(CANCER RESEARCH 40, 2061 -2068, June 198010008-5472/80/0040-0000$02.00

Selective Localizationand Growthof Bifidobacteriumbifidum in MouseTumorsfollowingIntravenousAdministration'

Noritaka T. Kimura, Shun'ichiro Taniguchi, Ken Aoki, and Tsuneo Baba

Laboratory of Cell Research, Cancer Research Institute, Faculty of Medicine, Kyushu University, Fukuoka 812, Japan

genic bacteria found in humans (3, 38, 39). Lac B p.o. hasbeen widely prescribed clinically for the control of intestinalflora.

In our preliminary experiments using Lac B, we found aremarkable colony formation of the bacterium in plates inoculated with tumor homogenate from a fibrosarcoma-beaningmouse given the bacterium i.v. 4 days before. No colonyformed in any of the plates inoculated with several homogenates of normal tissues such as the liver, kidney, spleen, andmuscle from the same animal. The scope of these experimentswas expanded, and our results are reported herein.

MATERIALS AND METHODS

Animals. We used inbred female DDD@H@2Smice, 6 to 8weeks old, which had been propagated in and supplied by theAnimal Center of Kyushu University.

Tumors. Ehnlich ascites tumor cells were used unless otherwise stated. The cefis were maintained in the DOD mice andpassaged routinely, in the ascites form, at weekly intervals. Asa rule, 4 x 106 tumor cells of 7- to 10-day-old ascites wereinoculated into the right thigh muscle of these mice. The solidtumors obtained at the end of the fourth week after inoculationwere then used for study. Two other tumors tested were atransplantable mouse fibrosancoma, originally induced by 3-methylcholanthrene in a female DDD mouse and maintaineds.c. for 50 generations in syngeneic mice, and a transplantablemammary carcinoma, originally developed spontaneously in afemale DDD mouse and maintained i.m. for 20 generations. Inthe case of these 2 tumor lines, one trocar unit of minced tumortissue was inoculated i.m. into the right thigh of syngeneicmice. The resulting tumors were used in the same way as werethe Ehrlich tumors at the end of the second week, becausethey grew more rapidly and at that time were almost as largeas that of the 4-week-old Ehrlich solid tumors.

Bacteria. Lac B was kindly provided by Nikken ChemicalsCo. , Ltd. (Tokyo, Japan). The bacilli had been lyophilized andampuled at 2 x 10@°onganisms/ampul, the air in the ampulsbeing replaced with CO2. When in use, 10 ml of PBS, pH 7.4,were added to an ampul, and the lyophilized bacilli weresuspended in PBS by pipetting. The original suspension thusprepared was diluted 10-fold with PBS; 0.2 ml of the dilutedsuspension was then injected into the tumor-bearing mice viaa tail vein. During the periods of administration, the suspensionwas kept in an ice box. Immediately after completion of injections to all mice, the suspension was quantitatively diluted withPBS and cultured as described below in order to determine theactual number of viable bacilli contained in the inoculum.Unless otherwise stated, the number of viable bacilli in theinoculum (inoculum size) in the present study was 5 to 6 X I 06organisms/mouse.

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ABSTRACT

A strain of domestic bacteria, Bifidobacterium bifidum (LacB), which is nonpathogenic and anaerobic, selectively localizedand proliferated in several types of mouse tumors following iv.administration. None of the same bacilli could be detected inthe tissues of healthy organs such as the liven, spleen, kidney,lung, blood, bone marrow, and muscle 48 or 96 hr after i.v.administration into tumor-bearing mice.

Proliferation of Lac B in the tumor was artificially stimulatedby i.p. administration to DDD@H@2Smice of a synthesized disacchanide, lactulose (4-O-f.@-D-galactopyranosyl-D-fructofuranose), a sugar which is not metabolized by mammalian tissuecells.

Lac B, which survives and proliferates selectively in thetumor following i.v. administration into the tumor-beaning host,should aid in diagnosis and selective therapy for cancer.

INTRODUCTION

Regardless of the type of prescribed therapy, it is of cardinalimportance in treating cancer to distinguish neoplastic fromhealthy tissue. However, the basic criterion for a clear distinction is yet to be established. Chemotherapy has the advantageof being able to attack cancer cells scattered in undetectablesites. It also has a disadvantage, in that it is capable of attackingrapidly growing normal cells such as those of gastrointestinalepithelia on of bone marrow. Although studies on the possibleaffinity of certain microorganisms to cancer have been done(24, 27—29),a practical approach has been awaited. In ourinvestigations on cancer, we concluded that a new approachtoward distinguishing between cancer cells and nonmalignantcells was required.

