Shinshu M ed J, 63⑹ :375〜384, 2015

Obligate Anaerobic Lactobacillus Casei KJ686
Selectively Targets Solid Tumors and Exhibits an Antitumor Eﬀect
Michihiko HARADA

and Jun AMANO

1) Department of Molecular Oncology, Institute of Pathogenesis and Disease Prevention,
Shinshu University Graduate School of Medicine
2) Department of Surgery, Shinshu University School of Medicine

Hypoxia in solid tumors is known to confer treatment resistance and lead to rapid progression and poor
prognosis. As a novel cancer drug‑delivery candidate, Lactobacillus casei KJ686 (L.casei KJ686) is a mutant
strain of the facultatively anaerobic L.casei ATCC393 that was transformed into an obligate anaerobe to suitthe hypoxic conditions within solid tumors.In the present study,we intravenously administered L.casei KJ686
to treat tumor‑bearing mice and investigated the in vitro cytotoxicityof L.casei KJ686 and its culture medium
containing bacterial excretion products. We discovered that L.casei KJ686 localized exclusively in tumor
tissues and suppressed tumor growth in our animal experiments. Furthermore, the culture supernatant of L.
casei KJ686 exhibited a strong cytotoxic eﬀect in vitro. The speciﬁc cytotoxic substances presented in its
culture supernatant may have a pivotal role in direct anti‑tumor eﬀect in solid tumors. Shinshu Med J 63 :375 ―384, 2015
(Received for publication May 19, 2015;accepted in revised form August 17, 2015)
Key words:obligate anaerobe, hypoxia, Lactobacillus casei, cytotoxicity, culturing‑supernatantAbbreviations:LLC, Lewis lung carcinoma

Introduction

or more selective delivery systems of antitumor
drugs to tumor tissues are needed.

Cytotoxic chemotherapy is useful for systemic

Living bacteria have recently been considered as

antitumor treatment, but the large doses of drugs

a drug delivery tool in cancer gene therapy; onethat are often needed for eﬀective results may also

such example is lactic acid bacteria (LAB).LAB is

induce many adverse side eﬀects, such as nausea,

a nonpathogenic anaerobic bacteria widely used invomiting, hair loss, anemia, agranulocytosis,

food and intestinal health supplements

stomatitis, diarrhea, and dehydration. Although

ous reports have documented the safety and func-

many molecular targeted agents have been devel-tional characteristics of LAB.

oped up to the present,they also have manyadverse. Numer-

Hypoxic conditions are characteristic of solid

side eﬀects such as hand‑foot syndrome and inter-tumors in both rodent

stitial pneumonitis, because target molecules exist

tumors . The oxygen pressure in human tumors

in both tumor and normal tissues.

has been reported to be 0‑20 mmHg,whereas that inand many types of humanAccordingly,more speciﬁc antitumor eﬀects and/normal tissues shows a range of 20‑100 mmHg

Corresponding author:Michihiko Harada

Tumor hypoxia, mostly stemming from poor per-Department of M olecular Oncology, Institute of Path-fusion of fresh blood, causes the development ofogenesis and Disease Prevention, Shinshu University
Graduate School of M edicine, 3‑1‑1 Asahi, M atsumoto,
Nagano 390‑8621, JapanE‑mail:mrharada2000＠yahoo.co.jp

No. 6, 2015.

aggressive cell clones and leads to rapid progression
and poor prognosis.

Futhermore, hypoxia is known to directly and375

Harada.Amano

indirectly confer resistance to radiation and somebeen transformed to be obligately anaerobic. The

chemotherapies, which often results in treatmentmutant was maintained in MRS broth (OXOID,

failureHampshire,England)with erythromycin (0.2μg/ml).

Recently, Biﬁdobacterium longum (B.longum), a

at 37°
C without O .L.casei KJ686 in the mid‑loga-

strain of obligated LAB,has been reported to selec-rithmic growth phase was used for all experiments.

tively germinate and grow in the hypoxic regions of

２ Preparation of tumor‑bearing micesolid tumors following intravenous injection andLewis lung carcinoma (LLC) cells were cultured

was demonstrated to be a safe and eﬀective drugin medium containing DM EM (Sigma, Louis, M O,

carrier to solid tumors.

