Vol. 21, No. 3
INFECTION AND IMMUNITY, Sept. 1978, p. 914-917 0019-9567/78/0021-0914$0200/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Antitumor Activity of Mycobacterial Glycolipid Al ZULEMA REGGIARDO
Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201 Received for publication 13 June 1978
Glycolipid Al isolated from Mycobacterium bovis BCG, when dissolved in olive oil and injected together with Line 10 transplantable hepatoma cells, is able to elicit a host response which results in the abrogation or retardation of tumor growth in syngeneic guinea pigs. Glycolipid Al does not have adjuvant activity for delayed type hypersensitivity, and antibodies to Al have not been detected in the sera of guinea pigs during or after the tumor abrogation induced by Al injection. Glycolipid Al does not share antigenic determinants with Line 10 cell lipid fractions. The possible role of the granuloma response elicited by Al in controlling tumor growth is discussed. Since the first observations (5, 15) that showed that living bacillus Calmette-Guerin (BCG) was active in inhibiting the growth of neoplastic cells in mice, many other studies have reported the antitumor effect of BCG in different animal species and in humans (1). The isolation and characterization of BCG components responsible for the antitumor effect are necessary not only to have a better understanding of the BCG tumor suppression-host response complex but also for the optimal use of these components in the therapy of cancer. Glycolipid Al is a serologically active lipid isolated and purified from Mycobacterium bovis BCG (17). Recently, it has been shown that glycolipid Al dissolved in olive oil is able to elicit a strong granulomatous response in the skin of guinea pigs (19). The inflammatory reaction appears early (24 h) and is long lasting (approximately 3 to 4 weeks), and it was thought that this property of Al might endow it with antitumor effect. This paper describes the effect of Al on the growth of hepatoma transplants in syngeneic guinea pigs.
MATERIALS AND METHODS Serologically active glycolipids. Glycolipids Al, Bi, and a mixture of Cl, C2, and C3 referred to as C were extracted from Mycobacterium bovis BCG (Research Foundation, University of Illinois, Chicago) and purified by silicic acid chromatography as previously described (17). The indicated amount of the glycolipids was dissolved in olive oil (highly refined, Sigma Chemical Co., St. Louis, Mo.) and heated for 15 min at 56"C before use. Line 10 tumor cells. Line 10 is a transplantable hepatocarcinoma originally produced by oral administration of diethylnitrosamine to guinea pigs. The ascites variant of Line 10 was used following the model developed by Rapp et al. (16, 27). In this model the
intradermal inoculation of 10" Line 10 cells results in progressive tumor growth, with death of the guinea pig in 7 to 8 weeks. Line 10 cells, passage 16, were originally supplied by B. Zbar. A fresh suspension of Line 10 cells was prepared as follows. Line 10 cells that had been stored in 10% dimethylsulfoxide at -70'C were thawed, washed once with saline, and resuspended in saline. Each guinea pig was injected intraperitoneally with 4 x 1I0 Line 10 cells. Approximately 2 weeks later, the cells were recovered by paracentesis, washed once with saline, and resuspended in saline to give a concentration of 2 107 viable cells per ml. Viability was determined by trypan blue exclusion. Animals. Sewall Wright inbred strain 2 male guinea pigs weighing 500 to 700 g, which have been bred in these laboratories from a breeding stock from the National Institutes of Health, were used. Inoculation of guinea pigs. Guinea pigs were shaved at least 2 h prior to the injections. All of the inoculations were done by the intradermal route. For the tumor studies, equal volumes of Line 10 cell suspensions, containing 2 x 107 cells per ml and the glycolipids in olive oil, were mixed well in a syringe with an 18 gauge needle. Each guinea pig received 0.1 ml of this mixture in the right flank. Guinea pigs received 10' Line 10 cells in combination with one of the following: 5 or 20 ,g of Al, 100 ,g of B1, or 100 jig of C. Control animals were injected with a mixture of X