Focal centers of necrosis with accompanying zones of lowoxygen tension are frequently seen in cancer lesions. Whenanaerobic bacteria are given i.v. to the tumor-beaning host, thebacteria will proliferate freely in those zones of low oxygentension (24, 29); there is no proliferation in healthy tissues richin oxygen, as supplied by an adequate blood flow. Thus, thepresence of anaerobic bacteria in tumor tissues may be a cluewhich would aid in distinguishing tumor and normal tissues, amarker which could be used effectively in tumor diagnosis ortreatment. For such purposes, the anaerobic bacterium mustbe nonpathogenic.

Lac B2 is anaerobic and is one of the domestic, nonpatho

, Supported In part by a Grant-in-Aid for Cancer Research from the Ministryof Education, Science and Culture, Japan; by the Princess Takamatsu CancerResearch Fund; and by the Cancer Association of Fukuoka Prefecture, Japan.

2 The abbreviations used are: Lac B, Lactobacillus bifidus, a synonym of the

new nomenclature, Bifidobacterium bifidum; PBS, phosphate-buffered saline (2.7mM KCI, 8.1 mM Na2HPO4,1.5 mM KH2PO4,and 0.8% NaCI, pH 7.4); FITC.fluorescein Isothiocyanate-conjugated.

Received May 31. 1978; accepted March 11, 1980.

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N. T. Kimura et a!.

Lactulose Solution. Lactulose (4-O-06-D-galactopynanosylo-fnuctofuranose) was also kindly provided by Nikken Chemicals Co., Ltd., and used as a 20% water solution after stenilization by passing through a 0.45-jzm Millipore filter (MilliporeCo.,Bedford,Mass.).

Treatment of Mice with Lactulose. Preliminary experimentsrevealed that the number of bacilli per g of tumor tissues ofEhrlich solid tumor-beaning mice given in a single i.v. administration of Lac B 7 days before, followed by daily i.p. administrations of 1 ml 20% lactulose solution, was about 1000-fold

more than that of the mice given daily i.p. administrations of 1ml PBS.

Thus, throughout the present study, the bacterium suspension was given i.v. to the mice only once on Day 0, andthereafter 1 ml of 20% lactulose was given i.p. daily startingfrom Day 0 to the day of sacrifice. In the control groups, thesame volume of PBS instead of lactulose was given by thesame way and for the same period of time.

Preparation of Tissue Homogenate. Both the tumor-beaningand non-tumor-bearing mice were sacrificed by cervical dislocation. Organs and tissues were excised and minced thoroughly with scissors, and a sample was weighed and placed ina Potten-Elvehjem homogenizer to prepare a 10% homogenatewith cold PBS.

The above procedure was carried out under aseptic conditions at 0°.As samples of nonmalignant tissues, the muscles(pectoral, deltoid, biceps, and triceps bnachii of both sides;gluteal and thigh muscles of the non-tumor-beaning side); bothkidneys; spleen; median lobes of liver (excluding the gallbladden); both lungs; blood; and bone marrow of bilateral humerus,radius, and ulna and of femur, tibia, and fibula of the nontumor-bearing side were used. The whole tumor, which grewat the right thigh, was enucleated and weighed after removal ofthe bony material. Then the tumor tissue was minced thoroughly, and a sample was processed as described above.Since bone marrow could be neither obtained in an adequateamount nor prepared easily from bony material, the abovementioned bones were mixed together and weighed, crushedwith scissors, and then homogenized with cold PBS.

Blood was obtained aseptically from the femoral vessels ofnon-tumor-beaning thigh. An aliquot (200 @sl)of blood waspipetted quickly into a test tube containing 50 @slof hepaninsolution (1000 units/mI). The hepanin in this blood-hepaninmixture was preliminarily confirmed to have no effect on thegrowth of Lac B in vitro.