USA),10% fetal bovine serum and streptomycin (10Lactobacillus casei (L.casei), another strain of

μg/ml) at 37°
C with 5% CO . Male 8‑week‑old

LAB,is a gram‑positive facultativelyanaerobicrod‑

C57BL/6 mice (Japan SLC, Hamamatsu, Japan) of

shaped bacterium. L.casei is believed to have in-similar size and weight were used and maintained

direct antitumor eﬀects due to its ability to modu-

with a standard rodent diet (Oriental Yeast,Tokyo,

late host immune responses, but no direct

Japan) at the Shinshu University animal center

antitumor properties have been shown to date .

(Matsumoto, Japan). The animal experiments in

However, numerous recent reports have noted that

this study were carried out in accordance with thethe cytoplasmic fractions, proteins, and peptidog-

Guidelines for Animal Experimentation of Shinshu

lycans of L.casei may suppress the proliferation of

University School of M edicine. Aliquots of 10

tumor cell lines

tumor cells in 100μl HanksʼSolution (Nissui Phar-.

We directed our attention to the fact that L.casei
has thin polysaccharides in its wall and may easily

maceutical Co.,Ltd.,Tokyo,Japan)were inoculated
under the femoral skin of the mice.

excrete proteins synthesized via an expression

The solid tumors were palpable 5‑8 days after

vector,since B.longum with cytosine deaminase has

inoculation. For each tumor, the maximum diame-

successfully exhibited antitumor eﬀects throughter (A)and diameter at a right angle to A (B)wereintravenous injection in combination with 5‑FC.

measured daily using a sliding caliper. Tumor vol-We examined whether obligated L.casei could

ume was estimated as 1/2×A×B . All mice sur-also be a speciﬁc delivery tool to target tumor

vived the testing period.

tissues,and produced L.casei KJ686, a novel L.casei

３ Intravenous injection of L.casei KJ686 into

strain that was transformed to be obligately anaerobic, to germinate and grow in the hypoxic regionsof solid tumors after intravenous injection.

tumor‑bearing miceL.casei KJ686 was harvested by centrifugation(3000 rpm, 1 min at 4°C), washed twice with PBS

In the present study,we found that L.casei KJ686(−),and then resuspended in PBS (−)at 5×10 c.f.

was a tolerated and selective carrier to solid tumors

u./100μl. The solid tumor‑bearing mice were ran-and exhibited suppressive eﬀects on tumor growth.

domly separated into 2 groups and used for subse-

We also assessed the direct cytotoxic eﬀect of L.

quent studies.When tumors reached approximately

casei KJ686 and/or the substances excreted from65.4 mm (5 mm in diameter),L.casei KJ686［5×10

this bacterium in vitro.

c.f.u./100μl PBS (−)］was injected into a tail vein

M aterials and M ethodsof one group once a day for 2 days.The other mice
underwent no treatment as the control group. The
tumor growth ratio was calculated every day as

１ Growth of L.casei KJ686
L.casei KJ686 with the erythromycin resistancefollows:(tumor volume) / (tumor volume on the ﬁrst day ofgene was developed as a mutant strain of theintravenous injection).

facultatively anaerobic L.casei ATCC393 that had

４ Localization of L.casei KJ686

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Direct anti‑tumor eﬀect of L.casei KJ686

All tumor‑bearing mice were sacriﬁed 17 days

eral concentrations (20‑100%) of methyl alcohol

after the ﬁrst injection of L.casei KJ686. Normal

were threaded into the hydrophobic resin andtissue samples from the lung,liver,and tumor wererecovered (20‑100% methyl alcohol fraction).Each

excised and placed into a homogenizer with 9 ‑foldaqueous supernatant with alcohol was lyophilized inanaerobic diluent. The samples were plated on ana desiccator and diluted with sterilized water.

