Localization of the Vegetative Form of Clostridium tetani inMouse Tumors Following Intravenous Spore Administration

RICHARD A. MALMGREN AND CLYDE C. FLANIGAN

(National Cancer Institute and Division of Pathology and Microbiology, University of Tennessee, Memphi8, Thin.)

Studies of the effect of bacteria on neoplasmsare among the oldest approaches to the problem ofcancer therapy. In 1868, Busch (2) reported temporary clinical improvement in two patients withsarcoma who developed concurrent erysipelas.Subsequent studies, identifying streptococci andother micro-organisms involved in tumor destruction, ascribed their effect to a toxic bacterial product (3) and established an investigative patternthat has dominated the bacterial approach to thetumor problem in the intervening years. Perhapsas a result of this emphasis on bacterial productscapable of eliciting a deleterious effect on the tumor cell, little attention has been given to the possibility of localizing1 the bacteria per se in the tumor site. Although it is well recognized that bacteria are sometimes found in tumors, no consistency regarding their presence or type has been observed, and there are, to the best of our knowledge,no previous reports demonstrating that eitherthese chance contaminants or the intentional systemic administration of bacteria result in exclusivelocalization of the organism in the tumor site.

The cancer studies involving fungi and protozoahave been similarly oriented to the concept of atumor-destructive microbiologic product, and theexclusive localization of the test organism in tumors has not been reported. All these variousmicrobiologic technics have recently been reviewed by Reilly (7).

In recent viral studies, on the other hand, theestablishment of the virus in the tumor tissue hasbeen an important aspect of the investigation, andthere are numerous reports of attempts to adaptthe virus to the malignant cell (6, 8). This adaptation, based on the concept of an intrinsic differencebetween normal and neoplastic cells, has been

3 As used in this report “exclusive localization,― “restric

tion,―and synonymous terms denote the presence of the vegetative (antigenic) form of the bacteria in the tumor and, conversely, the absence of the vegetative (antigenic) form of thebacteria in the normal tissues of the tumor-bearing animal.

Received for publication March 21, 1955.

achieved in a number of instances, and the desiredobjective of tumor cell destruction has met with anencouraging degree of success. However, the unfavorable systemic response resulting from treatment with virus would suggest that the adaptationattained does not result in the exclusive confinement of the organism within the tumor tissue.Moreover, in studies designed to demonstrate thein vivo distribution of certain tumor tropic viruses,restriction of the organism to the tumor area wasnot demonstrable (1, 9).

The desirability of such restriction in relation tomicro-organisms or their products is, however,generally acknowledged. With this in mind, thepresent studies were conceived on the premise thatthe cancerous area in its entirety constitutes a distinctive in vivo lesion which might be exploited toachieve the exclusive tumor localization of systemically administered bacteria.

Basically, this intratumoral milieu is characterized by an increased rate of cellular proliferation.In addition, it has also been well established byWarburg (10) that anaerobic glycolysis is significantly increased in tumor tissue with a resultingaccumulation of lactic acid in the cancerous area.Focal centers of necrosis with accompanyingzones of low oxygen tension are also very frequently found in cancer sites. That there maybe alteration in the vascular system in tumortissues is suggested by the work of Youngner andAlgire (11). Cumulatively the in vivo environmentresulting from these distinctive tumor characteristics closely approximates the known growth requirements for certain bacteria of the genusClostridium.

Therefore, in the present study, the spore formof Cl. t4ani, one of the well characterized organisms of this group known to be nonpathogenic for

normal tissues, was administered systemically todetermine whether the vegetative (antigenic) formof this bacterium would localize exclusively in thecancerous areas.

473

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474 Cancer Research

In one instance, four mice bearing mammary tumor C3HBAreceived intracardiac injections of the spores.

Commercial tetanus antitoxin, when used, was administered intraperitoneally beginning with 300 units (0.2 ml.) 24hours after spore injection, followed by 150 units (0.1 ml.) at48 hours and 75 units (0.05 ml.) each day thereafter until theanimals were killed.

One group of animals received Cl. tetani spores only. Whenthese animals developed tetanus their tumors and organswere harvested for microscopic examination.

