Ini J. Radiamn Oncology Em/. Phys., Vol. Printed in the U.S.A. All rights reserved.
0360-3016/90 $3.00 + .oO Copyright 0 1990 Pergamon Press plc
18, pp. 1415-1420
0 Original Contribution COMBINATION THERAPY OF PIONS AND SPG (SONIFILAN, SCHIZOPHYLLAN), A BIOLOGICAL RESPONSE MODIFIER FOR MOUSE TUMOR SYSTEMS
DAVIDJ. CHAPLIN,PH.D.,~ YASUHIRO OGAWA, M.D.,‘T~GEORGE B. GOODMAN, F.R.C.P.C.,' WILLIAM GRULKEY,B.SC.~ANDGABRIELK.Y.LAM,PH.D.~ ‘DevelopmentalRadiotherapy,Cancer Canada;
Control Agency of British Columbia, 600 West 10th Avenue, Vancouver, B.C., V5Z 4E6, ‘Kochi Medical School, Oko-cho, Nankoku-shi, Kochi 783-5 1 Japan; and ‘British Columbia Cancer Research Center, 601 West 10th Avenue, Vancouver, B.C., V5Z 4E6, Canada
Female C3H mice aged 8-10 weeks with transplanted KHT sarcoma or SCCVII tumor were used to investigate the antitumor effect of SPG (Sunifilan, Schizophyllan) alone and in combination with local irradiation of pions with 4 fractions of 400 cGy (total 1600 cGy). Daily doses of 10 mg/kg of SPG were given intramuscularly to the mice bearing KHT sarcoma for 14 consecutive days from day 7, and to mice bearing SCCVII tumor for 20 consecutive days from day 7 and thereafter three times a week for another 2 weeks. The antitumor effect was evaluated by the changes in tumor volume, survival curves, and the number of pulmonary metastatic nodules on the surface of the lungs. SPG failed to exert any antitumor effect and any life-prolonging effect for the KHT sarcoma. As for SCCVII tumor, in the group treated with pions and SPG, tumor growth decreased significantly (p < 0.01) compared with the group treated with pion only, and life prolonging effect and metastasis-suppressing effect were also observed (p < 0.05). In conditions of minimal residual disease brought about by pion irradiation, the adjuvant effect of a Biological Response Modifier (BRM) SPG may prove to be a promising method of cancer therapy for some tumors. Pion, SPG (sonifilan), KHT sarcoma, SCCVII tumor, BRM (biological energy transfer) radiation, Radiotherapy.
response modifier), High LET (linear
of pion and SPG, a BRM (Biological Response Modifier) for mouse tumor systems in this study. Pions are used to reduce the tumor burden to a minimum. SPG, which can restore immune competence, may enhance this effect by controlling the residual tumor and controlling distant micrometastases (24).
INTRODUCTION
Today, surgery, radiotherapy, and antitumor chemotherapy are the three major methods of cancer treatment. Conventional radiotherapy using X rays from linear accelerators or gamma rays from 6oCo teletherapy units has been extensively used for three decades. Although conventional radiotherapy has contributed to improvements in the results of cancer treatment, there is still a considerable number of cases which are not cured. Recently, new methods of radiotherapy (high Linear Energy Transfer radiations) have been developed; pion radiotherapy is one of these new methods (4). Despite the introduction of new radiations with high LET (Linear Energy Transfer) such as pions, failure to eradicate local disease still remains a problem (4). It seems that in a proportion of patients local failure will lead to the development of distant metastases. Therefore, treatment with combination therapy appears sound. For this reason, we have examined the effect of combined therapy
METHODS
AND
MATERIALS
Mice and tumors
All experiments were performed using RI-IT sarcoma or SCCVII tumor in female C3H mice* 8-10 weeks old weighing on average 20 g. Each experimental group consisted of 15 animals housed in 3 cages. All mice were kept in plastic cages on sawdust bedding with five animals per cage. The mice were kept in a small animal facility where the temperature and humidity were constant. Food and water were freely available at all times. RI-IT sarcoma cells and SCCVII tumor cells were transplanted by sub-
Reprint requests to: Dr. Yasuhiro Ogawa, Department of Radiology, Kochi Medical School, Oko-cho, Nankoku-shi, Kochi 783-5 I, Japan. Acknowledgements-The authors would like to thank Mr. Ivan Liu for his technical assistance, Ms. Ksenia Koric for secretarial assistance, and Dr. Dorianne E. Rheaume and Dr. Julie Bowen
for valuable discussion of the experiments. We also appreciate Kaken Pharmaceutical Co., Japan for providing us with SPG (Sonifilan) and information on SPG. Accepted for publication 20 December 1989. * Charles River Inc.
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June 1990, Volume 18, Number 6 see
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Fig. 1. Tumor growth curves of KHT sarcoma implanted in C3H mice treated with pion irradiation and/or SPG. The error bars are 95% confidence intervals.