MRS agar medium at 37°
C without O to assess the

２ Gramʼs stainbacterial numbers in each tissue.The localization of

For both living and heat‑killed L.casei KJ686, theL.casei KJ686 in tumor tissue sections was visual

neo B & M (Wako Pure Chemical Industries, Ltd.,

ized microscopicallywith Gramʼs and Hematoxylin‑

Osaka, Japan)method was performed according to

Eosin staining.

the manufacturerʼ
s instructions.
３ Cell viability assay

１ Preparation of L.casei KJ686 and culture super-

The Premix WST‑1 Cell Proliferation Assaynatant of L.casei KJ686 to examine for cytotoxic

System (Takara Bio Inc.,Shiga,Japan)is based on

eﬀect

the cleavage of the tetrazolium salt WST‑1 by cells

LLC cells were maintained at 37°
C with 5% CO

to form a red formazan dye. Cell viability was

and routinely cultured in DM EM supplemented withmeasured with this assay according to the manufac-

10% fetal bovine serum with or without streptomy-

turerʼ
s instructions. LLC cells (1×10 in 90μl/well)cin (10μg/ml). L.casei KJ686 was harvested byon a 96‑well microplate were incubated for 24 hr at

centrifugation (3000 rpm, 1 min at 4°C)and washed37°
C with 5% CO with various concentrations of

twice with PBS (−).The bacteria were resuspended

L.casei KJ686 culture supernatants (1×10 −1×10

in DMEM without streptomycin at 1×10 c.f.u./ml

in 10μl/well of living or heat‑killed L.casei KJ686).

(living L.casei KJ686). Heat‑killed L.casei KJ686

WST‑1 reagent (1:10)was then added.After 60 min

was prepared by boiling living L.casei KJ686 at 95

of incubation at 37°
C with 5% CO ,the absorbance°
C for 90 min. After 24 hr of anaerobic culture inof the colored formazan was determined using a

DM EM without antibiotics,the culture supernatant

Vmax automated microplate reader (Wako,Osaka,

of L.casei KJ686 was harvested by centrifugation

Japan) at 450 nm and 650 nm wavelengths. The(12000 rpm, 10 min at 4°C). The supernatant wasmean absorbance of control wells (cells without

divided into 6 fractions by molecular weight (oversupernatant)represented 100% cell viability.

100 kDa,under 100‑50 kDa,under 50‑10 kDa,under

４ Colony‑forming assay

10‑3 kDa, under 3‑1 kDa and under 1 kDa) with

LLC cells (500/well) on a 6‑well plate were in-Amicon Ultra‑0.5 ml Centrifuged Filter devices (100cubated for 24 hr at 37°
C with 5% CO . Various

k, 50 k, 10 k,and 3 k ;Merck Millipore,Darmstadt,

conditions of L.casei KJ686 culture supernatants

Germany), and a Microsep Centrifugal device (1 k ;were then added to the LLC cells, which were thenPall Life Sciences, M ichigan, USA). Each fraction

incubated further. After 8 days, the supernatants

was diluted to its original concentration with

were removed from the wells and the cells wereDM EM. Furthermore,the under 1 kDa supernatant

ﬁxed and stained with 10% formalin (Wako,Osaka,(1 ml) was vortexed with ethyl acetate (1 ml) andJapan) and crystal violet solution (Wako, Osaka,

separated into aqueous and organic layers by

Japan)according to the manufacturerʼ
s instructions.

centrifugation. To remove the ethyl acetate, theThe toxic activityof supernatants was estimated byorganic layer was desiccated in draft for 48 hr.

the viability of LLC cells.

After desiccation, the organic layer powder was

Statistical analysis

vortexed with 990μl DM EM and 10μl DM SO.The

Experimental data are expressed as the mean±aqueous layer was threaded into a hydrophobic

SE of the mean.Statistical analysis was performed

resin (non‑methyl alcohol fraction), and then sev-

using the Mann‑Whitney U ‑test. A p＜0.05 was

No. 6, 2015

377

Harada.Amano

Fig. 1Germination of L.casei KJ686. Seventeen days after intravenous inoculation of L.casei KJ686,
organs and tumors were homogenized with a 9 ‑fold anaerobic diluent. Samples of 100μl of
homogenized tissues were cultured on an M RS agar medium at 37°
C without O for 3 days.

considered to be statistically signiﬁcant.
Results

KJ686 group was signiﬁcantly less than that of thecontrol group. We next estimated RNA expressionof IL‑2, IL‑4, IL‑12p, IL‑1β, and IFN‑γ by realtime RT‑PCR of the border areas of tissue samples,

１ Selective localization ofL.casei KJ686 in tumors

but no signiﬁcant diﬀerences were noted (data not

We examined the germination of L.casei KJ686 inshown). Cytokine levels in the serum of KJ686 ‑the lungs,livers and tumors of KJ686 ‑injected mice.

injected mice were also estimated on days 3 and 9,

Another obligate anaerobic bacterium, B.longum

but no signiﬁcant ﬁndings were seen except for IL‑

was not at a detectable level in any normal tissues

1βon day 3 (data not shown). Although IL‑1βwas

after seven days . So we sacriﬁed KJ686 ‑injectedtransiently increased, enhancement of host im-

mice nine days after intravenous injection, L.casei

munological defenses by L.casei KJ686, such as an

KJ686 was selectively detected in tumors, with noincrease in the number of NK cells in tumors, wasbacterial growth in either the lungs or livers (Fig.

not observed (data not shown).