In another group of animals treated with both spores andantitoxin, the mice were killed at varying time intervals afterthe spore administration. The tumor and the normal organswere removed aseptically. One portion was homogenized forculturing, and the remainder was fixed in 10 per cent formalin,imbedded, sectioned at 6@ and stained with hematoxylin andeosin and with Gram stain. Direct smears were taken from thetumor before fixation and were Gram-stained.

Culture technics.—The tumor and the normal organs, removed aseptically from the treated mice, were weighed andhomogenized with sterile sand in a sterile Ten Broeck homoge

No. dead nizer. For each animal the organs (liver, lungs, spleen, andof teta- kidneys) were combined with 10 ml. of sterile physiological

nus5t saline solution and prepared as a one homogenate; the tumor,

6/6 combined with 5 ml. of sterile saline solution, was prepared as3/3@ a separate homogenate.2/2 Homogenates thus prepared were immediately diluted2/2 from 1 : 10 to 1 : 10,000 and inoculated on the surface of dupli

@ cate blood agar plates. The 0.25 ml. inoculum was spread

2/2 over the agar surface with a sterile glass spreader. In some2/2 tumor homogenates the dilution was carried to 1:10,000,000.2/2 After inoculation of the unheated specimens, the same tubes2/2 were heated at 78°C. for 20 minutes and inoculated in the2/2 same manner. No trouble was experienced with spreading4/4 colonies, as is often seen in strains of Cl. tekini.2/2 Colony counts were made on the plates after a 48-hour0 ‘8 anaerobic incubation period. Following the colony counts,0/6 direct morphologic examination of representative colonies0/6 was made. Other typical colonies were cultured in anaerobic0/9 thioglycolate broth and observed for typical spore formation.

In addition to the plates cultured anaerobically, a secondseries of blood agar plates were, in a number of representativecases, set up with the same inocula and cultured aerobically.

RESULTS

MATERIALS AND METHODS

Preparation of spores.—An alkaline deep meat infusionbroth, pH 7.4, was inoculated with Clostridium tetani2 andand incubated at 37°C. for 5 days. The liquid portion of thecultures was decanted, and after centrifugation at 900 X g for10 minutes the sediment was discarded. The supernatantwas recentrifuged at 3,000 X g for 10 minutes. The resultingsediment was suspended in 0.85 per cent saline solution andcentrifuged at 3,000 X g for 10 minutes. The last procedurewas repeated 2 more times. Following the final centrifugation,the sediment was resuspended in 0.85 per cent saline solution,heated at 73°C. for 20 minutes, and used for mouse inoculation.

Determination of the spore concentration administered tothe mice was made by inoculating 0.25 ml. of a serial twofold

TABLE 1

EFFECT OF THE INTRAVENOUSINJECTION OF Cl. tetaniSPORES ON TUMOR-BEARING AND NoR

a

Spontaneous mammary98/15 HepatomaHE 8971 fibro

sarcomaNone

aa

MAL CSH/He MICE

Tumor size(gm.)

2—72—52—4

a Allanimalsdyingof tetanusexpiredat approximately48hoursafterthe spores were injected.

t The numerator represents animals which died.The denominator represents animals injected.

t Spores administered intracardially.I Strain BALB/c.

dilution of the spore suspension on blood agar plates. Colonycounts were made on the plates after 5 days of anaerobic culture at 37°C.

Adminietra@ion of apores.—Male and female mice, strainC3H/He, over 2 months old, were used as test animals. Thetransplanted tumors tested were: (a) mammary tumorC8HBA, (b) fibrosarcoma HE 8971, and (a) Hepatoma 98/15.In addition, two female mice of the BALB/c strain bearingspontaneous mammary tumors and several female mice of theC3H/He strain also bearing spontaneous mammary tumorswere studied. The test tumors ranged in size from 0.8 gm. to10 gm. Nontumor-bearing mice of appropriate age, strain, andsex were used as controls.

Washed tetanus spores suspended in saline solution were injected intravenously into test animals. All the animals received a constant volume (0.5 ml.) of the spore suspension,although the spore count per injection ranged from 2,400,000to 9,375 spores per mouse, depending on the group studied.

