Cancer Immunol Immunother (1988) 26:209-214 a ncer mmunology mmunotherapy

© Springer-Verlag 1988

Antitumor effect of intrapleural administration of Lactobacillus casei in mice

Takeshi Matsuzaki, Teruo Yokokura, and Masahiko Mutai

Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186, Japan

Summary. The antitumor effect of intrapleural (i.pl.) ad- ministration of Lactobacillus casei YIT 9018 (LC 9018) on Meth A sarcoma in BALB/c mice was examined. Inocula- tion of Meth A cells into the thoracic cavity of BALB/c mice caused growth of the cells and the mice died from the tumor with an increased amount of pleural fluid. LC 9018 was given i. pl. to BALB/c mice before or after i. pl. inocu- lation of Meth A cells and the survival of the mice was de- termined. The i.pl. administration of LC 9018 was effec- tive in prolonging the survival of the mice after i. pl. inocu- lation of Meth A tumor, and pretreatment with LC 9018 i.pl. also prolonged survival. Moreover, i.pl. administra- tion of LC 9018 not only increased the number of thoracic exudate cells (TEC) but also augmented both cytolytic ac- tivity of thoracic macrophages and natural killer cell activ- ity of TEC. Furthermore, phagocytic activity of thoracic macrophages against sheep red blood cells was enhanced and Ia antigen-positive cells in TEC were increased by the i.pl. treatment with LC 9018. These results showed that TEC induced by i.pl. administration of LC 9018 had anti- tumor activity against Meth A tumor inoculated i. pl. into BALB/c mice.

Introduction

Tumor growth occasionally causes exudation of fluid in various organs and cavities in the late tumor-bearing stage of experimental animals. In lung cancer with malignant pleurisy, the diminution of the effusion containing tumor cells is one of the most important problems. Bacillus Cal- mette-Gu6rin (BCG) and Nocardia rubra cell wall skeleton (N-CWS) are among the immunotherapeutic agents that have been evaluated in lung cancer in experimental ani- mals and clinical trials [2, 4, 6, 7, 20-24]. In particular in malignant pleurisy, attempts have been made to utilize treatment with such immunoadjuvants. It has been report- ed that intrapleural (i.pl.) inoculation of tumor cells into the thoracic cavity of C57BL/6 mice causes death of the mice, with an increased amount of effusion fluid contain- ing the tumor cells, and that i.pl. treatment with BCG- CWS is effective in inhibiting this effusion [22]. Yasumoto et al. [24, 25] reported that BCG-CWS treatment had a marked therapeutic effect when given to lung cancer pa-

Offprint requests to: T. Matsuzaki

tients in stages I and II. However, in advanced stages of lung cancer, BCG therapy in clinical trials has not shown a beneficial effect. On the other hand, N-CWS therapy was reported to be benefical in malignant pleurisy in lung can- cer [23, 26]. These results suggest that a trial of i.pl. im- munotherapy is appropriate for lung cancer in some stages.

Recently, much attention has been focused on the mi- croorganisms which exhibit strong antitumor activity in experimental systems and in clinical trials [1, 12, 15, 16, 19, 27]. Heat-killed cells of Lactobacillus casei YIT 9018 (LC 9018), a gram-positive and nonpathogeneic organism, have been reported to exhibit strong antitumor activity and to inhibit metastasis in allogeneic and syngeneic mouse or guinea pig systems [5, 9-11]. It has been report- ed also that injection of LC 9018 protects mice against Pseudomonas aeruginosa and Listeria monocytogenes in- fection [13, 14]. In the present work, we studied the antitu- mor activity of LC 9018 using an experimental tumor model in syngeneic mice, and investigated the cytolytic ac- tivity of thoracic macrophages and natural killer (NK) cell functions of thoracic exudate cells (TEC) elicited with LC 9018. The thoracic macrophages induced by the i. pl. treat- ment with LC 9018 were also examined for phagocytic ac- tivity and Ia expression.

Materials and methods

Animals. Inbred male BALB/c mice, 7 -10 weeks old, were purchased from the Shizuoka Agricultural Coopera- tive Association for Laboratory Animals, Hamamatsu, Ja- pan. They were housed in plastic cages and given food and water ad libiturn.

Tumor. The Meth A fibrosarcoma syngeneic in BALB/c mice was used and maintained in ascites form by weekly i.p. passage. YAC-1 lymphoma and P-815 mastocytoma cells used as target cells for N K cell assay were maintained in vitro in RPMI 1640 medium (Nissui Seiyaku Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS).