1).Each tumor had necrotic areas in its center (Fig.
2).Gramʼs staining of tumor tissues showed that L.

１ Cytotoxic eﬀect of L.casei KJ686 on LLC cells

casei KJ686 was focally scattered in the border

The direct cytotoxic eﬀect of L.casei KJ686 on

areas between necrotic and viable regions (Fig. 2).

LLC cells was assessed by the WST‑1 method.The

Neutrophils existed in necrotic and viable regions.

morphology of L.casei KJ686 was not visibly alter-

The number of L.casei KJ686 in solid tumors esti-

ed by heat treatment (Fig. 4), which indicated thatmated by tissue homogenation appeared to reﬂect

the bacterial body was not ruptured. The viability

the bacterial number in border areas.

of LLC cells with living L.casei KJ686 decreased in

２ Inhibition of tumor growth with L.casei KJ686

a concentration‑dependent manner, while LLC in-

Fig. 3 shows the tumor growth ratio after intra-

cubated with heat‑killed L.casei KJ686 exhibitedvenous injection of L.casei KJ686 (n＝25) and inmuch the same viability as controls (Fig. 5).

controls (n＝36). After administration of L.casei

２ Cytotoxic eﬀect of L.casei KJ686 culture super-

KJ686, neither suppression of activity nor deathnatant on LLC cells

were observed in the test group. From day 5 after

Conditioned media in which L.casei KJ686 had

the ﬁrst injection, the tumor growth ratio of the

been cultured for 24 hr and split into 6 fractions by

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Direct anti‑tumor eﬀect of L.casei KJ686(×1000)(×40)(×1000)Fig. 2

Localization of L.casei KJ686 in tumors. On day 9, Gramʼs staining of histological sections
indicated that L.casei KJ686 was scattered in the border areas between necrotic and viable regions.

Fig. 3In vivo tumor growth ratio.Controls were not given any treatment after tumor inoculation,while
the test group was intravenously injected with L.casei KJ686. The tumor growth ratio became
signiﬁcantly diﬀerent from day 5 ( p＜0.01).

No. 6, 2015379

Harada.Amano(×1000)Fig. 4(×1000)Gramʼ
s staining by the neo B & M method.The morphologyofliving and heat‑killed
L.casei KJ686 was not apparently altered.

Fig. 5

Cytotoxic eﬀect of L.casei KJ686 on LLC cells. The survival of cells treated with living L.casei
KJ686 ( )was signiﬁcantly less than those treated with heat‑killed L.casei KJ686 ( ).Untreated
LLC cells exhibited much the same viability as heat‑killed L.casei KJ686 cells.

molecular weight were evaluated for cytotoxic

shown). When conditioned medium and cytotoxic

eﬀect.The fraction with a molecular weight of lessfractions were boiled at 100°
C for one minute,than 10 kDa exhibited a comparable cytotoxic

cytotoxicity showed only a slight decrease(data noteﬀect as the original conditioned medium,while theshown). To test for pH‑mediated cytotoxicity, weother fractions did not (Fig. 6). We then compared

added a comparatively acidic pH4.0 lactic acidthe aqueous layer with the organic layer of thesolution instead of the supernatant,but no cytotox-

supernatant under 1 kDa, and found that only the

icity was observed (data not shown).

aqueous layer had a signiﬁcant cytotoxic eﬀect(Fig. 7). However, no fraction of the under 1 kDa

Discussion

supernatant further separated by a hydrophobic

Hypoxia in solid tumors is known to confer resis-

resin column exhibited cytotoxicity (data nottance to radiation and some chemotherapies,which

380

Shinshu Med J Vol.63

Direct anti‑tumor eﬀect of L.casei KJ686

Fig. 6

Cytotoxic eﬀect of the conditioned medium of L.casei KJ686 on LLC cells.The concentration ofeach fraction was adjusted to that of the original supernatant with DM EM ( p＜0.01 vs. control).