Sporedose

2,400,000

I,200,000600,000300,000150,00075,00037,50018,7509,375

150,000

a

2,400,000150,000

75,000

Tumor

CSHBA mammary

a

aa

a

a

2—43—10

43—63—93

2.50.8-31—32—11

In the first series of tests washed Cl. tetanispores, derived from a strain known to be highlytoxic in the vegetative state, were injected intra..venously into 22 CSH/He mice bearing transplanted mammary tumor C3HBA. These tumorsranged in size from 2 to 10 gui., and in no instancewas superficial ulceration present. The spore dosewas varied from 9,375 to 2,400,000 spores per injection, although no attempt was made to correlate the number of spores administered with thetumor size. As shown in Table 1, all the animalsthus treated died from tetanus approximately 48hours after the spore injection. in all cases theirdeath was preceded by the convulsion, the musclespasms, the fiexion of the forelegs, and the extension of the hind legs characteristic of the development of tetanus.

Similar results were also obtained in three2 Cl. tetani j@8033 was obtained from the American Type

Culture Collection,2029 M. Street, N.W., Washington, D.C.

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MALMGREN AND Fi@IGAN—C1. tetani Localization in Mouse Tumors 475

C8H/He mice bearing 2-5-gm. transplantedCSHBA mammary tumors when the Cl. tetanispores were administered intracardially. In viewof the greater experimental hazards involved inthe latter procedure, however, the intracardiacroute was abandoned, and all subsequent spore injections were made intravenously.

Typical tetanic death also occurred in 48 hoursin both spore-treated C3H/He mice bearing spontaneous mammary tumors weighing 0.3 and 3 gm.,respectively. in these animals the number ofspores per injection was 150,000.

The administration of 150,000 spores to each offour C3H/He mice bearing Hepatoma 98/15 (1 to3 gm.) also resulted in typical tetanic death in 48hours. Two C3H/He mice bearing transplantedHE 8971 fibrosarcoma (2 and 11 gm.) were alsotested and died with tetanic symptoms approximately 48 hours after the injection of 150,000spores.

In all the latter tumors, as in all tumors studied,superficial ulceration was absent.

As a control group, fifteen normal C3H/Hemice and nine BALB/c mice were given injectionsintravenously of washed Cl. tetani spores derivedfrom the same spore preparation as that used intreating the tumor-bearinganimals. Three of thesemice received 2,400,000 spores each, six received150,000 spores each, and fifteen received 75,000spores each. All these animals survived and remained free from tetanic symptoms in the 30—40-day period during which they were observed.

As summarized in Table I , it can therefore beconcluded that the injection of washed Cl. tetanispores, which was without untoward eff@ectin normal animals, uniformly resulted in tetanic death inthe tumor-bearing host in approximately 48 hours,regardless of the tumor size, the tumor type, or thespore dose.

The 100 per cent incidence of tetanus in thespore-treated, tumor-bearing animals was indicative of the germination of the Cl. tetani spores inthis group. At 48 hours, microscopic examinationof both the tumor and the organs revealed thepresence of the vegetative form of the bacteria inthe cancerous area only. Subsequently, othergroups of tumor-bearing C8H/He mice weretreated with tetanus antitoxin to protect themagainst the lethal effects of the bacterial toxin sothey might@be observed over a longer period oftime. All these antitoxin-treated mice, bearing thesame tumors previously described, received intravenous injections of 600,000 spores each. Following the spore injection, microscopic examination of Gram-stained direct smears, and hema

toxylin and eosin- and Gram-stained sectionstaken from the tumor on the 3d through the 13thday revealed abundant growth of the vegetativeforms of the bacteria in this area. It was also ohserved, when the tumors were harvested, that themalignant area possessed the brownish color, thepeculiar odor, and the edematous reaction commonly associated with lesions supporting thegrowth of Cl. tetani. Focal areas of necrosis weregrossly evident in some and microscopically cvident in all tumors at the time of harvesting. Microscopic examination of the tumor sections alsorevealed that the vegetative forms of Cl. tetaniwere largely, although not exclusively, confined tothe necrotic areas. No vegetative forms of the tetanus organism were observed at any time in thecourse of the experiment in the parenchyma of theorgans (liver, lungs, spleen, and kidneys) harvested from the spore-treated, tumor-bearing host.