Culture Medium. CTJ medium utilized in this study waskindly provided by Nikken Chemicals Co., Ltd. The compositionof the medium was as follows: pancreatmn-hydnolyzedcasein,5 g (as casein); sodium acetate anhydrous, 25 g; K2HPO4, 2.5g; lactose, 35 g; ammonium acetate, 2 g; adenine-HCI, guanine-HCI, uracil, and xanthine, 10 mg each; DL-tryptophan,DL-alanine,and L-cystine, 200 mg each; L-asparagine, 100 mg;fumanic acid, 500 mg; sodium ethyloxaloacetate, 500 mg;thiamine-HCI and riboflavin, 200 @tgeach; calcium pantothenate, 400 ;Lg;pynidoxine-HCI, 1200 @sg;nicotinic acid, 600 @tg;p-aminobenzoic acid and folic acid, 10 @geach; biotin, 4 sg;cyanocobalamin, 5 @g;Salts B (1 0 g of MgSO4 . 7H20, 0.5 g ofFeSO4.7H2O, 0.5 g of NaCI, and 0.335 g of MnSO4.H20 in250 ml of H2O), 5 ml; Tween 80, 1 ml; agar, 10 g. pH wasadjusted to 6.4 by adding 10% NaOH, and the whole volume

was adjusted to 1000 ml with distilled water. The medium wasautoclaved at 15 pounds for 10 mm, then cooled rapidly, andstoned in a refrigerator.

Culture Conditions. Refrigerated solidified culture medium(CTJ medium without vitamin C) was melted thoroughly inboiling water, and vitamin C (1 mg/mI, final concentration;Takeda Chemical Industries, Ltd., Osaka, Japan) was added tothe medium when the temperature dropped below 550•Thereafter, the medium was kept in a 48°water bath ready for use.Also, P-3 Petnidishes, prewarmed at 48°in an incubator, wereplaced on a thermocontrolled table-type heater prewanmed andkept at 48°during the procedure of pouring and mixing themedium and homogenates. The medium was poured into thedishes, 4 mI/dish. Usually, appropriately diluted tissue homogenate, 50 @xl/dish,was inoculated into 4 dishes pen sample andthoroughly mixed with the medium using a glass rod spreader.After the agar medium was solidified at room temperature, allthe dishes were placed in a completely airtight desiccator. Theair in the desiccator was replaced with 0.5 atm of C02, andincubation was carried out at 37°under these anaerobic condition for a given period. On Day 3 of culture, colonies grownin one of the 4 dishes were morphologically and bacteniologically identified; those in the remaining 3 dishes were counted.

Under this culture condition, the number of colonies rangingfrom 40 to 400 per plate (P-3 Petni dish) closely correspondsto the number of Lac B in the materials.

Test of the Time Course Distribution of i.v.-admlnisteredLac B in Various Organs or Tissues. Forty tumor-bearing miceand 8 non-tumor-bearing mice were given 5 x 106 viable bacillii.v. and lactulose solution i.p. as described above. At 12, 24,48, 96, and 168 hr (7 days) after injection of the bacilli, 8tumor-beaning mice each were sacrificed. Another group of 8non-tumor-beaning mice was sacrificed at 168 hr to observethe bacilli in normal tissues of the non-tumor-bearing mice atthe same time when bacilli were not detectable in normaltissues of the tumor-bearing mice.

Confirmation of the Growth-promoting Effect of Lactuloseon Intratumoral Lac B. Fifty tumor-bearing mice were given 1ml of 20% lactulose solution i.p. daily for the first week afterreceiving Lac B. At the beginning of the second week, the micewere divided into 2 groups: Group A, 20 mice; and Group Bplus C, 30 mice. To the mice in Group A, lactulose administration was continued for 2 more weeks. At the end of the secondand third week, 10 mice each were sacrificed for the determination of intratumoral Lac B. For the remaining 30 mice (GroupB plus C), lactulose administration was suspended during thesecond week. At the end of the second week, 10 mice weresacrificed for the determination of the intratumoral Lac B. Theremaining 20 mice were then divided into 2 groups of 10 each(Groups B and C). To the mice in Group B, lactulose was againgiven i.p. daily during the third week. The mice in Group Cwere not given lactulose injection during the third week. At theend of the third week, all the mice of Groups B and C weresacrificed and examined for intratumoral Lac B in the sameway that the mice in Group A were examined.