Preparation of LC 9018, 0K432, C. parvum and BCG. The LC 9018 was cultured for 24 h at 37 ° C in Rogosa's medi- um as described by Kato et al. [5]. After cultivation, LC 9018 cells were collected by centrifugation, washed with ion-exchanged water, killed by heating at 100°C for 30

210

min, and lyophilized. LC 9018 was suspended in sterile sa- line before use. The OK432, Corynebaterium parvum (C. parvum), and BCG were purchased from Tyugai Seiya- ku Co., Ltd., Tokyo, Japan, the Institute Merieux, Lyon, France and Kyowa-yakuhin Inc., Co., Ltd., Tokyo, Japan, respectively.

Antitumor experiment. Mice (10/group) were inoculated i.pl. with Meth A cells at a dose of l × 10 4, 5 × 10 4, 1 × 105, 5 x 105 or 1 x 106/mouse and with LC 9018, OK432, C. par- rum, or BCG was given i. pl. and their survival was moni- tored. The survival rate was calculated using the following formula:

T/C (%) = mean survival days of treated group mean survival days of control group x 100

Preparation of TEC. The TEC were collected by washing the thoracic cavity of mice (10/group) with 2.5 ml of Hanks' balanced salt solution (HBSS). The TEC suspen- sion was centrifuged (500 g, 10 min) and resuspended in HBSS. The cells were counted using a hemocytometer, and adjusted to the desired concentration.

Population of TEC. Mice (10/group) were given LC 9018 (100 p.g/mouse) i.pl. on day 0 and TEC were collected on days l, 3, 5, 7, 10, and 14. The population of TEC was de- termined microscopically in cytocentrifuge preparations stained with May-Grfinwald and Giemsa solutions.

Cytolytic activity of thoracic macrophages. Mice (10/group) were injected with LC 9018 (100 txg/mouse) i.pl. on day 0, and TEC were obtained on days 1, 3, 5, and 7. The TEC suspension was incubated in a plastic dish at 37 ° C for 90 min twice and the plastic adherent or nonadherent cells were obtained. The cytolytic activity of the adherent or non- adherent cells for Meth A cells was assayed using a 51Cr release assay. In brief, Meth A cells (1 x 106) as target cells were labeled by incubation with 100 ~tCi of Na 2 51CRO4 (New England Nuclear, Boston, Mass., USA) for 1 h at 37°C in a shaking water bath. After incubation, the cells were washed three times with HBSS to remove unbound radiolabel. The target cells (1 × 104/well) were placed in 96-well microtiter plates (Nunc, Roskilde, Denmark) and effector cells in a volume of 100 lxl were added to triplicate cultures. The plates were centrifuged for 5 min at 400 g and then incubated for 18 h at 37°C in a humidified at- mosphere containing 5% CO 2. After incubation, the plates were centrifuged for 7 min at 500 g. The percent specific release of SlCr was determined by removing 0.1 ml of su- pernatant from each well and counting the activity in a gamma-scintillation counter (400CGD, Packard Instru- ment Co., Inc., Downer's Grove, Ill., USA). Percent cyto- toxicity mediated by the thoracic macrophages was calcu- lated as follows:

Cytotoxicity (%) = experimental 5~Cr release - spontaneous 51Cr release x 100

total 5 J Cr release - spontaneous 5z Cr release

NK cell activity. Mice (10/group) were injected with LC 9018 (100 Ixg/mouse) i.pl. on day 0, and TEC were collect- ed on days 1, 3, 5, and 7. The NK activity of the TEC was determined as described by Herberman et al. [3]. It was as- sessed using a 4-h 51Cr release assay with YAC-1 target

cells. Whole TEC were incubated in a plastic dish (Falcon 3003 culture dish) twice at 37°C for 90 min to deplete them of plastic adherent cells. Then the plastic nonadher- ent cells were collected and used as the effector cells for the NK assay. The cytotoxicity was determined as de- scribed for assay of the cytolytic activity of TEC.