Fig. 3. The tumor growth curves of SCCVII tumor in C3H mice treated with pion irradiations and/or < 0.0 1 vs all other groups.
cutaneous injections of approximately 2 X 1O5viable cells (obtained from enzymatic disaggregation) into the foot and the irradiations were started when the tumors attained a mean diameter of 5 mm. These two tumors and the method of cell suspension preparation ( 1) have been fully described elsewhere (9, 20).
irradiated in the stopping peak region (2). The space for the mouse foot within the device (2) was enlarged to provide enough room to prevent tumor hypoxia. A schedule with 4 fractions of 400 cGy was used, and pion irradiations were given on day 0, day 1, day 4, and day 7. The total treatment was given in 7 days.
Irradiation procedures,for pion Pion studies were done using an irradiation system described previously which uses no anesthesia (3). A horizontal beam with a field size of 2.0 X 5.0 cm and a primary proton beam current of 160 PA was used. This resulted in an effective dose rate of pions of approximately 20 cGy per min. The field size used encompassed one foot tumor of each of the three mice stacked vertically. The irradiation set up was mounted on a scanning couch system (lo), which delivered the required field size using 6 beam spots. The dosimetry for pion beam has been described elsewhere (11). The mice were positioned in the pion beam with the required amount of bolus, so that the foot tumor was
Schedule of the administration of SPG (sonifilan) SPG is a simple beta-glucan isolated from the fermentation broth of Schizophyllum commune Fries and has a molecular weight of about 450,000 (14). SPG was administered in daily doses of 10 mg/kg in 20 ~1 by intramuscular injections in the leg opposite to the tumor-bearing foot. In the experiment for KHT sarcoma, SPG was injected from day 4 to day 7 in the group which received only SPG (group 2) and from day 7 to day 20 in the group which received pion irradiations (group 4). The control group (group 1) and the group which received pion irradiations alone (group 3) were injected daily with 20 wl/ mouse of sterilized PBS (Phosphate Buffered Saline)
10: t
KHTsar
implanted SPG. *p
coma
-
PION ----_I
PION
00
& Sffi 4 SbC.
I
O0
I
10
I
20
days
Fig. 2. The survival rate of C3H mice with implanted sarcoma treated with pion irradiations and/or SPG.
KHT
20
40
60
day8
Fig. 4. Survival rate of C3H mice with implanted SCCVII tumor treated with pion irradiations and/or SPG. Improvement of survival rate is noted in group 8 compared to group 7 from day 72 to day 78 (p < 0.05).
Combination therapy of pions and SPG 0 Y.
with the same schedule as group 2 and group 4, respectively (16). In the experiment for SCCVII tumor, SPG was injected daily for 20 consecutive days from day 7, and three times a week for 2 weeks, thereafter, for the SPG group (group 6) and for the SPG and pion irradiation group (group 8). The control group (group 5) and the group which received pion irradiations alone (group 7) were injected daily with 20 &mouse of sterilized PBS with the same schedule as groups 6 and 8, respectively (16). Evaluation of antitumor eflect The largest and the smallest diameter and the thickness of the tumor were measured with a micrometer caliper three times a week. The result of these values was recorded as volume (mm3) ( 15). The mortality of mice was recorded daily for 28 days and 78 days after the initiation of pion irradiations for KI-IT sarcoma and SCCVII tumor, respectively. Autopsies were performed to examine the development of metastatic changes. Specifically, in the experiments for SCCVII tumor, mice were sacrificed on day 78 and pulmonary metastases were estimated grossly by counting the number of metastatic nodules on the pulmonary surface after fixing the lungs in Boin’s solution (24). The statistical differences among the groups were analyzed by the Student’s “t” test or the X2 test. RESULTS Figure 1 shows the tumor growth curves of KHT sarcoma implanted in C3H mice treated with pion irradiations and/or SPG. There was no difference in the tumor
OGAWA
et
al.
1417
growth between group 1 and group 2, nor between group 3 and group 4. SPG failed to exert any antitumor effect for KHT sarcoma. Figure 2 shows the survival rate of C3H mice with implanted IQ-IT sarcoma treated with pion irradiations and/ or SPG. There was no difference in the survival rate between group 1 and group 2 nor between group 3 and group 4. SPG failed to exert any life-prolonging effect for KHT sarcoma. Figure 3 shows the tumor growth curves of SCCVII tumor implanted in C3H mice treated with pion irradiations and/or SPG. While no inhibition of the tumor growth was observed in the group which received SPG alone (group 6), remarkable inhibition was noted in the group which received SPG after pion irradiations (group 8) compared to the group which received pion irradiations alone (group 7) (p < 0.01, Student’s “t” test). Figure 4 shows the survival rate of C3H mice with implanted SCCVII tumor treated with pion irradiations and/ or SPG. Statistically significant improvement of survival rate was noted in group 8 compared to group 7 during the last 7 days of a 78 day follow-up period (p < 0.05, X2 test). As shown in Figures 5 and 6, a comparison of tumor sizes on day 78 showed that tumors were very big in all of the five surviving mice in group 7 (Fig. 5) while 2 out of 10 mice in group 8 still had a small sized tumor (Fig. 6). Figures 7 and 8 show the pulmonary metastases of SCCVII tumor in groups 7 and 8 on day 78, respectively. In 4 out of 5 lungs in group 7, pulmonary metastases were observed easily and the average number of metastatic
Fig. 5. Photograph of the 5 surviving mice in group 7 on day 78. Tumor sizes were very big in all of the 5 mice.