Assessment of Possible Proliferation of I.v.-admlnisteredLac B in the Traumatized or Inflammatory Lesion. To determine whether the i.v.-administered Lac B would be detectablein the traumatized or inflammatory lesions, hematomas weremade artificially by injecting 1 ml of blood obtained aseptically

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Se!ective Growth of B. bifidum in Tumor

for 1 hr. After incubation, the sections were rinsed thoroughlywith the buffer and stained with goat FITC-anti-rabbit yglobulin(Boehninger Institute, Marburg, West Germany) at a 1-mg/mIconcentration of y-globulin (fluonescein:pnoteinmolar ratio, 3.2)at room temperature for 20 mm. After staining, the sectionswere again thoroughly washed with the buffer and mounted ina buffered glycerol (pH 7.2). The preparations were examinedunder a Leiz Ortholux microscope. As control, some sectionswere stained with goat FITC-anti-rabbit yglobulin without preincubation with anti-Lac B serum.

Histology. Tumors of mice given Lac B i.v. 7 days beforewere fixed in 10% fonmalin solution and embedded in paraffin.A Gram stain was applied to 5-@.om-thicksections, as reportedby Brown and Hopps (6).

RESULTS

Distribution of Lac B in Various Organ Tissues. Chart 1shows the number of Lac B organisms per g of various tissuesat various time intervals after i.v. administration of 5 x 106viable bacilli into tumor-bearing mice. The number of Lac B inthe tumor continued to increase for 1 week (1 68 hr).

Contrastingly, the number of Lac B organisms in nonmalignant tissues, such as the liven, spleen, kidney, lung, blood,bone marrow, and muscle from the tumor-bearing mice, beganto decrease immediately after Lac B injection. The bacilli completely disappeared in 24 to 96 hr. Disappearance of Lac Bfrom normal tissues of non-tumor-bearing mice was also confirmed at 168 hr.

Growth-promoting Effect of Lactulose on Intratumoral LacB. This experimentwas performedto confirmthe growth-promoting effect of lactulose on intratumoral Lac B. Since all thetumor-bearing mice given injections of Lac B were a part of the

from the donor mice into crushed thigh muscle lesions of 8recipient mice. Four days later, Lac B (5 x 106 viable orgaflame/mouse) was given i.v. Lactulose treatment was alsogiven as in the other experiments for 7 days. On Day 7 afterLac B injection, the hematoma tissues were harvested andexamined for Lac B.

Teat on the Effect of i.v.-admlnlstered Lac B on the LifeSpan of Tumor-bearing Mice. To determinethe effect of i.v.injected Lac B on the survival of Ehrlich solid tumor-bearingmice, 3 experIments were repeated. Female 6- to 7-week-oldDDD mice were inoculatedwith Ehrlich ascites tumor cells inthe right thigh muscle. On Day 28 (Experiment a), 33 (Expeniment b), or 26 (Experiment C)of tumor growth, 2 x 10@viableLac B bacilli were given i.v. only once; 20% lactulose solution,1 mI/mouse, was given i.p. once a day for 7 days (Experimenta) or for 21 days (Experiments b and C), the first injection being

given on Day 0 immediately after the Lac B injection.As controls, one group of tumor-beaning mice was not

treated; another group was given lactulose solution (1 mI/mouse) alone i.p. for the period corresponding to that of eachtest group. The mice were inspected daily, and their bodyweight and tumor size were recorded once a week.

Immunofluorescence Studies. Anti-Lac B rabbit serum (titer, I :256) was a gift from Ohmiya Research Institute, NikkenChemicals Co., Ltd. (Ohmiya, Japan). For indirect immunofluorescence tests, tumor slices were frozen in dry ice:99.6%ethanol at —78°,and 5-sm-thick sections were prepared in acryostat (Lipshaw Manufacturing Co., Detroit, Mich.) at —18°.The sections were dried and fixed in 95% ethanol at —20°for20 mm and then dried again. After a rinsing with a cold stainingbuffer (2.9 mM NaH2PO4,9.0 mM Na2HPO4,and 0.8% NaCI,pH 7.2), the sections were covered with 30-fold diluted antiLac B rabbit serum and incubated in a moist chamber at 37°

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Chart 1. Organ distribution of Lac B following a single iv. administration of 5 x 106viable bacilli into Ehrlich solid tumor-bearingmice. Each point represents the mean of thenumber of bacilli per g tissue of 8 mice; bars,SE.