Phagocytic activity against opsonized sheep red blood cells. Sheep red blood cells (SRBC) and anti-SRBC lgG were purchased from Nippon Bio-Test Laboratories Inc. and Japan Immunoresearch Laboratories Co., Ltd., Takasaki, Japan, respectively. The SRBC were washed twice with phosphate-buffered saline (PBS) and then incubated with an equal volume of 1:50 diluted anti-SRBC IgG for 30 rain at 37 ° C. The TEC were obtained from mice by wash- ing out the thoracic cavity with HBSS. The TEC were resus- pended in RPMI 1640 medium containing 10% FBS. The TEC suspension was placed on cover slips, and after incu- bation at 37 ° C for 90 min in 5% CO 2 in air nonadherent cells were washed off with PBS and then the cover slips were incubated with anti-SRBC IgG-coated SRBC at 37°C for 60 min. After treatment with 0.83% ammonium chloride to lyse the nonadherent phagocytic red cells, the number of SRBC phagocytosed by thoracic macrophages was determined microscopically. Phagocytic activity was expressed as the numbr of SRBC phagocytosed per 100 macrophages.

Expression of la antigen in thoracic macrophages. The TEC were harvested as described and resuspended in RPMI 1640 medium. The TEC suspension was allowed to stand on glass cover slips for 2 h at 37 ° C in 5% CO 2 in air and then nonadherent cells were removed by gentle washing with HBSS. The glass-adherent cells were fixed with 0.1% paraformaldehyde and the cover slips were incubated with 100 p~l of 1:500 diluted anti-I-A d antibody (Becton Dickin- son Monoclonal Center, Inc., Mountain View, Calif., USA) for 30 min on ice. After treatment with 100 lxl of 1:100 diluted fluorescin isothiocyanate-conjugated F(ab')2 fragment rabbit anti-mouse IgG antibody (Cappel Labo- ratories, Cochranville, Pa., USA), the cover slips were washed with PBS and Ia antigen-positive cells counted us- ing a fluorescence microscope.

Statistical analysis. The experimental results were analyzed for their statistical significance by Student's t-test.

Results

Growth of Meth A cells in the thoracic cavity

We first determined the growth rate of Meth A cells in the thoracic cavity of BALB/c mice. Meth A cells were inocu- lated i.pl. into mice at a dose of 1 × 104 , 5 x 104 , 1 x l0 s , 5 × 105, or 1 x 106/mouse and the survival time was calcu- lated (Table 1). The duration of survival of the mice de- pended on the inoculum size. We also observed an in- crease in the effusion containing tumor cells in the thora- cic cavity of the dead mice.

Antitumor effect of LC 9018

The effect of LC 9018 on the survival of mice following in- oculation of Meht A i. pl. was determined. Based on the re- sults shown in Table 1, an inoculum of 5 x 105 tumor cells

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Table 1. Survival time of BALB/c mice inoculated with Meth A cells i.pl.

No. of Meth A cells Survival time inoculated (per mouse) a (days, mean _+ SD)

1 x 106 6.6 -+ 0.5 5 x 105 7.0+0.6 1 x 10 s 13.3 + 2.4 5 × 104 18.6 + 1.0 1 x 104 22.6 _+ 2.6

a Meth A cells were inoculated i.pl. into BALB/c mice (10 mice/ group) and survival monitored

was used because we obtained reproducible results in the survival of tumor-bearing mice inoculated with that num- ber of cells.

Meth A cells (5x 105/mouse) were inoculated into mice i.pl. on day 0. LC 9018 was given i.pl. on days 1 to 5 or 1, 3, and 5 at a dose of 100 or 250 lxg/mouse. The i.pl. t reatment with LC 9018 significantly prolonged the surviv- al of the mice (Table 2) and the tumor cells in the thoracic cavities of the mice decreased compared with the control group, while a single injection of LC 9018 on day 1, 3, or 5 was slightly effective (data not shown). It was more effec- tive in prolonging survival of the mice when used at a dose of 100 ~tg/mouse than at 250 gg/mouse. Pretreatment with LC 9018 given i.pl. also significantly prolonged the surviv- al of the mice inoculated with Meth A i.pl. (Table 3). In

Table 2. Effect of LC 9018 on the survival of Meth A-bearing mice

Group a Treatment b Survival time T/C with LC 9018 (days, mean -+ SD) (%)

1 - 7.4+0.9 100 2 250 lag x 5 9.6-+2.8 130" 3 250 lag x 3 8.6-+ 1.1 116 4 100lag x 5 10.9+3.0 147"* 5 100 lag x 3 10.1+1.9 136"

a Meth A cells (5 x 105/mouse) were inoculated i.pl. into BALB/c mice (10 mice/group) on day 0 b LC 9018 was injected i.pl. on days 1 to 5 (groups 2 and 4) or on days 1, 3, and 5 (groups 3 and 5) Statistical significance of difference from group 1: * P <0.05, • * P < 0.01

