Cancer Immunol Immunother (1985) 20:18-22 ancer mmunolggy mmunotherapy

© Springer-Verlag 1985

Anti-tumour activity of Lactobacillus casei on Lewis lung carcinoma and line-10 hepatoma in syngeneic mice and guinea pigs.

Takeshi Matsuzaki 1, Teruo Yokokura 1, and Ichiro Azuma 2

l Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186, Japan : Institute of Immunological Science, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060, Japan

Summary. The anti-tumour activity of Lactobacillus casei YIT 9018 (LC 9018) on Lewis lung carcinoma (3LL) in C57BL/6 mice and line-10 hepatoma in strain-2 guinea pigs was examined. Intravenous injection of LC 9018 was effective for inhibition of pulmonary metastases in C57BL/6 mice after s.c. inoculation with 3LL tumours. In- tralesional (i.1.) injection of LC 9018 was also effective for both prolongation of the survival period and inhibition of pulmonary metastases in 3LL tumour-bearing mice. The combination treatment of i.1. and i.v. injections of LC 9018 before or after surgical excision of the primary tumour re- markably inhibited the pulmonary metastases after inocu- lation with 3LL tumour. Intralesional injection of LC 9018 was effective for regression of the established tumours of line-10 hepatoma inoculated i.d. and for induction of sys- temic tumour immunity in strain-2 guinea pigs.

Introduction

The formation of cancer metastasis is known to consist of various steps and the mechanism of metastasis has been in- vestigated using experimental metastasis models, Lewis lung carcinoma (3LL) has been widely used as one of the haematogenous metastasis model~ and the metastasis oc- curred markedly into lungs of C57BL/6 mice after s.c. or i.d. implantation of 3LL tumour cells [4, 15, 19, 27, 28]. Following i.d. inoculation of line-10 cells into a syngeneic host, a tumour grows progressively at the inoculated site and animals die after the development of palpable metas- tases in regional and distant lymph nodes. The mechanism of metastasis of line-10 hepatoma into regional lymph nodes was examined by Hanna Jr. et al. [6-9] in detail. Zbar et al. [34-37] have reported that intralesional (i.l.) in- jection of living BCG into established line-10 hepatoma caused turnout regression and induced systemic and spe- cific tumour immunity in syngeneic strain-2 guinea pigs. Since then, the line-10 hepatoma model has been used in studies of specific and non-specific immunotherapy of cancer [7, 13, 14, 21, 25, 26, 29, 35, 36]. These 3LL and line-10 tumour models have provided an opportunity for the study of immunotherapy in experimental systems anal- ogous to human tumour metastasis.

In recent years, there have been many reports that mic- ro-organisms such as living BCG [1, 6, 9, 14, 18, 19], the cell-wall skeletons of Propionibacterium acnes [2, 30, 31]

Offprints request to: T. Matsuzaki

and Nocardia rubra [22-24, 32, 33] have considerable anti- tumour activities in experimental animals and clinical tri- als. The heat-killed cells of Laetobacillus easei YIT 9018 (LC 9018), a gram-positive and non-pathogeneic organ- ism, have been widely used as a kind of yogurt preparation (Yakult) and have also been reported to exhibit potent an- ti-tumour activity in allogeneic and syngeneic mouse tu- mour systems [16, 20]. In the present paper, we describe the anti-tumour activity of LC 9018, with special reference to the anti-metastatic effect, using two experimental trans- plantable tumour models, 3LL and line-10 hepatoma in syngeneic mice and guinea pigs.

Materials and methods

Animals. Inbred male C57BL/6J mice, 7 -10 weeks old, were purchased from Shizuoka Agricultural Co-operative Experimental Animals, Hamamatsu, Japan. They were housed in plastic cages and given food and water freely. Inbred female strain-2 guinea pigs were purchased from Nisseiken Co., Ltd., Ohme, Japan. They were fed standard animal diet and tap water ad libitum.