Line 10 cells and olive oil. To find out whether Al was toxic for Line 10 cells, the following experiment was carried out. Equal volumes of Line 10 cells in saline and Al in olive oil were mixed. This mixture, containing 10' cells and 200 jig of Al per ml, was incubated at room temperature for 1 h, then centrifuged, and the oil phase containing Al was removed. An approximately equal volume of olive oil was added to the cell suspension, mixed, and centrifuged, and the oil phase was discarded. This process was repeated two times more to remove Al and finally the suspension was adjusted to contain 107 Line 10 cells per ml of saline. A control suspension of Line 10 cells was prepared in similar fashion with Line 10 cells and olive oil alone. Six guinea pigs were injected intradermally with 0.1 914
VOL. 21, 1978
ANTITUMOR ACTIVITY OF GLYCOLIPID Al
ml of the Line 10 suspension that had been treated with Al, and six guinea pigs received the control suspension. Line 10-Al-inoculated animals that were capable of abrogating tumor growth were rechallenged intradermally on the left flank with 10 Line 10 cells 20 or 24 weeks after the first inoculation. For the adjuvant studies, one group of 10 guinea pigs was inoculated with 0.1 ml of a mixture of equal volumes of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.) and bovine serum albumin (BSA) (Fraction V, Armour Pharmaceutical Co., Phoenix, Ariz.) in saline. Another group of 10 guinea pigs was injected with 0.1 ml of a mixture of equal volumes of 400 ,g of Al per ml of olive oil and 10 mg of BSA per ml of saline. Skin testing. Half of the guinea pigs in each group of the adjuvant studies were skin tested 15 days after inoculation and the other half at 30 days. The skin tests were done with 10 and 50Mg of BSA; the reactions were read at 24 and 48 h, and the size (millimeters) of erythema and induration were recorded. As a control, 0.1 ml of saline was used. All injections were done in
duplicate. Preparation of lipids from Line 10 cells. Six guinea pigs were injected intraperitoneally with 4 x 10" Line 10 cells. Approximately 2 weeks later, the Line 10 cells were recovered by paracentesis and washed two times with saline. A sample was suspended at 10' cells per ml in saline containing 0.5% phenol, and the rest of the cells were washed with acetone. The acetone-washed cells were then extracted, first three times with methanol at 560C and then three times with chloroform at 560C. These extracts were specially treated as previously described (17) to eliminate the nonlipidic contaminants and cell debris. The methanol extract was chromatographed on silicic acid columns as described (17). The chloroform extract was not chromatographed and tested as such. Serological procedures. Antibodies against Al were investigated in the sera of guinea pigs by indirect hemagglutination (17). Guinea pigs were bled 3, 6, and 10 weeks after the injection of the Line 10-Al mixture. To investigate whether Line 10 and Al share antigenic determinants, the Line 10 lipid fractions, suspended at 1,000 and 100 /g/ml, and the Line 10 cell suspension were tested by hemagglutination inhibition (17).
RESULTS Effect of glycolipid Al on tumor growth. (i) In the group injected with 106 Line 10 cells and 20 ,tg of Al in olive oil, 50% (13) of the animals were able to abrogate tumor growth, and in the remaining guinea pigs tumor growth was delayed. In four guinea pigs the tumor began to grow in week 2 to 3 postinoculation, and the animals died between weeks 11 and 12. In four other guinea pigs, the tumor began to grow between weeks 4 and 6 after injection, and the animals died between weeks 12 and 15 postinoculation. In three guinea pigs, the tumor began to grow between weeks 7 and 11 postinoculation. Metastases were palpable in the axillary regional nodes when the tumor nodules were approxi-
915
mately 12 to 15 mm in diameter. In two guinea pigs, there was no tumor growth at the site of injection, but a regional lymph node metastasis was palpable at week 9 to 10 postinoculation and the guinea pigs died between weeks 15 and 16. The 13 surviving guinea pigs remained tumor free and well 5 and 6 months after the inoculation, at which time 10 of them were rechallenged with 10" Line 10 cells. No observable tumor appeared in the skin, no palpable regional metastases were present, and these animals are living and healthy 3 months after the rechallenging. (ii) In the group injected with 10" Line 10 cells and 5 Iug of Al in olive oil, four guinea pigs were used, and only one was able to abrogate