Table 3. Effect of pretreatment with LC 9018 on the survival of Meth A-bearing mice

Group a Treatment b Survival time T/C with LC 9018 (days, mean -+ SD) (%)

1 - 7.3 _+ 0.8 100 2 250 tag x 5 14.3 +3.4 196" 3 250 lag x 3 17.1 +6.6 234* 4 100 lag x 5 21.0_+3.9 288* 5 100 lag x 3 20.8_+5.2 285*

a Meth A cells (5 x 105/mouse) were inoculated i.pl. in BALB/c mice (10 mice/group) on day 0 b LC 9018 was injected i.pl. on days - 5 to - 1 (groups 2 and 4), or on days - 5, - 3, and - 1 (groups 3 and 5) Statistical significance of difference from group 1: * P < 0.001

Table 4. Effect of i.pl. injection of LC 9018, Corynebacterium parvum (C. parvum) and bacillus Calmette-Gtierin (BCG) on the survival of Meth A-bearing mice

Treatment Dose Survival time T/C with adjuvants (days, mean _+ SD) (%)

- (control) - 10.8 + 1.5 100 LC 9018 500 lag 18.2_+4.0 169"* LC 9018 250 lag 20.0_+3.6 185"** LC 9018 100 lag 27.0_+0.7 250*** OK432 250 txg 15.7+2.2 145"* OK432 100 lag 15.7-+3.6 145" C. parvum 1 mg 11.2+ 1.2 104 C. parvum 500 lag 11.2-+2.3 104 C. parvum 250 lag 9.6_+ 1.2 89 C. parvurn 100 lag 13.0 _+ 1.1 120" BCG 1 mg 9.6 -+ 1.5 89 BCG 500 Ixg 14.2-+3.1 131" BCG 250 lag 13.8-+4.2 128 BCG 100 lag 15.5_+1.1 144"*

Meth A cells (1 x 105/mouse) were inoculated i.pl. in BALB/c mice (10 mice/group) on day 0. LC 9018, OK432, C. parvum, or BCG was injected i.pl. on days 1, 3, 5, 7, and 9, and survival of the mice was monitored Statistical significance of difference from control: *P <0.05, ** P <0.01, *** P <0.001

this case, a single injection of LC 9018 on day - 5 or - 3 was also effective. Since administrat ion of LC 9018 i.pl. into Meth A-bearing mice prolonged the survival, we com- pared the ant i tumor effect of OK432, C. parvum, or BCG with that of LC 9018. We determined the survival effect of an inoculum of less than 5 x 105 tumor ceils because long- term and multiple administrat ion could be given. Meth A cells (1 x 105/mouse) were inoculated into mice i.pl. on day 0, and LC 9018, OK432, C. parvum, or BCG was given on days 1, 3, 5, 7, and 9 after tumor cell inoculation, and the T / C values were calculated (Table 4). Maximum T / C values for the OK432-, C. parvum-, and BCG-treated groups were 145 (P <0.01), 120 (P <0.05), and 144 (P < 0.001), respectively, while that of the LC 9018-treated group was 250 (P < 0.001). These results suggested that LC 9018 was as effective as OK432, C. parvum, or BCG.

Effect of LC 9018 on the number of TEC

The effect of i.pl. injection of LC 9018 on the number of TEC in mice was studied. LC 9018 (100 gtg/mouse) was given i.pl. on day 0 and TEC were harvested on days 1, 3, 5, 7, 10, and 14. The total number of TEC induced by i.pl. injection of LC 9018 was increased on day 1 and a high level was mainta ined for 14 days (Fig. 1). The peak of the number of TEC was observed on day 1. The main popula- tion of the TEC in the early period after LC 9018 injection were polymorphonuclear leukocytes (PMN) and after that the relative populat ion of macrophages increased.

In vitro cytolytic activity of thoracic macrophages and NK cell activity of TEC

We determined the cytolytic activity of thoracic macro- phages and N K function of TEC induced by i. pl. injection of LC 9018. Mice were given LC 9018 (100 gg/mouse) i.pl. on day 0 and TEC were collected on days 1, 3, 5, and 7.