Tumours. Lewis lung carcinoma (3LL) was maintained s.c. by serial transplantation in C57BL/6 mice. Line-10 hepa- toma was maintained in ascites form by serial passage in strain-2 guinea pigs. Following aseptic removal 3LL tu- mours were minced in Hanks' balanced salt solution (HBSS) and the cell suspension was filtered through a stainless mesh. Turnout cells were collected by centrifuga- tion (1,000rpm, 10min) and resuspended in HBSS. Line-10 tumours were harvested from the peritoneal cavity of strain-2 guinea pigs, collected by centrifugation (1,000 rpm, 10 rain) and resuspended in phosphate-buf- fered saline (PBS). The tumour cells were counted using a haemocytometer and adjusted to the desired concentra- tion.

Preparation of Laetobaeillus easel YIT 9018 (LC 9018) and Propionibaeterium anees C7 (P. aenes)

Lactobacillus easei YIT 9018 (LC 9018) was cultured for 24 h at 37 °C in Rogosa's medium as described by Kato et al. [16]. After cultivation, LC 9018 cells were collected by centrifugation, washed with ion-exchanged water, killed with heat (100 ° C, 30 min) and lyophilyzed. Propionibacterium aenes strain C7 was cultured at 37 °C for 2 weeks on thioglycolate medium as described by Azu-

ma et al. [2]. After cult ivat ion the cells were collected by centrifugation, and washed with saline and water. The cell- wall skeleton of P. aches C7 (P. acnes-CWS) was prepared as descr ibed by Azuma et al. [2]. These heat-ki l led whole cells of P. acnes and P. acnes-CWS were used as reference adjuvants in this study.

Effect o f LC9018 on 3LL. Lewis lung carc inoma cells (5 x 105) were inoculated s.c. into the left groin in C57BL/6 mice. LC 9018 was injected i.v. (Exper iment 1, result will be shown in Table 1) or i.1. (Exper iment 1, result will be shown in Table 1) or i.1. (Experiment 2) on days 7, 10, 13 and 16 after tumour cell inoculat ion. The mice in Experi- ment 2 were d ivided into two groups (Groups A and B) and the mice of Group A were kil led on day 28 and the metastat ic nodules in the lungs counted (Table 2). The sur- vival of Group B was moni to red for 90 days (Fig. 1). In Exper iment 3, the mice were injected with 5 x 105 cells of 3LL into a left h ind footpad. LC 9018 was given i.1. on days 3, 6 and 9 after tumour cell inoculat ion and the pri- mary tumour was amputa ted on day 14. After amputa t ion , LC 9018 was injected i.v. and all mice were kil led on day 28. The number of metastat ic foc~ was determined by counting the surface colonies (Table 3).

Effect o f LC 9018 on line-lO hepatoma. Line-10 cells (5 × 106) were inoculated i.d. in strain-2 guinea pigs. LC 9018 was injected i.l., at which t ime the tumours were 9 - 1 0 m m in diameter , and the tumour sizes and lymph node metastases in surviving animals were examined every week. On day 73, tumour-free aniixmls were inoculated with line-10 cells (1 .5x 106) i.d. on the opposi te site in

19

strain-2 guinea pigs. Skin react ion at 24 h after tumour in- oculat ion and tumour growth at inocula ted site were ex- amined.

Statistical analysis. Student 's t-test was used to compare the number of pu lmonary metastases and weight of lungs. Mann-Whi tney U-test was used to compare survival time. Fisher exact test was used to compare survival rates.