To further confirm the apparent localization ofthe vegetative form of Cl. tetani in the tumor area,a cultural technic, based on the differential susceptibility of the spore and the vegetative forms ofthe organisms to heat, was used. Because both thevegetative and the spore form of Cl. tetani will produce typical colonies when anaerobically culturedon an appropriate medium such as blood agar, thecolony count resulting from inoculation of anunheated material represents the combined totalof both forms of the organism present. Since heating at 73°C. for 20 minutes destroys all the vegetative forms without altering the viability of thespores, those Cl. tetani colonies arising from inoculations of heated material originate solely from thespores present in the inocula. Therefore, if a comparison of the colony count obtained from inoculaof the same material before and after heating results in a significant decrease in the number ofcolonies cultured from the heated fraction, it mdicates that the organisms present in the unheatedfraction were primarily vegetative organisms.

Applying this principle to the results, it is apparent that a comparison of the heated and unheated fractions of homogenates made from tumors and organs of antitoxin-protected, sporetreated mice reflects a marked localization of thevegetative form of the tetanus organism in the tu

mor site. In all the homogenates prepared from theseveral tumors previously described, heating uniformly and strikingly reduced the high bacterialcount of the unheated fraction of the homogenatesto nearly zero, indicating that nearly all the organisms in the unheated fraction were in the vegetative state. On the other hand, a comparison ofthe heated and unheated homogenates of the or

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The primary purpose of the present study wasto demonstrate that tumor tissue in vivo constitutes a distinctive lesion conducive to the selectivelocalization of systemically administered bacteria.In evaluating the phenomenon of germination ofthe spores and resultant localization of the vegetative form of Cl. tetani in tumor tissue, it is important to consider the relationship of several properties inherent in both the tumor and the bacterium.First, the tumor lesion in vivo possesses an environment which is clearly distinct from the environment of normal tissue. Secondly, the propertieswhich characterize the tumor environment are, forthe most part, constant for all malignant areas,and cumulatively they result in an intratumoralmedium which is very similar to the environmentnecessary for germination of Clostridium spores.Essentially, these tumor properties considered tobe important in the present study are anaerobicglycolysis, increased lactic acid concentration, focal zones of necrosis with associated low oxygentension, and possible vascular alterations in thetumor area.

Of equal, and possibly even greater, importancein attaining this tumor localization are certainproperties of the Cl. tetani organism itself. Beforeconsidering the properties of the bacterium in detail, however, it should perhaps be noted that, tothe best of our knowledge, the use of the intrave

0 120,000 216 304 nous route has not been previously reported for theadministration of Cl. tetani spores. However, the

0 528,000 230 250 work of Francis (5) and Fildes (4) demonstrates582 626 that the presence of spores in any normal tissue is

@@ completely without local or systemic ill effect in48 58 experimental animals and in humans; and, further,

60 72 that spores migrate from the site of subcutaneous

40 40 or intramuscular injection. The work of Francis

(5) would indicate, however, that the migration ofthe spore form injected subcutaneously or intramuscularly is a somewhat uncertain process.Therefore, the intravenous method was selected inthe present study because it seemed the most certhin manner of insuring deposition of the Cl. tetanispores in the tumor site.

As seen in the results, injection of the Cl. tetanispores by the intravenous route proved, despitethe undoubtedly wide dissemination of the organism, to be an innocuous procedure, since all thenoncancer-bearing animals so treated remainedwell, appeared normal, and exhibited no tetanicsymptoms. The nonpathogenicity of the sporeform of Cl. tetani and its inability to germinate innormal hosts or the normal tissues of cancer-bearing hosts should be emphasized, because it is one ofthe most important bacterial properties respon

476 Cancer Research

gans reveals no significant difference between thebacterial counts of these fractions, indicating thatin both the heated and unheated organ homogenates the colonies arose solely from the sporeform of Cl. tetani.