212

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Fig. 1. Effect of intrapleural (i. pl.) injection of Lactobacillus casei YIT 9018 (LC 9018) on the number of thoracic exudate cells (TEC). The LC 9018 (100p.g/mouse) was injected i.pl. into BALB/c mice (10 mice/group) on day 0, and TEC were collected on days 1, 3, 5, 7, 10, and 14, and the population of the TEC ex- amined. Bars represent the SD of the total number of TEC. (1~): macrophages, ( l l ) : lymphocytes, ([]): PMNs

Table 5. In vitro cytolytic activity of TEC from mice injected with LC 9018 i.pl. against Meth A cells

Days after LC 9018 injection a

Cytolytic activity b, c

Whole Adherent Nonadherent

Untreated (control) 0.2±1.8 d -3 .7±1 .9 1.8±1.5 1 2.5±1.5 1.0±4.3 4.7±0.2 3 32.7±4.8 37.9±4.4 -1 .8±1 .6 5 28.6±6.1 22.6±3.7 3.3±0.8 7 27.4±4.6 18.9±6.4 0.7±2.7

a LC 9018 was given i.pl. to BALB/c mice (10 mice/group) at a dose of 100 I.tg/mouse b Cytolytic activity was measured by the 5~Cr release assay de- scribed in Materials and methods 0 Effector: target ratio was 25:1 d Mean ± SD of triplicate cultures

The cytolytic activity against Meth A cells and N K cell act ivi ty were assessed by the 51Cr release method descr ibed in "Mater ia ls and methods". The cytolytic activity of the TEC, especial ly the thoracic macrophages , against Meth A was increased on day 3 and was main ta ined for 7 days (Table 5). The N K cell act ivi ty o f TEC against YAC-1 l y m p h o m a was also enhanced on days 3 to 7 by the treat- ment with LC 9018 (Table 6).

Phagocytic activity of thoracic macrophages

LC 9018 (100 ~tg/mouse) was injected i .pl. into mice on day 0 and TEC were collected on days 1, 3, 5, and 7, and examined for phagocyt ic activity against SRBC. The activ- i ty was marked ly increased on days 1 and 3 and then de-

Table 6. Natural killer cell activity of TEC from mice injected with LC 9018 i.pl.

Days after LC 9018 injection a

Cytolytic activity against b

YAC-1 P-815

100:1 c 50:1 100:1

Untreated (control) 1.8±1.3 d 1.7±1.8 -1 .6±1.9 1 2.6±1.8 1.3±0.6 -0 .2±4 .4 3 52.4±3.0 47.0±7.7 1.4±1.5 5 47.0±9.2 34.6±5.0 -2 .8±3.7 7 57.0±7.0 50.6±6.9 2.3±1.3

" LC 9018 was given i.pl. to BALB/c mice (10 mice/group) at a dose of 100 p-g/mouse b Cytolytic activity was measured by the 51Cr release assay described in Materials and methods o Effector: target ratio d Mean + SD (%) of triplicate cultures

creased gradual ly (Fig. 2). However , the activity after 7 days was significantly higher than that of resident macro- phages obta ined from normal mice.

Ia expression of thoracic macrophages

The effect of LC 9018 given i.pl. on the induct ion of Ia ex- press ion of thoracic macrophages was determined. LC 9018 (100 p~g/mouse) was injected i.pl. into mice on day 0 and TEC were collected on days 1, 3, 5, 7, 10, and 14, and then the number of Ia-posi t ive thoracic macrophages was de termined (Fig. 3). The resident thoracic macrophages were 40% Ia positive, and Ia-posi t ive cells gradual ly in- creased after the inject ion of LC 9018. On day 7, 90% of

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Fig. 2. Phagocytic activity of thoracic macrophages against sheep red blood cells (SRBC). The LC 9018 (100 p-g/mouse) was inject- ed i.pl. into BALB/c mice (10 mice/group) on day 0, and thoracic macrophages were collected on days 1, 3, 5, and 7 and examined for phagocytic activity against SRBC

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Fig. 3. Ia expression of thoracic macrophages. The LC 9018 (1001xg/mouse) was injected i.pl. into BALB/c mice (10 mice/group) on day 0. Thoracic macrophages were collected on days 1, 3, 5, 7, 10, and 14 and Ia-positive cells were counted

the thoracic macrophages were Ia positive and this high percentage was maintained for 14 days after LC 9018 in- jection.