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Fig. 6. Photograph
of the 2 of 10 surviving
mice in group 8 on day 78. These two mice still had small sized tumors.
nodules identified macroscopically on the pulmonary surface was 9.6 k 6.9 (mean + 1 S.D.). In group 8 the corresponding numbers were 2.1 + 1.8. There is a significant difference in the number of nodules between these two groups (a < 0.05, Student’s “t” test). Moreover, each nodule of metastatic change observed in group 8 is relatively small compared to that observed in group 7. It is concluded that the combined therapy of pion and SPG inhibited pulmonary metastases compared to treatment with pions alone. DISCUSSION Recently, extensive cerning the effect of immunotherapy using mentally ( 12, 19, 22).
June 1990, Volume 18, Number 6
studies have been reported concombined radiation therapy and BRM both clinically and experiThe importance of timing of the
Fig. 7. Photograph of the lungs of the 5 surviving metastases are easily observed.
combination of local irradiation and BRMs, such as PSK, OK-432, and SPG has been reported (13, 16). BRMs demonstrated significant antitumor activity when they were received after irradiation. It was for this reason that we started the administration of SPG after pion irradiations. SPG is a simple glucan composed of glucose alone prepared from a highly viscous polysaccharide isolated from a culture filtrate of Schizophyllum commune Fries, a species of Basidiomycetes. The viscous polysaccharide is treated ultrasonically at highspeed extrusion to adjust its molecular weight to 450,000. The resulting SPG preparation is chemically homogeneous and its physico-chemical properties have been well characterized (14). In a recent study (16) using MM46 tumor inoculated into C3H/He mice, we have demonstrated that tumor
mice in group 7 on day 78. In 4 out of 5 lungs, pulmonary
Combination therapy of pions and SPG 0 Y.
OCAWA
Fig. 8. Photograph of the lungs of the 10 surviving mice in group 8 on day 78. Fewer and smaller metastatic are noted compared with Figure 7.
growth was most suppressed in the group treated with radiation and SPG. There was a marked infiltration of macrophages into tumor tissue in addition to the heavy infiltrate of T lymphocytes (7). This suggests that the mechanism of enhanced effect from the combined use of SPG with radiotherapy was exerted by immunological responses of the host such as that of T lymphocytes and macrophages. More than 700 patients with malignancy have now been treated with pion beams at LAMPF (Los Alamos Meson Physics Facility) in USA (23) SIN (Swiss Institute for Nuclear Research) in Switzerland (5) and TRIUMF (TriUniversity Meson Facility) in Canada (4). As pions have increased biological effectiveness and lower OER (Oxygen Enhancement Ratio), this beam can kill hypoxic tumor cells more efficiently than conventional radiotherapy (2 1). SPG should therefore exhibit its antitumor effect maximally after the tumor cells are depleted by pion irradiations to numbers controllable by this drug. In the experiment using KHT sarcoma, SPG failed to exert any antitumor effect or life-prolonging effect. The reason for this failure could be that KHT sarcoma has no tumor antigenicity and it is a tumor which grows very rapidly (9). The host-mediated immunological responses
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et al.
lesions
augmented by SPG cannot exert their effect against tumors lacking tumor antigenicity. Moreover, a BRM, such as bestatin, has been demonstrated to be more effective for tumors of slow growth (8). In the experiment using SCCVII tumor, the administration of SPG combined with pion irradiations was found to exert a significant antitumor effect, life-prolonging effect, and metastasis-suppressing effect. As radiotherapy has been demonstrated to promote immune responses in some kinds of tumor tissue (6, 17, 18) and the tumor was of relatively slow growth (20) the combined effect of SPG and pion irradiation should have been fully exerted for this tumor system. This report is the first study demonstrating a significantly slower regrowth of tumors after irradiation using a combination of pions and a BRM. It seems reasonable that pions and BRM through dual mechanisms produce minimal residual tumor cells in the local lesion and augment host-mediated immune responses. Therefore, it is possible that a combined therapy of pion and a BRM such as SPG will prove to be a promising method of cancer treatment in the near future. Further investigations using other tumor systems will be needed to clarify those tumor characteristics most likely to be beneficial.
