novisible alteration couídbe detected in the cultures addedwith germinated M55 spores when they were comparedwith the control cultures.

DISCUSSION

In the light of these results, it appears that germinatedspore suspensions of oncolytic clostridia (M55, C185) ex

ert an inhibitory effect on malignant cells, as well as oncells which, although originating from tumor tissue andhaving lost their malignancy through propagation in vitro,nonetheless maintain their heteroploid character. In contrast, diploid cells derived either from animal or humanembryos or from immature or adult animals, do not exhibit such a sensitivity to germinated oncolytic spores. Itwould thus be reasonable to conclude that sensitivity tooncolytic spores in vitro, as well as in vivo, is related tothe heteroploid character, whereas resistance is linkedwith the diploid character. Since no vegetative clostridiawere isolated in the cell cultures at the end of the experiment, and since no germinated spores could be foundwithin the cells placed under experiment, it is reasonableto assume that this specific activity seems to result fromthe action of a soluble substance given off by the germinated spores of Cloxtridium M55 into the surroundingmedium.

REFERENCES

1. Chagnon, A., and Pavilanis. V. L'Etablissement de Souches Cellu

laires DiploïdesHumaines m Vitro. Rev. Can. Biol., 74:35 43, 1965.2. Chagnon, A., and Pavilanis. V. Freezing and Storing of Monkey

and Rabbit Kidney Cells for Tissue Cultures. Cryobiology, 3(2): 8184, 1966.

3. Engelbart, K., and Gericke, D. Oncolysis by Clostridia. V. Transplanted Tumors of the Hamster. Cancer Res., 24: 239-243. 1964.

4. Gericke, D., and Engelbart. K. Oncolysis by Clostridia. II. Experiments on a Tumor Spectrum with a Variety of Clostridia in Combination with Heavy Metal. Cancer Res., 24: 217 221, 1964.

5. Malmgren. R. A., and Flanigan. C. C. Localization of the Vegetative Form of Ctosiridium tetani in Mouse Tumours Following Intravenous Spore Administration. Cancer Res., 15: 473 478, 1955.

6. Mose. J. R.. and Möse,G. Onkolyseversuche mit apathogenen an-aeroben Sporenbildern am Ehrlich Tumor des Maus. Z. Krebsforsch., 63: 63 74, 1959.

7. Möse,J. R., and Möse,G. Oncolysis by Clostridia. I. Activity ofClostridium butyrícum(M-55) and other Nonpathogenic Clostridiaagainst the Ehrlich Carcinoma. Cancer Res., 24: 212 216, 1964.

8. Prévôt.A. R., and Boorsma. H. J. Répartitionde l'A/.ote et Mé

tabolisme Azotédans la ToxinogénèseTétanique.Ann. Inst. Pasteur. 63: 600 610, 1939.

9. Thiele. E. H.. Arison. R. N., and Boxer, G. E. Oncolysis by Clostridia. HI. Effects of Clostridia and Chemotherapeutic Agents onRodent Tumors. Cancer Res., 24: 222-233, 1964.

10. Thiele, E. H., Arison, R. N., and Boxer, G. E. Oncolysis by Clostridia. IV. Effect of Nonpathogenic Clostridial Spores in Normaland Pathological Tissues. Cancer Res., 24: 234-238, 1964.

11. Vinel, G., and predette, V. Apparatus for the Culture of Bacteriain Cellophane Tubes. Science, 114: 549 550, 1951.

12. Warburg. O. Metabolism of Carcinoma Cells. J. Cancer Res., 9:148-163, 1925.

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P. Rousseau, A. Chagnon, and V. Predette

Fig. I. Swiss mice bearing Ehrlich carcinoma. Top, control animal, bottom, 1 days after i.v. injection of M55 spores.Fig. 2. Histológica!section of border of Le solid tumor 3 days after i.v. injection of M55 spores.Fig. 3. Histológica!section of center of Le solid tumor 7 days after i.v. injection of M55 spores.Fig. 4. Le cells, 35-hr-old control culture. X 40.Fig. 5. Le cells, 120-hr-old control culture. X 50.Fig. 6. Le cells, 36-hr-old, 50 hr after addition of M55 spores. X 50.Fig. 7. Le cells, 36-hr-old, 120 hr after addition of C185 spores. X 50.

Fig. 8. Mouse embryonic cells (Swiss) after 120 hr of contact with M55 spores. X 40.

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1970;30:849-854. Cancer Res   P. Rousseau, A. Chagnon and V. Fredette  Effect of Oncolytic Anaerobic Spores on Animal Cell Cultures

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