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N. T. Kimura et a!.

same material used in the preceding experiment, the resultsobtained (Chart 2) represent a continuation insofar as thenumber of the intratumoral Lac B is concerned. Consequently,in Chart 2, the data within 7 days after i.v. administration of

Lac B are those of the preceding experiment. The number ofLac B in the tumor decreased after the interruption of lactuloseadministration (Group B plus C on Day 14 and Group C on Day21 ), as compared with Group A in which daily lactulose injections were given for 14 or 21 days ( p < 0.05, t test) withoutinterruption. However, the number of Lac B organisms in thetumors of Group B began to increase again with resumption oflactulose administration ( p < 0.05, t test).

Detectability of Lac B in the Traumatized or InflammatoryLesion. This experiment was done to determine whether thei.v.-administened Lac B would be detectable in a traumatizedor inflammatory lesion at the time when the injected Lac B wasdetectable in tumor but not in any normal tissues. On Day 7after injection of Lac B (5 x 106viable organisms/mouse), thisbacterium was not detectable in any hematoma-containingcrushed tissue of the thigh muscles, induced 4 days before LacB injection.

Effect of Lac B on the Life Span of Tumor-bearing Mice.As shown in Chart 3, 3 repeated experiments showed neithersignificant prolongation nonshortening of the life span of tumorbeaning mice after a single i.v. administration of Lac B followedby a daily i.p. administration of lactulose. There was no significant difference in body weight or tumor size among the 3groups.

Detectability of Intratumoral Lac B In Relation to TumorSize. Detectabilityof Lac B in the Ehrlichsolidtumorfollowinga single administration of 5 x 106 bacilli i.v. and with a dailyi.p. lactulose administration into the tumor-bearing mice wasexamined on Day 7 in relation to the tumor size. As shown inTable 1, the detectability of Lac B was 90.6% (58 of 64) whenthe tumor diameters were more than 15 mm. A decrease to64.3% (9 of 14) occurred when the tumor diameters were lessthan 15 mm. However, when 1.2 x 108 bacilli were administered, the detectability increased to 100% (1 0 of 10) evenwhen the tumor diameters were less than 15 mm. Bacilli were

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Chart 2. Effect of lactulose administration or of its interruption on intratumoralgrowth of Lac B in Ehrlich solid tumor-bearing mice. , period when lactulosewas given; ————,period when lactulose was not given. Each point representsthe mean of the number of Lac B per g tumor tissue of 10 mice; bars, SE. Themean value of Group A is significantly different from that of Group B plus C onDay 14 ( p < 0.05, t test). The differences between Groups A and C and betweenGroups B and C on Day 21 are also significant ( p < 0.05, t test). Differencebetween the Groups A and B on Day 21 is not significant (p > 0.05, t test).

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Days after Tumor InocuLationChart 3. Effect of iv. administration of Lac B on life span of Ehrlich solid tumor-bearing mice. Number of mice per group was 6, 8, and 7 for Experiments a, b, and

c, respectively. Doses of Ehrlich tumor cells were 1.4, 3.6, and 3.7 x 106 for Experiments a, b, and c, respectively. Dose of viable Lac B was 2.0 x 1O@bacilli forExperiment a and 2.4 x 1o@bacilli for Experiments b and c. Hatched bars, period of lactulose (LTS) administration. Curves are survival curves.

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Tumor diameter6(mm)Inoculum

of bacilli(X106/mouse)Detectability(%)<15120100

(10/10)b<15564.3(9/14)15—25590.6

(58/64)

Se!ective Growth of B. bifidum in Tumor

ever, these are not universally effective against all forms ofcancer. Furthermore, their usefulness is limited because theydo not strictly distinguish cancer cells from normal cells andhave serious side effects.

Little attention was paid to the selective affinity per se ofmicroorganisms to a tumor tissue until Malmgren and Flanigan(24) reported the selective germination of the vegetative formof C!ostridium tetani in the locus of a mouse tumor after i.v.administration of its spores. They established that all tumorbearing mice died of tetanus intoxication within a few days,while all mice without tumor remained healthy. On the otherhand, nonpathogenic and anaerobic Clostridium butyricum isreportedly oncolytic when given i.v. to tumor-bearing mice (29).The oncolytic action in this case apparently depended on theaction of H202 (21) and kininase (5, 27, 28) produced by thebacteria. A clinical application of the bacteria has apparentlynot been reported. The above-mentioned workers attempted totreat tumor using bacteria but did not utilize the bacteria forspecific diagnostic detection of cancer.