REFERENCES 1. Chaplin, D. J. Potentiation of RSU- 1069 tumour cytotoxicity by 5hydroxytryptamine (5-HT). Brit. J. Cancer 54: 727-73 1; 1986. 2. Chaplin, D. J.; Douglas, B. G.; Grulkey, W.; Skarsgard, L. D.; Lam, G. K. Y.; Denekamp, J. The response of mouse epidermis to fractionated doses of a mesons. Int. J. Radiat. Oncol. Biol. Phys. 13:1199-1208; 1987. 3. Douglas, B. G.; Fowler, J. F. The effects of multiple small
doses of x-rays on skin reactions in the mouse and a basic interpretation. Radiat. Res. 66:40 l-426; 1976. 4. Goodman, G. B.; Dixon, P.; Lam, G. K. Y.; Harrison, R.; Komelsen, R. 0.; Ludgate, C. M.; Flores, A. D. Preparatory clinical studies of pi-mesons at TRIUMF. Radiat. Res. 104: S-279-S-284; 1985. 5. Greiner, R. Six years pion treatment. SIN Med. Newsletter 9:12-18; 1987.
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6. Hirota, S.; Ogawa, Y.; Seguchi, H. Histological analysis of
7.
8.
9.
10.
1 I.
12.
13.
14.
15.
lymphocyte subsets infiltrated into mouse tumour tissue exposed to local irradiation. Oncology 44:3 12-3 18; 1987. Inomata, T.; Ogawa, Y.; Terashima, M.; Nishioka, A.; Yoshida, S.; Maeda, T.; Seguchi, H. An experimental study on the effects of radiation therapy and a long term administration of Sonifilan (SPG) on MM46 tumour inoculated into C3H/He mice. Analysis with immunohistochemical method. Radiat. Syst. Res. 5(Suppl.):229-232; 1988. Ishizuka, M.; Aoyagi, T.; Takeuchi, T.; Umezawa, H. Activity of bestatin: enhancement of immune responses and antitumour effect. In: Umezawa, H., ed. Small molecular immunomodifiers of microbial origin. Oxford: Pergamon Press; 1981:17-38. Kallman, R. F.; Silini, G.; van Putten, L. M. Factors influencing the quantitative estimation of the in vivo survival of cells from solid turnouts. J. Natl. Cancer Inst. 39:539-549; 1967. Lam, G. K. Y.; Harrison, R. W.; Kornelsen, R. 0.; Skarsgard, L. D. Beam scanning system for a clinical pi-meson beam. Rev. Sci. Instrum. 57:329-335; 1986. Lam, G. K. Y.; Skarsgard, L. D. Physical aspects of the pion beam at TRIUMF. In: Skarsgard, L. D., ed. Pions and heavy ion radiotherapy: Preclinical and clinical studies. New York. N.Y.: Elsevier Science; 1983:83-90. Milas, L.; Hunter, N.; Withers, H. R. Combination of local irradiation with systemic application of anaerobic corynebacteria in therapy of murine fibrosarcoma. Cancer Res. 35:1274-1277; 1975. Miyaji, C.; Ogawa, Y.; Imajo, Y.; Imanaka, K.; Kimura, S. Combination therapy of radiation and immunomodulators in the treatment of MM46 tumour transplanted in C3H/ He mice. Oncology 40: 115- I 19; 1983. Norisuye, T.; Yanaki, T.; Fujita, H. Triple helix ofa Schizophyllum commune polysaccharide in aqueous solution. J. Polymer Sci. 18:547-558; 1980. Ogawa, Y.; Imanaka, K.; Ashida, C.; Takashima, H.; Imajo, Y.; Kimura, S. Active specific immunotherapy using the immune reaction of a low-dose irradiated tumour tissue. Int. J. Radiat. Oncol. Biol. Phys. 9:533-537; 1983.