tumor growth. In the other three animals, tumor growth was retarded and death occurred in week 10 postinoculation. (iii) In the group injected with 10" Line 10 cells and olive oil, all the guinea pigs showed evidence of tumor growth during the first week after inoculation, and the animals died in week 7 to 8 postinoculation. Effect of glycolipids Bi and C on tumor growth. Guinea pigs injected with mixtures of Line 10 cells with B1 or C failed to abrogate tumor growth. The animals died in week 7 to 8 postinoculation. Effect of glycolipid Al on Line 10 cells. Glycolipid Al was not toxic for Line 10 cells. In the guinea pigs that were inoculated with Line 10 cells preincubated with Al (which was subsequently removed), the tumor grew as in the control guinea pigs that had received Line 10 cells preincubated with olive oil alone. All the animals died in week 7 to 8 postinoculation. Adjuvant studies. Glycolipid Al in olive oil failed to act as an adjuvant for delayed type hypersensitivity to BSA. The guinea pigs inoculated with the mixture of BSA and Al in olive oil did not show delayed type hypersensitivity to BSA when skin tested 15 and 30 days after inoculation. Guinea pigs injected with BSA in Freund complete adjuvant showed hypersensitivity of 11- to 12-mm induration with central necrosis when skin tested with 10 jig of BSA at 30 days but not at 15 days. Studies on common antigenic determinants between Line 10 lipids and glycolipid Al. No antigenic cross-reactions were observed between glycolipid Al and Line 10 cells or Line 10 lipid extracts and fractions. None of the Line 10 preparations was capable of inhibiting the hemagglutination reactions between Al and human serum antibodies. Studies on antibody response to Al. Antibodies against glycolipid Al were not detected in the sera of guinea pigs during or after tumor
916
REGGIARDO
abrogation as a consequence of Al injection. No Al antibodies were detected in the sera of those guinea pigs which failed to abrogate tumor growth. DISCUSSION Living BCG injected together with Line 10 cells or into 7- to 8-day established tumors is able to suppress or regress hepatoma transplants in strain 2 guinea pigs (25, 26). The studies presented in this paper show that mycobacterial glycolipid Al, when dissolved in olive oil and injected together with Line 10 cells, is able to elicit a host response which results in the abrogation or retardation of tumor growth in guinea pigs. Al was not toxic for Line 10 cells. In some guinea pigs there was no evidence of tumor growth for several weeks, although eventually tumors began to grow 7 to 11 weeks postinoculation. It is reasonable to speculate that the majority of Line 10 cells were killed at the beginning and later on the few surviving cells began to multiply. Why the host was unable to control the growth of these remaining cells after the initial killing is not understood. It is possible that some Line 10 cells were in a dormant state and could not be killed by the host. Glycolipids B1 and C did not have any effect on tumor growth. Other purified mycobacterial lipid fractions have been shown to control Line 10 growth in syngeneic guinea pigs. Mycobacterial trehalose 6,6'-dimycolate (TDM), so-called "cord factor," has been reported effective when combined with cell walls or dead BCG and injected into 6- to 10-mm tumors (2). P3, a highly purified TDM preparation, has been shown to be active when mixed with mycobacterial cell walls or bacterial endotoxins and injected on mineral oil droplets either together with Line 10 or into 7- to 8-day tumors (13, 20, 21). Neither of these trehalose mycolate preparations had an antitumor effect in guinea pigs when administered alone. Both of them have been reported to have adjuvant effects, TDM for antibody formation in mice and rats (4) and P3 for delayed-type hypersensitivity in guinea pigs (8). Also P3 is reported to be a mild granulomagenic agent in guinea pigs (12). TDM is granulomagenic in mice but not in guinea pigs when given intradermally (2, 22). Recently, using guinea pig macrophages, Kelly has shown that glycolipid P3 is able to generate chemotactic activity from plasma (11). TDM is said to be directly chemotactic for mouse leukocytes and macrophages and able to stimulate mouse macrophages (14, 24). All of the abovementioned properties could contribute to the antitumor effect(s) of P3 and TDM.