R e s u l t s

Anti-tumour effect o f LC 9018 on 3LL

We compared the effect of the t iming of t reatment with LC 9018 against 3LL tumour inocula ted s.c. in C57BL/6 mice. In t ravenous or i.1. injection of LC 9018 from the 1st or the 3rd day after tumour cell inoculat ion slightly pro longed the survival per iod of the mice and inhibited the pulmon- ary metastases (data not shown), while it was effective to treat from the 7th day following inoculat ion of tumour cells. As shown in Table 1, i.v. injection of LC 9018 signifi- cantly inhibited pu lmonary metastases as effective as P. acnes-CWS as compared with the control group in 3LL tu- mour-bear ing mice (P<0.001). Intrales ional injection of LC 9018 was also effective for inhibi t ion of pu lmonary metastases (Table 2) and resulted in pro longat ion of the survival per iod of 3LL tumour-bear ing mice (Fig. 1). In part icular , i.1. injection of LC 9018 cured two out of seven mice on day 90 in group 2 (Fig. 1).

The combinat ion t reatment of i.1. and i.v. injections of LC 9018 on the inhibi t ion of pu lmonary metastases in 3LL tumour-bear ing mice is repor ted in Table 3. Intrales ional

Table 1. Anti-metastatic effect of i.v. injection of LC 9018 on 3 LL

Expt. a Adjuvant Dose b No. oftumour-free group

No. of mice tested

No. of pulmonary metastases c (median)

Weight of lungs (mg) (mean + SE)

1 - saline ×4 0/7 2 LC 9018 250 gg × 4 0/7 3 LC 9018 100 ~g x 4 0/7 4 LC9018 50 ~g x ~L 0/7 5 P. acnes-CWS 250 gg × ~ 0/7

68,65,65,58,55,52,48 (58) 480 ± 58 19, 17,8,7,5 ,3 ,2 (7)* 170 ± 6* 23,23,18, 13, 12, 12,6 (13)* 175 ± 8* 35,32,23,19,14,10 (19)* 205 ± 12" 26,19,16,15,9,7 (15)* 196 ± 21"

a 3LL cells (5 x 105) were inoculated s.c. in C57BL/6 mice on day 0 b LC 9018 or P. acnes-CWS was injected i.v. on days 7, 10, 13 and 16 c The number of pulmonary metastases was counted on day 28 Statistical significance by Student's t-test from control: * P < 0.001

Table 2. Anti-metastatic effect of i.1. injection of LC 9018 on 3 LL

Expt. a Treatment b No. of tumour-~ree No. of pulmonary metastases c Weight of lungs (mg) group with LC 9018 (median) (mean + SE)

No. of mice tested

1 saline x 4 0/7 44, 40, 36, 27, 27, 14, 13 (27) 248 ___ 28 2 2501xg ×4 2/7 24, 3 ,1 ,0 ,0 ,0 ,0 (0)*** 148___ 8** 3 100 lxg x 4 0/7 21, 17, 17, 12, 11, 6, 6 (12)** 178 _ 15" 4 50 txg x 4 0/7 30, 22, 18, 13, 11, 7, 4 (13)* 207 + 26

a 3LL cells (5 x 105) were inoculated s.c. in C57BL/6 mice on day 0 b LC 9018 was injected i.1. on days 7, 10, 13 and 16 c The number of pulmonary metastases was counted on day 28 Statistical significance by Student's t-test from control: *P < 0.05, **P < 0.01, ***P < 0.001

20

"~ so

loo II

I,, I I

i ~ P..C 0.001 I, P,0.001

~- II. o (ZlT}

I P'(O.O1 l - I/ = (1/7~

' ' ' ' l ' '

30 40 50 60 90

Days after tuiiior inoculation

Fig. 1. Effect of LC 9018 on the survival rate of 3LL tumour-bearing mice. 3LL cells (5 x 105) were inoculated s.c. in C57BL/6 mice on day 0. LC 9018 was injected i.l. on days 7, 10, 13 and 16 at a dose of 250 l~g (©), 100 p.g (11), and 50 Ixg (n) , respectively. Control group ( 0 ) was injected with saline. The survival was monitored for 90 days. PValue was determined by Mann-Whitney U-test. Numbers in parentheses indicate tumour-free animals on day 90