June 1990, Volume 18, Number 6
16. Ogawa, Y.; Maeda, T.; Hirota, S.; Yoshida, S.; Ohara, S.; Morita, M.; Hamada, F.; Inomata, T.; Morita, S.; Sawada, A.; Seguchi, H. Combined effect of radiotherapy and SPG (schizophyllan) in treatment of mouse MM46 tumour. J. Jpn. Sot. Cancer Ther. 21:87-95; 1986. 17. Ogawa, Y.; Maeda, T.; Seguchi, H.; Hamada, F.; Inomata, T.; Yoshida, S.; Kishimoto, S.; Saito, H. Infiltration of Leu3a+3bpositive lymphocytes and expression of human leukocyte antigen DR on tumour tissue induced by radiation therapy. Oncology 45: 18-20; 1988. 18. Ogawa, Y.; Maeda, T.; Seguchi, H.; Inomata, T.; Yoshida, S.; Kishimoto, S.; Saito, H. Application of the immunohistochemical method as predictive assay in radiotherapy of squamous cell carcinoma of oropharynx and hypophatynx. Oncology 47: 155- 159; 1990. 19. Okamura, K.; Suzuki, M.; Chihara, T.; Fujiwara, A.; Fukuda, T.; Goto, S.; Ichinose, K.; Jimi, S.; Kasamatsu, T.; Kawai, N.; Mizuguchi, K.; Mori, S.; Nakano, H.; Noda, K.; Sekiba, K.; Suzuki, K.; Suzuki, T.; Takahashi, K.; Takeuchi, K.; Takeuchi, S.; Yajima, A.; Ogawa, N. Clinical evaluation of Schizophyllan combined with irradiation in patients with cervical cancer. A randomized controlled study. Cancer 58: 865-872; 1986. 20. Olive, P. L.; Chaplin, D. J.; Durand, R. E. Pharmacokinetics, binding and distribution of Hoechst 33342 in spheroids and murine tumours. Brit. J. Cancer 52:739-746; 1985. 21. Skarsgard, L. D.; Henkelman, R. M.; Eaves, C. J. Pions for radiotherapy at TRIUMF. J. Can. Assoc. Radiol. 3 1:3-12; 1980. 22. Suit, H. D.; Sedlacek, R.; Wagner, M.; Orsi, L.; Silobrcic, V.; Rothman, K. J. Effect of Corynebacterium parvum on the response to irradiation of a C3H fibrosarcoma. Cancer Res. 36:1305-1314; 1976. 23. von Essen, C. F.; Bagshaw, M. A.; Bush, S. E.; Smith, A. R.; Kligerman, M. M. Long term results of pion therapy at Los Alamos. Int. J. Radiat. Oncol. Biol. Phys. 13:13891398; 1987. 24. Yamamoto, T.; Yamashita, T.; Tsubura, E. Inhibition of pulmonary metastasis of Lewis lung carcinoma by a glucan, schizophyllan. Invasion Metas. 1:7 l-84; 198 1.
0360-3016/90 $3.00 + .oO Copyright 0 1990 Pergamon Press plc
18, pp. 1415-1420
0 Original Contribution COMBINATION THERAPY OF PIONS AND SPG (SONIFILAN, SCHIZOPHYLLAN), A BIOLOGICAL RESPONSE MODIFIER FOR MOUSE TUMOR SYSTEMS
DAVIDJ. CHAPLIN,PH.D.,~ YASUHIRO OGAWA, M.D.,‘T~GEORGE B. GOODMAN, F.R.C.P.C.,' WILLIAM GRULKEY,B.SC.~ANDGABRIELK.Y.LAM,PH.D.~ ‘DevelopmentalRadiotherapy,Cancer Canada;
Control Agency of British Columbia, 600 West 10th Avenue, Vancouver, B.C., V5Z 4E6, ‘Kochi Medical School, Oko-cho, Nankoku-shi, Kochi 783-5 1 Japan; and ‘British Columbia Cancer Research Center, 601 West 10th Avenue, Vancouver, B.C., V5Z 4E6, Canada
Female C3H mice aged 8-10 weeks with transplanted KHT sarcoma or SCCVII tumor were used to investigate the antitumor effect of SPG (Sunifilan, Schizophyllan) alone and in combination with local irradiation of pions with 4 fractions of 400 cGy (total 1600 cGy). Daily doses of 10 mg/kg of SPG were given intramuscularly to the mice bearing KHT sarcoma for 14 consecutive days from day 7, and to mice bearing SCCVII tumor for 20 consecutive days from day 7 and thereafter three times a week for another 2 weeks. The antitumor effect was evaluated by the changes in tumor volume, survival curves, and the number of pulmonary metastatic nodules on the surface of the lungs. SPG failed to exert any antitumor effect and any life-prolonging effect for the KHT sarcoma. As for SCCVII tumor, in the group treated with pions and SPG, tumor growth decreased significantly (p < 0.01) compared with the group treated with pion only, and life prolonging effect and metastasis-suppressing effect were also observed (p < 0.05). In conditions of minimal residual disease brought about by pion irradiation, the adjuvant effect of a Biological Response Modifier (BRM) SPG may prove to be a promising method of cancer therapy for some tumors. Pion, SPG (sonifilan), KHT sarcoma, SCCVII tumor, BRM (biological energy transfer) radiation, Radiotherapy.
response modifier), High LET (linear
of pion and SPG, a BRM (Biological Response Modifier) for mouse tumor systems in this study. Pions are used to reduce the tumor burden to a minimum. SPG, which can restore immune competence, may enhance this effect by controlling the residual tumor and controlling distant micrometastases (24).