INFECT. IMMUN.
Several different mechanisms may be involved in the antitumor activity of glycolipid Al. It is possible to assume that antibodies against Al could contribute to the killing of Line 10 cells. Antibodies against Al have been found in the sera of tuberculous and leprous patients (18) and in the sera of patients undergoing BCG immunotherapy (23). However, Al antibodies have not been detected in the sera of strain 2 guinea pigs during or after tumor abrogation as a consequence of Al injection. It is possible that Al antibodies are cytophilic and could specifically direct the macrophages toward the tumor target cell if Al and Line 10 cells have a common antigenic determinant. It has been reported that BCG shares antigenic determinants with Line 10 cells that are tumor specific and could provide one possible mechanism for the antitumor effect of living BCG (6). In the experiments reported in this paper, glycolipid Al did not antigenically cross-react with lipid fractions and extracts from Line 10 cells, providing further evidence for a nonspecific effect of Al and ruling out a role for Al antibodies in this model. Numerous observations indicate that macrophages may play an important role in controlling tumor growth (7). The granuloma elicited by Al consists of an extensive accumulation of histiocytes (19), and it is reasonable to speculate that these macrophages are activated in some fashion and capable of killing the neoplastic cells, allowing the development of tumor-specific immunity and enabling the guinea pig to resist subsequent tumor challenge. Histological studies at the skin site of Line 10 and living BCG injection suggested that the granulomatous response may be important for the regression of tumor growth (10). BCG-activated macrophages may nonspecifically kill tumor cells by a different mechanism(s) than Al-activated macrophages. In the former, sensitized lymphocytes may play a crucial role. For instance, immunosuppressed guinea pigs which were unable to develop tuberculin sensitivity failed to control tumor growth at the site of BCG infection (9). On the other hand, the granuloma elicited by Al seems to be of the nonallergic type, with lymphocytes being rare (19). Furthermore, Al when injected in olive oil does not have adjuvant activity for delayed type hypersensitivity to an unrelated antigen, which also suggests that T lymphocytes or "specific arming" of macrophages by immune T cells may not be involved in this model. Whether some other components of the inflammatory reaction induced by Al besides granuloma formation may participate in controlling tumor growth remains to be studied. Finally, if Al activates macrophages by a nonimmunolog-
VOL. 21, 1978
ANTITUMOR ACTIVITY OF GLYCOLIPID Al
ical pathway, it would be expected that immunosuppressed guinea pigs would be able to control tumor growth after Al injection. This hypothesis and studies on regression of established tumors are under investigation. ACKNOWLEDGMENTS This work was supported in part by research grants from the American Cancer Society, Maryland Division, Inc. LITERATURE CITED 1. Bast, R. C., Jr., B. Zbar, T. Borsos, and H. J. Rapp. 1974. BCG and cancer. N. Engl. J. Med. 290:1413-1420. 2. Bekierkunst, A. 1968. Acute granulomatous response produced in mice by trehalose-6,6'-dimycolate. J. Bacteriol. 96:958-961. 3. Bekierkunst, A., L. Wang, R. Toubiana, and E. Lederer. 1974. Immunotherapy of cancer with nonliving BCG and fractions derived from mycobacteria. Role of cord factor (trehalose-6,6'-dimycolate) in tumor regression. Infect. Immun. 10:1044-1050. 4. Bekierkunst, A., E. Yarkoni, I. Flechner, S. Morecki, E. Vilkas, and E. Lederer. 1971. Immune response to sheep red blood cells in mice pretreated with mycobacterial fractions. Infect. Immun. 