Table 3. Inhibition of lung metastases by LC 9018 in 3LL

Expt. a Treatment b No. of pulmonary metastases ~ Weight of lungs (mg) group with LC 9018 (median) (mean ± SE)

i.1. i.v. 1 - - 132,121,118,89,87,78,47 (89) 6 5 6 ± 6 4 2 + - 91,73,59,56,55,42,31 (56)* 512 ± 63 3 - + 81,71,47,44,33,32,23 (44)** 388 ± 16" 4 + + 30,16,14,12,10,10,6 (12)*** 341 ± 20***

a 3LL cells (5 × 105) were inoculated into a footpad of C57BL/6 mice on day 0 b LC 9018 (100 lxg/mouse) was given i.1. on days 3, 6 and 9, and the primary tumour was amputated on

day 14. LC 9018 (250 lxg/mouse) was given i.v. on days 15, 18, 21, and 24 c The number of pulmonary metastases was counted on day 28 Statistical significance by Student's t-test from control: * P < 0.02, ** P < 0.01, *** P < 0.001

or i.v. injection of LC 9018 alone was effective for inhibi- t ion of pu lmonary metastases and the combinat ion treat- ment with LC 9018 was most effective for inhibi t ion of pu lmonary metastases compared with i.v. or i.1. injection of LC 9018 alone ( P < 0.05).

Regressive activity o f L C 9018 on line-lO hepatoma

We examined the regressive activity of LC 9018 on line-10 tumour cells inocula ted i.d. in strain-2 guinea pigs. As shown in Table 4, i.1. injection of LC 9018 was effective for regression of established tumour and prevented the metas- tases into regional lymph nodes of guinea pigs. Mult iple i.l. injections of 400 Jig or 1 mg of LC 9018 were markedly effective for the regression of line-10 hepatoma. Delayed type skin react ion was observed following i.d. reinocula- t ion of line-10 tumour cells in guinea pigs in which the tu- mour had been cured by the i.l. injection of LC 9018, and tumour growth at the re inoculated site was completely re- jected.

D i s c u s s i o n

We have descr ibed here the ant i - tumour activity of LC 9018, conducted using two experimental metastasis mod-

els, 3LL and line-10 hepatoma. It has been demonst ra ted that i.1. injection of viable BCG exhibited the regression of line-10 tumours inoculated i.d. in strain-2 guinea pigs and el iminated the regional lymph nodes metastases [8, 34]. It was also repor ted that i.1. or i.v. injections of P. acnes-CWS was effective for both the survival per iod and the prevent ion of pu lmonary metastases of C57BL/6 mice in 3LL tumour-bear ing mice [30, 31]. The mechanism of living BCG and P. acnes-CWS on anti-metastat ic activity has been repor ted to be dependent on the act ivated macro- phages accumulated in the inoculated site of the immu- noadjuvants [2, 3]. Our present study indicated that i.1. or i.v. injection of LC 9018 was effective for both the survival per iod and the inhibi t ion of pu lmonary metastases in C57BL/6 mice inoculated s.c. with 3LL tumour (Tables 1, 2 and Fig. 1). The combinat ion t reatment of i.1. and i.v. in- ject ions of LC 9018 was effective for the inhibi t ion of pul- monary metastases in C57BL/6 mice with surgically ex- cised 3LL tumours (Table 3). As shown in Tables 1 and 4, LC 9018 was as effective as P. acnes whole cells or P. acnes-CWS in preventing metastases. Intrales ional injec- t ion of LC 9018 caused regression of established line-10 hepa toma in strain-2 guinea pigs (Table 4). Systemic tu- mour immuni ty was demonst ra ted by the subsequent rein-

Table 4. Anti-tumour activity of LC 9018 on line-10 hepatoma in strain-2 guinea pigs

21

Expt. a Adjuvant b Dose Results at 73 days group

Results at 24 h (Rechallenge)

Survivol ~ Tumour-free d Lymph node Mean diameter of erythaema metastasis e (mm) e