Recently, the distribution of killed Corynebacterium parvumlabeled with 1251(10) or with FITC (36) was studied after its i.v.administration into tumor-beaning mice. A similar study using99mTc-labeled C. parvum was done by other researchers (4). Inthese investigations, the distribution of C. parvum was determined indirectly by assaying the radioactivity emanating fromthe isotopes which had been conjugated to the bacteria. However, the radiological method can merely show the presenceand localization of the labeled bacteria in animals. The viabilityand rate of proliferation of the bacteria in vivo cannot beassessed by this method. We examined organ distribution andproliferation of Lac B following iv. administration into tumorbearing mice directly by quantitative assay of the living microorganisms in various organs and the tumors.

Bifidobacterium bifidum (Lactobaci!!us bifidus) was first doscnibed by Tissier (38) as constituting almost the entire flora ofthe stools of breast-fed infants. The nature of this microorganism was extensively studied by Gyargy et a!. (3, 15, 17, 18,30, 39, 40). The nonpathogenicity and importance of Lac B inmaintaining the health of infants is now generally acknowledged. In Japan, Lac B has been widely prescribed clinicallyas one of the most popular drugs for the control of intestinalflora.

When we gave Lac B to tumor-bearing mice i.v. , the distnibution was first seen over the whole body, but after 48 to 96 hrit was detectable only in the tumor tissue (Chart 1). The factthat the bacilli are not only detected but can also proliferate inthe tumor tissue implies that this tissue possesses an environment suitable for the growth of this bacterium. The suitabilitymay be ascribed to either an anaerobic or hypoxic environment.We found in cultures that the tumor homogenate per se nevealed no growth-promoting effect on Lac B (data not shown).

It is interesting that the growth of Lac B in the tumor tissuecan be remarkably stimulated artificially by systemic administnation of lactulose to the tumor-bearing host (Chart 2). Thissynthesized sugar, lactulose (19, 32), is not found in naturebut can be prepared from lactose by alkaline isomenization(26). Since the sugar cannot be metabolized by humans, rats,and pigs (8, 12, 14, 35), it is assumed that lactulose in vivostimulates the growth of Lac B in the tumor without otherwiseaffecting the mammalian tissues.

Here, a discussion on the utility of this nonpathogenic and

Table 1Detectability of intratumoral Lac B in relation to tumor size and enhancement of

the detectability by a higher dose of the bacilliEhrllch solid tumors were harvested on Day 7 following iv. administration of

the bacIlli Into tumor-bearing mice.

a Geomefric mean of tumor diameters measured In 3 dimensions.b Numbers in parentheses, number of cases in which presence of intratumoral

Lac B was positive/number of cases tested.

not detected in the normal tissues even with this higher doseof Lac B.

Detectability of Lac B in Other Kinds of Tumor. Detectabilityof intratumoral Lac B in other kinds of tumor was also tested asdescribed above on Day 7, following a single administration of5 X I 06 bacilli i.v. and daily lactulose administration. Thediameters of the tumor in these experiments were 20 to 25 mm[22.1 ±I .2 (S.D.) mm] with transplantable fibrosarcoma and20 to 27 mm (24.7 ±2.0 mm) with transplantable mammarycarcinoma. Detectabilities were I 00% (11 of 11) for fibrosarcoma and 73% (8 of 11) for mammary carcinoma, respectively.

Immunofluorescence Studies. The presence of i.v.-administered Lac B in the tumor tissue was examined by an immunofluorescence technique using specific antiserum against LacB in vitro. A large number of fluorescent bacilli were seendispersed throughout the tumor sections (Fig. 1). No bacilliwere observed in the surrounding normal tissues or in sectionsstained without pneincubation with anti-Lac B serum. We alsofound that intratumonal Lac B could react with its antiserum invivo. Fluorescent bacilli were detected even in cases where thetumor sections were prepared from mice which had been given0.5 ml of 2-fold diluted anti-Lac B serum i.v. 40 mm beforefixation, although all procedures that were performed in vitrowere limited to the FITC-globulmnstaining.

HIstology. Numerousbacilli, some representinga typical Ashape, were scattered or clustered either on the border between the necrotic and nonnecrotic regions (Fig. 2a) or in thenecrotic region (Fig. 2b) of Ehrlich tumor tissues.