INTRODUCTION
Today, surgery, radiotherapy, and antitumor chemotherapy are the three major methods of cancer treatment. Conventional radiotherapy using X rays from linear accelerators or gamma rays from 6oCo teletherapy units has been extensively used for three decades. Although conventional radiotherapy has contributed to improvements in the results of cancer treatment, there is still a considerable number of cases which are not cured. Recently, new methods of radiotherapy (high Linear Energy Transfer radiations) have been developed; pion radiotherapy is one of these new methods (4). Despite the introduction of new radiations with high LET (Linear Energy Transfer) such as pions, failure to eradicate local disease still remains a problem (4). It seems that in a proportion of patients local failure will lead to the development of distant metastases. Therefore, treatment with combination therapy appears sound. For this reason, we have examined the effect of combined therapy
METHODS
AND
MATERIALS
Mice and tumors
All experiments were performed using RI-IT sarcoma or SCCVII tumor in female C3H mice* 8-10 weeks old weighing on average 20 g. Each experimental group consisted of 15 animals housed in 3 cages. All mice were kept in plastic cages on sawdust bedding with five animals per cage. The mice were kept in a small animal facility where the temperature and humidity were constant. Food and water were freely available at all times. RI-IT sarcoma cells and SCCVII tumor cells were transplanted by sub-
Reprint requests to: Dr. Yasuhiro Ogawa, Department of Radiology, Kochi Medical School, Oko-cho, Nankoku-shi, Kochi 783-5 I, Japan. Acknowledgements-The authors would like to thank Mr. Ivan Liu for his technical assistance, Ms. Ksenia Koric for secretarial assistance, and Dr. Dorianne E. Rheaume and Dr. Julie Bowen
for valuable discussion of the experiments. We also appreciate Kaken Pharmaceutical Co., Japan for providing us with SPG (Sonifilan) and information on SPG. Accepted for publication 20 December 1989. * Charles River Inc.
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June 1990, Volume 18, Number 6 see
i
O
I 5
I 0
r
.cONTROL
rrad.
I miays
I 15
I 10
VII t “Ill0
Fig. 1. Tumor growth curves of KHT sarcoma implanted in C3H mice treated with pion irradiation and/or SPG. The error bars are 95% confidence intervals.
Fig. 3. The tumor growth curves of SCCVII tumor in C3H mice treated with pion irradiations and/or < 0.0 1 vs all other groups.
cutaneous injections of approximately 2 X 1O5viable cells (obtained from enzymatic disaggregation) into the foot and the irradiations were started when the tumors attained a mean diameter of 5 mm. These two tumors and the method of cell suspension preparation ( 1) have been fully described elsewhere (9, 20).
irradiated in the stopping peak region (2). The space for the mouse foot within the device (2) was enlarged to provide enough room to prevent tumor hypoxia. A schedule with 4 fractions of 400 cGy was used, and pion irradiations were given on day 0, day 1, day 4, and day 7. The total treatment was given in 7 days.
Irradiation procedures,for pion Pion studies were done using an irradiation system described previously which uses no anesthesia (3). A horizontal beam with a field size of 2.0 X 5.0 cm and a primary proton beam current of 160 PA was used. This resulted in an effective dose rate of pions of approximately 20 cGy per min. The field size used encompassed one foot tumor of each of the three mice stacked vertically. The irradiation set up was mounted on a scanning couch system (lo), which delivered the required field size using 6 beam spots. The dosimetry for pion beam has been described elsewhere (11). The mice were positioned in the pion beam with the required amount of bolus, so that the foot tumor was
Schedule of the administration of SPG (sonifilan) SPG is a simple beta-glucan isolated from the fermentation broth of Schizophyllum commune Fries and has a molecular weight of about 450,000 (14). SPG was administered in daily doses of 10 mg/kg in 20 ~1 by intramuscular injections in the leg opposite to the tumor-bearing foot. In the experiment for KHT sarcoma, SPG was injected from day 4 to day 7 in the group which received only SPG (group 2) and from day 7 to day 20 in the group which received pion irradiations (group 4). The control group (group 1) and the group which received pion irradiations alone (group 3) were injected daily with 20 wl/ mouse of sterilized PBS (Phosphate Buffered Saline)
10: t
KHTsar
implanted SPG. *p
coma
-
PION ----_I
PION
00
& Sffi 4 SbC.
I
O0
I
10
I
20
days
Fig. 2. The survival rate of C3H mice with implanted sarcoma treated with pion irradiations and/or SPG.
KHT
20
40
60
day8
Fig. 4. Survival rate of C3H mice with implanted SCCVII tumor treated with pion irradiations and/or SPG. Improvement of survival rate is noted in group 8 compared to group 7 from day 72 to day 78 (p < 0.05).