1 PBS ( - ) × 4 0/6 0/6 6/6 - 2 LC 9018 400 l.tg x 1 3/6 3/6* 3/6 11 (11, 11, 11.5) 3 LC 9018 400 txg × 4 6/7 5/7** 2/7 13 (11.5, 11.5, 12, 15.5, 14) 4 LC 9018 1 I.tg x 1 2/7 2/7 5/7 13 (12.5, 14) 5 LC 9018 1 I.tg x 4 5/6 5/6*** 1/6 11 (11, 11, 12.5, 10.5, 11) 6 P. acnes 400 ~tg × 4 5/6 5/6*** 1/6 13 (13.5, 12, 12, 13.5, 11.5)

(whole cells)

a Line-10 tumour cells (5 × 106) were inoculated i.d. in strain-2 guinea pigs b LC 9018 or P. acneswas injected i.l., at which time the tumours were 9-10 mm in diameter c No. of survival animals/No, of tested animals d No. of tumour-free animals/No, of tested animals e NO. of animals with metastases/No, of tested animals f In group of tumour-free animals, line-10 cells (1.5 x 106) were inoculated i.d. on the opposite site in strain-2 guihea pigs and skin

reaction was measured 24 h after the rechallenge. Numbers in parentheses indicate the sizes of skin reaction in individual animals Statistical significance by Fisher exact test from control: *P < 0.05, **P < 0.01, *** P < 0.001

oculation of tumour cells in guinea pigs in which the tu- mour had regressed following i.1. injection of LC 9018.

On the mechanism for preventing metastases, another possible explanat ion of other effector cells may be the sti- mulat ion of natural killer (NK) cells [5, 10-12]. LC 9018 has been reported to augment the functions of N K cells as well as alveolar macrophages after kv. injection into var- ious mice [17]. However, N K cells from C57BL/6 mice in- jected with LC 9018 were found not to be cytolytic for 3LL tumour cells in vitro (data not shown). Therefore, we con- sidered that the effect of LC 9018 on the prolongat ion of survival period and the inhibi t ion of pulmonar3~ or lymph node metastases in 3LL tumour-bearing mice or line-10 tu- mour-bearing guinea pigs was due to the activated macro- phages induced by injection of LC 9018. Intralesional in- jection of LC 9018 prolonged the survival period after i.d. inoculat ion with B16-F10 or B16-BL6, which are highly metastatic variants of B16 melanoma, in C57BL/6 mice. We observed that i.1. injection of LC 9018 before surgical excision of the primary tumour was effective for the inhi- bition of both axillary lymph nodes and lung metastases in B16-BL6-bearing mice. On the other hand, i.v. injection of LC 9018 was effective for both prolongat ion of the surviv- al period and prevention against pulmonary metastases of C57BL/6 mice after i.v. inoculat ion with B16-F10 tumour. Intravenous injection of LC 9018 into C57BL/6 mice also could render their alveolar macrophages tumouricidal [Matsuzaki et al. Jpn J Cancer Res (Gann) submitted].

In conclusion, we have demonstrated ant i - tumour ac- tivity of LC 9018, especially an anti-metastatic effect, by using two experimental metastases models. Intralesional a n d / o r i.v. injections of LC 9018 were effective for the in- hibition of lung or lymph node metastases in syngeneic mice or guinea pigs, and the ant i -mmour activity of LC 9018 seems to be almost comparable with P. aenes whole cells and P. a e n e s - C W S . Hereafter, further investigations will be required to clarify the detailed mechanism in pre- venting cancer metastases with LC 9018.

Acknowledgements. We gratefully thank Ms. M. Araki for typing the manuscript. This work was supported in part by Grants-in-Aid

for Cancer Research from the Ministry of Education, Science and Culture, for Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, and for Scientific Re- search from the Ministry of Education, Science and Culture.

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Received February 6, 1985/Accepted March 22, 1985