These results, which have been expressed interms of comparable dilutions on the basis of original weight, are summarized in Table 2. It is also of

DISCUSSION

TABLE 2

SPORE

TUMOR INJECTION Heatedt

CSHBA mammary

aa

aaaaa

HE 8971 fibresarcoma

aaa

98/15 hepatoma

BALB/c spontaneousmammary

CSH/He spontaneousmammary

a

Noneaa

aaU

Tumor

a Organs included liver, spleen, kidneys, and lungs.fHeatedto 73' C. for 20 minutes.t Too numerous to count at a dilution of 1/10,000.I Too numerous to count at a dilution of 1/10 million.I 2,400,000spores injected instead of 600.000.

Cl. Tetani ORGANISMS/MG OF TISSUE HOMOGENATEDETERMINED BY CoLoNY PLATE COUNTS

All treated animals received an intravenous injection of600,000 spores.

DAYS OaossIeMs/MooriissuzAFTER Organs5

UnReatedf heated

32 48

156 138114 78400 54626% 294252 410190 210

48 3662 48

128 12020 170

160 206122 140

264 154

Unheated

4 0 TNC@

S 2 2,740,0007 0 2,220,0007 0 2,760,0009 0 4,340,0009 102 2,540,0009 8 2,000,00013 6 2,500,000

3 0 TNC@

4 22 TNC@5 60 1,710,0007 2 TNC@3 6 TNC@

5 4 58,000

4

43o#SWSW404040

interest to note that the colony counts obtainedfor organ homogenates derived from spore-treated,tumor-bearing animals are essentially the same asthose obtained from organ homogenates of nontumor-bearing, spore-treated mice.

No vegetative Cl. tetani organisms were demonstrated on microscopic and cultural examinationof the tissues of nontumor-bearing, spore-treatedmice.

Morphologic and cultural examination of representative colonies from each culture revealed typical Cl. tetani organisms. No other organisms wereseen in either aerobic or anaerobic blood agar cultures.

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MALMGREN AND Fi@NIG@—Cl. tetani Localization in Mouse Tumors 477

sible for the localization of the vegetative form ofthe organism within the tumor area.

Equally important is the fact that the rigid requirements which are necessary to stimulate Cl.tetani to convert from the spore to the vegetativeform exist in cancerous lesions. In the spore formCl. tetani is widely distributed in nature, and thebacterium has been studied extensively in vivo because of the clinical manifestations resulting fromthe accidental acquisition of the spore and the germination of the organism in certain lesions. Thesenumerous observations concerning the conditionsnecessary for this germination in both accidentallyand experimentally induced lesions in the hosthave been substantiated and well summarized byboth Fildes (4) and Francis (5) . Fildes states thatlow oxygen tension is the prime requisite for germination of the Cl. tetani spores. He also points outthat areas of necrosis and the presence of lacticacid are contributory factors. Francis (5) supportsthis general conclusion and adds that the presenceof agents which inhibit leukocytes may also be acontributing factor. Some authors have suggestedthat symbiosis is necessary for tetanus germination ; however, no consistent symbiont has beendemonstrated, and in many reports no concomitant organisms have been found. In the presentwork, both aerobic and anaerobic blood agar platecultures failed to demonstrate any other bacteriaassociated with the germination of the tetanus organism. Although the relative importance of theindividual properties of the bacteria and the tumor area cannot be evaluated in relation to thisphenomenon, it can be concluded that, consideredcollectively, the characteristics of both of the entities contribute to the situation essential to germination of Cl. tetani spores in tumor tissue.

In the present study Cl. tetani was selected asthe initial test organism, not only because of itswell characterized and desirable properties, butalso because its production of a lethal toxin in thevegetative state made determination of its germination both simple and conclusive in the earlyexperiments. As shown in the results (Table 1), oneof the most striking aspects of the phenomenon isthe seemingly broad scope of the response as evidenced by the germination of spores, toxin production, and tetanic death occurring in mice bearingspontaneous carcinomas, transplanted carcinomas,and transplanted sarcomas. This response was ap..parently independent of tumor size and spore dose,and tetamc death occurred in approximately 48hours in all spore-treated, tumor-bearing mice.The uniformity of these results indicates that Cl.tetani spores germinate' in the tumor site under awide variety of circumstances.