Combination therapy of pions and SPG 0 Y.
with the same schedule as group 2 and group 4, respectively (16). In the experiment for SCCVII tumor, SPG was injected daily for 20 consecutive days from day 7, and three times a week for 2 weeks, thereafter, for the SPG group (group 6) and for the SPG and pion irradiation group (group 8). The control group (group 5) and the group which received pion irradiations alone (group 7) were injected daily with 20 &mouse of sterilized PBS with the same schedule as groups 6 and 8, respectively (16). Evaluation of antitumor eflect The largest and the smallest diameter and the thickness of the tumor were measured with a micrometer caliper three times a week. The result of these values was recorded as volume (mm3) ( 15). The mortality of mice was recorded daily for 28 days and 78 days after the initiation of pion irradiations for KI-IT sarcoma and SCCVII tumor, respectively. Autopsies were performed to examine the development of metastatic changes. Specifically, in the experiments for SCCVII tumor, mice were sacrificed on day 78 and pulmonary metastases were estimated grossly by counting the number of metastatic nodules on the pulmonary surface after fixing the lungs in Boin’s solution (24). The statistical differences among the groups were analyzed by the Student’s “t” test or the X2 test. RESULTS Figure 1 shows the tumor growth curves of KHT sarcoma implanted in C3H mice treated with pion irradiations and/or SPG. There was no difference in the tumor
OGAWA
et
al.
1417
growth between group 1 and group 2, nor between group 3 and group 4. SPG failed to exert any antitumor effect for KHT sarcoma. Figure 2 shows the survival rate of C3H mice with implanted IQ-IT sarcoma treated with pion irradiations and/ or SPG. There was no difference in the survival rate between group 1 and group 2 nor between group 3 and group 4. SPG failed to exert any life-prolonging effect for KHT sarcoma. Figure 3 shows the tumor growth curves of SCCVII tumor implanted in C3H mice treated with pion irradiations and/or SPG. While no inhibition of the tumor growth was observed in the group which received SPG alone (group 6), remarkable inhibition was noted in the group which received SPG after pion irradiations (group 8) compared to the group which received pion irradiations alone (group 7) (p < 0.01, Student’s “t” test). Figure 4 shows the survival rate of C3H mice with implanted SCCVII tumor treated with pion irradiations and/ or SPG. Statistically significant improvement of survival rate was noted in group 8 compared to group 7 during the last 7 days of a 78 day follow-up period (p < 0.05, X2 test). As shown in Figures 5 and 6, a comparison of tumor sizes on day 78 showed that tumors were very big in all of the five surviving mice in group 7 (Fig. 5) while 2 out of 10 mice in group 8 still had a small sized tumor (Fig. 6). Figures 7 and 8 show the pulmonary metastases of SCCVII tumor in groups 7 and 8 on day 78, respectively. In 4 out of 5 lungs in group 7, pulmonary metastases were observed easily and the average number of metastatic
Fig. 5. Photograph of the 5 surviving mice in group 7 on day 78. Tumor sizes were very big in all of the 5 mice.
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I. J. Radiation Oncology 0 Biology 0 Physics
Fig. 6. Photograph
of the 2 of 10 surviving
mice in group 8 on day 78. These two mice still had small sized tumors.
nodules identified macroscopically on the pulmonary surface was 9.6 k 6.9 (mean + 1 S.D.). In group 8 the corresponding numbers were 2.1 + 1.8. There is a significant difference in the number of nodules between these two groups (a < 0.05, Student’s “t” test). Moreover, each nodule of metastatic change observed in group 8 is relatively small compared to that observed in group 7. It is concluded that the combined therapy of pion and SPG inhibited pulmonary metastases compared to treatment with pions alone. DISCUSSION Recently, extensive cerning the effect of immunotherapy using mentally ( 12, 19, 22).
June 1990, Volume 18, Number 6
studies have been reported concombined radiation therapy and BRM both clinically and experiThe importance of timing of the
Fig. 7. Photograph of the lungs of the 5 surviving metastases are easily observed.
combination of local irradiation and BRMs, such as PSK, OK-432, and SPG has been reported (13, 16). BRMs demonstrated significant antitumor activity when they were received after irradiation. It was for this reason that we started the administration of SPG after pion irradiations. SPG is a simple glucan composed of glucose alone prepared from a highly viscous polysaccharide isolated from a culture filtrate of Schizophyllum commune Fries, a species of Basidiomycetes. The viscous polysaccharide is treated ultrasonically at highspeed extrusion to adjust its molecular weight to 450,000. The resulting SPG preparation is chemically homogeneous and its physico-chemical properties have been well characterized (14). In a recent study (16) using MM46 tumor inoculated into C3H/He mice, we have demonstrated that tumor
mice in group 7 on day 78. In 4 out of 5 lungs, pulmonary
Combination therapy of pions and SPG 0 Y.