DISCUSSION

Coinciding with the embryonic achievements in bacteriologyas a natural science, use of microorganisms or their productsas possible therapeutic means against cancers began in thelatter part of the last century. The first report of this kindconcerning human tumors was made by Busch (7) in 1868. Onthe basis of his own clinical experience, he attempted to induceerysipelas infection in a 19-year-old woman with inoperablesarcoma; apparently, he obtained encouraging results. Sincethen, many varieties of microorganisms and their products havebeen tested to determine whether or not they are effectivetherapeutic agents. The history of these attempts has beenwell reviewed by Reilly (34).

Penicillin-attenuated Streptococcus hemo!yticus (OK-432)(23, 31) and Bacillus Ca!mette-Guërin (16, 25, 33, 37) arereportedly effective as nonspecific immunostimulators in cancer therapy. There are also antineoplastic fungal products orantibiotics, e.g. , actinomycin D, mitomycin C, bleomycin. How

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anaerobic bacterium, Lac B, in the field of cancer diagnosisand therapy may be useful. Our data suggest that Lac B canbe used profitably as a tool for diagnosis and selective therapyof cancer. For example, if nadioisotopically labeled antibody (1,2, 22) against Lac B or antibody-anticancer drug complex (9,11, 13, 20) is applied to the tumor-bearing and Lac B-treatedhost more than 96 hr after the administration of Lac B, theradioisotope or the drug complex would be selectively localizedin the tumor.

Since the presence of Lac B was demonstrated even intumors about 10 mm in diameter, detection of the locus of atumor or of a metastatic focus may be diagnostically feasiblewith the aid of radiology. In fact, colonies of Lac B wereobtained from a rice grain-sized inguinal lymph node withmetastasis of Ehrlich solid tumor of a mouse which had beengiven a single i.v. administration of Lac B 7 days before. Aselective tumor damage by means of a radioisotopically labeledantibody to Lac B could also be envisioned.

Although the nonpathogenicity of Lac B is generally acknowledged and no significant harmful effect on tumor-beaning micewas observed in the present study (Chart 3), further testsshould be done. In fact, a clinical case of bactenemia, whichwas caused by mixed infection with Eubacterium !entum andBifidobacterium species as a result of a perforated colonicdiverticulum with subsequent intraabdominal abscess formation and peritonitis, has been reported (41 ). However, the strainof Bifidobacterium was not specified in this report. Nonetheless, it should be kept in mind that even in the same species ofbacterium there are many strains displaying differences inpathogenicity [for instance, C!. tetani (24) and C!. butyricum(27—29)].In our tests using mice, hamsters, guinea pigs, andrabbits, no significant pathological changes in body weight,peripheral leukocyte counts, body temperature, the lie of fur,and life-span resulted from the i.v. administration of Lac B. Inhopes that genetic engineering may provide an oncolytic capacity by introducing a suitable plasmid on some such relatedcompound in Lac B, further investigations of the applicabilityof Lac B to cancer diagnosis and therapy are in progress.

ACKNOWLEDGMENTS

We are indebted to Dr. Ryoichi Mon. Professor of Microbiology, Faculty ofMedicine, Kyushu University, Japan, for pertinent suggestions, and to NikkenChemicals Co., Ltd. (Tokyo, Japan) for the generous gift of Lac B and technicalcooperation. Wethank Professor Kiyoshi Muto for suggestions on the manuscript,Kazuko Yasumatsu and Mihoko Kimura for technical and secretarial services,and Mariko Ohara, Kyoto University, for critical reading of the paper.

REFERENCES

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2. Bale, W. F., Spar, I. L., and Goodland, R. L. Experimental radiation therapyof tumors with ‘3l-carrying antibodies to fibrin. Cancer Ras., 20: 1488—1494, 1960.

3. Barbero, G. J.. Runga, G., Fischer, D., Crawford, M. N., Torres, F. E., andGyorgy, P. InvestigatIons on the bacterial flora, pH, and sugar content In theintestinal tract of Infants. J. Padlat., 40: 152—163, 1952.

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Fig. 1. Fluorescence photomicrograph of Lac B in Ehrlich solid tumor tissue stained by FITC-antibody method. x 400.Fig. 2. Photomlcrograph of Lac B In Ehrllch solid tumor tissue stained by a modified Gram stain method. a, part of the border between necrotic and nonnecrotic

Ehrllch tumor tissue; b, necrotic tissue of the tumor. x 400.

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