This localization of the vegetative form of Cl.tetani was confirmed both microscopically and culturally in all the tumors (Table @).It was furthernoted that almost all the organisms present in thetumor area were in the vegetative state. Sincemicroscopic examination revealed that these vegetative forms were associated with, but not confined entirely to, the necrotic areas in tumors, it isinteresting to speculate, in view of the nonnecrotictissues also present, whether this implies that theinjected spores were deposited only in the necrotictumor areas (which seems unlikely in view of theiroccurrence in normal organs), or whether the conditions existing throughout the tumor lesion resultin germination of all the spores reaching the siteregardless of the viability of the cells in the immediate vicinity.

Since examination of the tumor tissue for thepresence of necrosis prior to spore injection wasobviously not feasible, the degree of preinjectionnecrosis could not be determined in the treatedanimals. However, on the basis of examination ofcomparable tumors in untreated animals it appears likely, in the smaller tumors at least, thatnecrotic areas, if they existed, were minimal. Microscopic examination of the tumor sections aftertreatment would tend to bear this out, since, inmany of the animals, only microscopic evidence ofnecrosis was demonstrable. In other animals grossevidence of necrosis was present, and there appeared to be a positive correlation between thelength of the postinjection period and the degreeof necrosis noted.

Microscopic examination, which revealed numerous vegetative forms of Cl. tetani in the tumorarea, failed to disclose their presence in other organs of the spore-treated, tumor-bearing host. Cultural examination also failed to demonstrate thevegetative forms in these organs, suggesting thatthe vegetative forms resulting from the germination of Cl. tetani in tumor tissue remain confinedto the cancerous site.

This observation agrees with the previous conclusions made by both Fildes (4) and Francis (5),who were also unable to demonstrate the presenceof the vegetative form of Cl. tetani in the liver,spleen, and blood of animals with active tetanic lesions. Whether this is dependent upon the possibility that only the spores have migratory abilityor whether it involves a rapid reconversion to thespore form of those vegetative organisms thatescape the optimum growth site is uncertain, butthe fact remains that the vegetative forms are invariably confined to the site of germination which,in the case of a tumor-bearing host, is the cancerous tissue.

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478 Cancer Research

The selective localization of the vegetative formof Cl. tetani assumes added significance in view ofthe fact that this bacterium is antigenic only in thevegetative state. Therefore, regardless of howwidely the spore form of the organism may be dispersed throughout the host, the result of the selective localization of the vegetative form of Cl. tetaniis tantamount to the specific localization of anantigen in tumor tissue. That this antigenic localization not only occurs but also results in anextremely high antigen concentration in the tumorsite is indicated by the colony counts in Table @.

Studies are now in progress to determine whetherradioactive antibodies against this bacterial antigen will also localize selectively in the tumor site.Also on the basis of this observation, other nonpathogenic organisms are being tested for theirability to localize selectively in tumor areas. Theeffect of these organisms and the effect of antigenantibody combination on tumor tissue is also beingstudied with a variety of metastatic and primarytumors.

SUMMARYMice bearing spontaneous mammary carcino

mas, transplanted mammary carcinomas, hepatomas, and sarcomas were given intravenous injections of Cl. tetani spores in doses ranging from9,000 to 150,000 spores per mouse. All the treatedanimals died of tetanus 48 hours after spore injection, indicating the germination of the tetanusspores and the formation of the lethal toxin.

Nontumor-bearing mice similarly treated remained asymptomatic throughout the 40-day observation period.

At various time intervals after spore injection,microscopic examination of the tumors and organsof the Cl. tetani spore-treated, antitoxin-protected

mice bearing the previously described tumors revealed that germination of the Cl. tetani spores occurred only in the cancerous tissue and that thevegetative (antigenic) form of the organism remained within the tumor area.

Cultures made from portions of the tissues examined microscopically further confirmed sporegermination and the exclusive localization of thevegetative form of Cl. t4ani in the cancerous sitesand demonstrated that this antigen was present ina high concentration.

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1955;15:473-478. Cancer Res   Richard A. Malmgren and Clyde C. Flanigan  Mouse Tumors Following Intravenous Spore Administration

inClostridium tetaniLocalization of the Vegetative Form of

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