Fig. 7Comparison of the cytotoxic eﬀect of aqueous and organic layers of the conditioned medium
fraction under 1 kDa. The concentration of each fraction was adjusted to that of the original
supernatant with sterilized water ( p＜0.05 vs. control).

often results in treatment failure.To overcome

L.casei KJ686 would be safely used for systemic

this impasse, several new means of delivering

treatment. Interestingly, a signiﬁcant diﬀerence

agents to solid tumors are being explored

between L.casei KJ686 and B.longum is that the.

Intravenous injection of living bacteria is gener-

former has the ability to inhibit tumor growth byally understood to induce bacteremia and sepsis.

itself. We believe that this inhibition is due to theThis was surprisinglynot the case for B.longum,

local eﬀects of L.casei KJ686 and not immunomo-

nor was it for L.casei KJ686, which accumulated

dulation. In previous studies, inoculation of a large

speciﬁcally in tumor tissues without any adverse

dose of heat‑killed bacteria had no direct anti‑

symptoms in the mice. This fact demonstrates thattumor eﬀect, indicating that the property was not

No. 6, 2015

381

Harada.Amano

attributed to host immunoresponses. Thus, wethere have been no reported cases of patient deathnext investigated the direct anti‑tumor eﬀect of L.

if adequate treatment was performed . In conclu-

casei KJ686 in vitro. Following the addition ofsion, it appears that speciﬁc cytotoxic substances

living L.casei KJ686, the growth of LLC cells waspresent in the culture supernatant of L.casei KJ686

stronglyinhibited,but this was not the casefor heat‑and their mode of action may be pivotal as direct

killed L.casei KJ686. Both living and heat‑killed L.

anti‑tumor agents once they have been identiﬁed.

casei KJ686 exhibited the same morphology by

AcknowledgementsGramʼ
s stain, implying that heat treatment did notcause destruction of the cell wall. These results

Our deep appreciation goes to Prof. Shunʼichirosuggested that living L.casei KJ686 itself and/or

Taniguchi of the Department of M olecular On-

substances excreted from L.casei KJ686 imparted acology, Institute of Pathogenesis and Disease Pre-cytotoxic eﬀect on LLC cells.We further investigat-

vention, Shinshu University Graduate School ofed which fractions of the L.casei KJ686 culture

M edicine,and Prof.M inoru Fujimori of the Depart-supernatant had this eﬀect.Manjunath et al.report-

ment of Breast Surgery,Tokyo Medical University

ed that a 40‑fold concentrated bacterial culture

Ibaraki Medical Center, who provided carefully

supernatant exhibited cytotoxicity invitro .

considered feedback and valuable comments. We

Although we used a culture supernatant that wasalso owe a very important obligation to Prof.

not concentrated, we nonetheless witnessed thatTomoyuki Fujita of the Department of Sciences ofvery low molecular weight (＜1 kDa)excreted prod-

Functional Foods, Graduate School of Agriculture,ucts of L.casei KJ686 had an inhibitoryeﬀect on the

Shinshu University,and Akihiro Shou of the Depart-

growth of LLC cells.As the inhibitory properties ofment of Molecular Oncology, Institute of Path-the fraction were partially stable against both heat

ogenesis and Disease Prevention, Shinshu Univer-and lactic acid treatment,it can be said that L.caseisity Graduate School of M edicine, whose chemical

KJ686 had a direct suppressive eﬀect, possibly

analysis and technical assistance helped us greatly.

through several factors, on LLC cells rather thanFinally,we thank Dr.Shizunobu Igimi and Akinobu

indirect stimulation of host immunoresponses.

Kajikawa of the National Institute of Health Sci-

In terms of safety, L.casei may have immuno-

ences for provision of the original stock culture of

logical tolerance because it can be found in the

Lactobacillus casei KJ686 that was used in thishuman intestinal tract just after birth .Although it

study.

was reported that L.casei sometimes inducedThis study was supported in part by a grant‑in‑bacteremia in patients with cancer,diabetes,broad

aid for scientiﬁc research from the Japan Society

spectrum antibiotic therapy, and transplantation,

for the Promotion of Science (No. 21591678)

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