Discussion

We have demonstrated the antitumor effect of an immuno- potentiator, LC 9018, using an experimental model in mice. Azuma et al. [1] reported that N-CWS has a potent adjuvant effect on the induction of cell-mediated cytotox- icity in mice and also that it is useful as an immunothera- peutic agent for patients with malignant pleurisy. Yamamu- ra et al. [23] reported that malignant cells disappear from the pleural effusion in lung cancer patients after i. pl. treat- ment with N-CWS. These results suggest that the immu- noadjuvant has a very beneficial effect in lung cancer pa- tients with malignant pleurisy. Yamamura et al. [22] also reported that treatment with BCG-CWS prolonged the life of C57BL/6 mice inoculated with squamous cell carcino- ma of the lung induced by repeated i.pl. injection of ben- zo(a)pyrene along with charcoal powder. We previously reported that i.pl. administration of LC 9018 into Lewis lung carcinoma-bearing mice prevents lung metastasis [11].

In this study, we injected LC 9018 i.pl. into BALB/c mice inoculated with Meth A tumor cells i. pl. The i. pl. in- jection of Meth A cells resulted in subsequent growth of the tumor cells and the mice died from the tumor with in- creased pleural effusion (Table 1). This finding suggests that this experimental model is suitable for studying ex- udation into the thoracic cavity. Intrapleural injection of LC 9018 following the inoculation of Meth A cells into BALB/c mice prolonged the survival of the mice (Table 2) and treatment with LC 9018 i.pl. before i.pl. inoculation of Meht A cells also significantly prolonged the survival (Table 3). LC 9018 was as effective as OK432, C. parvum, or BCG when the agents were injected i.pl. (Table 4). Moreover, tumor cells in the pleural effusion disappeared

after i.pl. injection of LC 9018. In fact, i.pl. treatment with LC 9018 has been tried in a phase II study of patients with lung cancer and a strong beneficial effect was observed (unpublished data).

It has been reported that the cells accumulating in the pleural effusion after i.pl. injection of BCG-CWS are mainly neutrophils in the early phase and that lympho- cytes and histiocytes become predominant at 7 to 14 days [25]. The i.pl. injection of LC 9018 into BALB/c mice in- duced TEC and these cells were also mainly PMNs in the early phase, with macrophages dominant in the late phase (Fig. 1). In immunological studies of cancer patients, treat- ment with BCG-CWS enhanced the lymphocyte response to phytohemagglutinin and augmented the lymphocyto- toxic activity against allogeneic cultured lung cancer cells [8]. It has also been reported that i. pl. administration of a streptococcal preparation, OK432, prolongs the survival of lung cancer patients and that N K cells induced by i.pl. OK432 played an important role in killing tumor cells [17, 18]. We determined the cytolytic activity of thoracic mac- rophages and the N K cell activity of TEC from mice in- jected with LC 9018 i. pl. (Tables 5 and 6). Both the cytolyt- ic activity of thoracic macrophages and the N K cell activi- ty of TEC were augmented by treatment with LC 9018. These results suggest that TEC play a key role in killing tu- mor cells in the thoracic cavity. We also observed that the phagocytic activity against SRBC and Ia antigen expres- sion of thoracic macrophages were enhanced by i. pl. injec- tion of LC 9018. This suggests that the ability of thoracic macrophages to exclude tumor cells was augmented by i.pl. administration of LC 9018, resulting in inhibited tu- mor growth in the thoracic cavity. Macrophages induced by i.pl. administration of LC 9018 should also be expected to play an important role in destroying tumor cells and in other host immune responses.

In conclusion, we have demonstrated antitumor activi- ty of LC 9018 using an experimental system consisting of i. pl. inoculation of Meth A into mice and i. pl. administra- tion of LC 9018 into the tumor-bearing mice. The treat- ment resulted in prolongation of the survival of the mice, enhancement of the cytolytic activity of thoracic macro- phages and N K cell activity of TEC, and augmentation of the phagocytic function and Ia antigen expression of thor- acic macrophages. Thus, we consider that the thoracic macrophages induced by i.pl. administration of LC 9018 play an important role as one of the main effector cells for killing tumor cells in the thoracic cavity. Further studies will be required to reveal the detailed mechanisms by which tumor growth was prevented by LC 9018.

Acknowledgement. We thank Dr. M.L. Robbins for editing the manuscript.

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Received October 8, 1987/Accepted January 13, 1988