OCAWA
Fig. 8. Photograph of the lungs of the 10 surviving mice in group 8 on day 78. Fewer and smaller metastatic are noted compared with Figure 7.
growth was most suppressed in the group treated with radiation and SPG. There was a marked infiltration of macrophages into tumor tissue in addition to the heavy infiltrate of T lymphocytes (7). This suggests that the mechanism of enhanced effect from the combined use of SPG with radiotherapy was exerted by immunological responses of the host such as that of T lymphocytes and macrophages. More than 700 patients with malignancy have now been treated with pion beams at LAMPF (Los Alamos Meson Physics Facility) in USA (23) SIN (Swiss Institute for Nuclear Research) in Switzerland (5) and TRIUMF (TriUniversity Meson Facility) in Canada (4). As pions have increased biological effectiveness and lower OER (Oxygen Enhancement Ratio), this beam can kill hypoxic tumor cells more efficiently than conventional radiotherapy (2 1). SPG should therefore exhibit its antitumor effect maximally after the tumor cells are depleted by pion irradiations to numbers controllable by this drug. In the experiment using KHT sarcoma, SPG failed to exert any antitumor effect or life-prolonging effect. The reason for this failure could be that KHT sarcoma has no tumor antigenicity and it is a tumor which grows very rapidly (9). The host-mediated immunological responses
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augmented by SPG cannot exert their effect against tumors lacking tumor antigenicity. Moreover, a BRM, such as bestatin, has been demonstrated to be more effective for tumors of slow growth (8). In the experiment using SCCVII tumor, the administration of SPG combined with pion irradiations was found to exert a significant antitumor effect, life-prolonging effect, and metastasis-suppressing effect. As radiotherapy has been demonstrated to promote immune responses in some kinds of tumor tissue (6, 17, 18) and the tumor was of relatively slow growth (20) the combined effect of SPG and pion irradiation should have been fully exerted for this tumor system. This report is the first study demonstrating a significantly slower regrowth of tumors after irradiation using a combination of pions and a BRM. It seems reasonable that pions and BRM through dual mechanisms produce minimal residual tumor cells in the local lesion and augment host-mediated immune responses. Therefore, it is possible that a combined therapy of pion and a BRM such as SPG will prove to be a promising method of cancer treatment in the near future. Further investigations using other tumor systems will be needed to clarify those tumor characteristics most likely to be beneficial.
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16. Ogawa, Y.; Maeda, T.; Hirota, S.; Yoshida, S.; Ohara, S.; Morita, M.; Hamada, F.; Inomata, T.; Morita, S.; Sawada, A.; Seguchi, H. Combined effect of radiotherapy and SPG (schizophyllan) in treatment of mouse MM46 tumour. J. Jpn. Sot. Cancer Ther. 21:87-95; 1986. 17. Ogawa, Y.; Maeda, T.; Seguchi, H.; Hamada, F.; Inomata, T.; Yoshida, S.; Kishimoto, S.; Saito, H. Infiltration of Leu3a+3bpositive lymphocytes and expression of human leukocyte antigen DR on tumour tissue induced by radiation therapy. Oncology 45: 18-20; 1988. 18. Ogawa, Y.; Maeda, T.; Seguchi, H.; Inomata, T.; Yoshida, S.; Kishimoto, S.; Saito, H. Application of the immunohistochemical method as predictive assay in radiotherapy of squamous cell carcinoma of oropharynx and hypophatynx. Oncology 47: 155- 159; 1990. 19. Okamura, K.; Suzuki, M.; Chihara, T.; Fujiwara, A.; Fukuda, T.; Goto, S.; Ichinose, K.; Jimi, S.; Kasamatsu, T.; Kawai, N.; Mizuguchi, K.; Mori, S.; Nakano, H.; Noda, K.; Sekiba, K.; Suzuki, K.; Suzuki, T.; Takahashi, K.; Takeuchi, K.; Takeuchi, S.; Yajima, A.; Ogawa, N. Clinical evaluation of Schizophyllan combined with irradiation in patients with cervical cancer. A randomized controlled study. Cancer 58: 865-872; 1986. 20. Olive, P. L.; Chaplin, D. J.; Durand, R. E. Pharmacokinetics, binding and distribution of Hoechst 33342 in spheroids and murine tumours. Brit. J. Cancer 52:739-746; 1985. 21. Skarsgard, L. D.; Henkelman, R. M.; Eaves, C. J. Pions for radiotherapy at TRIUMF. J. Can. Assoc. Radiol. 3 1:3-12; 1980. 22. Suit, H. D.; Sedlacek, R.; Wagner, M.; Orsi, L.; Silobrcic, V.; Rothman, K. J. Effect of Corynebacterium parvum on the response to irradiation of a C3H fibrosarcoma. Cancer Res. 36:1305-1314; 1976. 23. von Essen, C. F.; Bagshaw, M. A.; Bush, S. E.; Smith, A. R.; Kligerman, M. M. Long term results of pion therapy at Los Alamos. Int. J. Radiat. Oncol. Biol. Phys. 13:13891398; 1987. 24. Yamamoto, T.; Yamashita, T.; Tsubura, E. Inhibition of pulmonary metastasis of Lewis lung carcinoma by a glucan, schizophyllan. Invasion Metas. 1:7 l-84; 198 1.