4:256-263. 5. Biozzi, G., C. Stiffel, B. N. Halpern, and D. Mouton. 1959. Effect de inoculation du bacille de CalmetteGuerin sur le developpement de la tumeur ascitique d'Ehrlich chez la souris. C. R. Soc. Biol. (Paris) 153:987-989. 6. Borsos, T., and H. J. Rapp. 1973. Antigenic relationship between Mycobacterium bovis (BCG) and a guinea pig hepatoma. J. Natl. Cancer Inst. 51:1085-1086. 7. Evans, R., and P. Alexander. 1976. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages, p. 535-575. In D. S. Nelson (ed.), Immunobiology of the macrophage. Academic Press, Inc., New York. 8. Granger, D. L, K. Yamamoto, and E. Ribi. 1976. Delayed hypersensitivity and granulomatous response after immunization with protein antigens associated with a mycobacterial glycolipid and oil droplets. J. Immunol. 116:482-488. 9. Hanna, M. G., Jr., M. J. Snodgrass, B. Zbar, and H. J. Rapp. 1973. Histopathology of tumor regression after intralesional injection of Mycobacterium bovis. IV. Development of immunity to tumor cells and BCG. J. Natl. Cancer Inst. 51:1897-1908. 10. Hanna, M. G., Jr., B. Zbar, and H. J. Rapp. 1972. Histopathology of tumor regression after intralesional injection of Mycobacterium bovts. I. Tumor growth and metastasis. J. Natl. Cancer Inst. 48:1441-1455. 11. Kelly, M. T. 1977. Plasma-dependent chemotaxis of macrophages toward BCG cell walls and the mycobacterial glycolipid P3. Infect. Immun. 15:180-183. 12. Meyer, T. J., E. Ribi, and I. Azuma. 1975. Biologically active components from mycobacterial cell walls. V. Granuloma formation in mouse lungs and guinea pig skin. Cell. Immunol. 16:11-24.
917
1;3. Meyer, T. J., E. Ribi, 1. Azuma, and B. Zbar. 1974. Biologically active components from mycobacterial cell walls. II. Suppression and regression of strain-2 guinea pig hepatoma. J. Natl. Cancer Inst. 52:103-111. 14. Ofek, I., and A. Bekierkunst. 1976. Chemotactic response of leukocytes to cord factor (trehalose-6,6'-dimycolate). J. Natl. Cancer Inst. 57:1379-1380. 15. Old, L. J., D. A. Clark, and B. Benacerraf. 1959. Effect of bacillus Calmette-Guerin infection on transplanted tumours in the mouse. Nature (London) 184:291-292. 16. Rapp. H. J., W. H. Churchill, Jr., B. S. Kronman, R. T. Rolley, W. G. Hammond, and T. Borsos. 1968. Antigenicity of a new diethylnitrosamine-induced transplantable guinea pig hepatoma: pathology and formation of ascites variant. J. Natl. Cancer Inst. 41:1-7. 17. Reggiardo, Z., and G. Middlebrook. 1974. Serologically active glycolipid families from Mycobacterium bovis BCG. I. Extraction, purification and immunological studies. Am. J. Epidemiol. 100:469-476. 18. Reggiardo, Z., and G. Middlebrook. 1974. Serologically active glycolipid families from Mycobacterium bovis BCG. II. Serological studies on human sera. Am. J. Epidemiol. 100:477-486. 19. Reggiardo, Z., and A. K. M. Shamsuddin. 1976. Granulomagenic activity of serologically active glycolipids from Mycobacterium bovis BCG. Infect. Immun. 14:1369-1374. 20. Ribi, E., R. Toubiana, S. M. Strain, K. C. Milner, C. McLaughlin, J. Cantrell, I. Azuma, B. C. Das, and R. Parker. 1978. Further studies on the structural requirements of agents for immunotherapy of the guinea pig Line-10 tumor. Cancer Immunol. Immunother. 3:171-177. 21. Ribi, E. E., D. L. Granger, K. C. Milner, and S. M. Strain. 1975. Tumor regression caused by endotoxins and mycobacterial fractions. J. Natl. Cancer Inst. 55:1253-1257. 22. Saito, R., A. Tanaka, K. Sugiyama, I. Azuma, Y. Yamamura, M. Kato, and M. Goren. 1976. Adjuvant effect of cord factor, a mycobacterial lipid. Infect. Immun. 13:776-781. 23. Wile, A., Z. Reggiardo, F. C. Sparks, and D. L. Morton. 1976. Monitoring BCG immunotherapy with serologically active mycobacterial glycolipids. Surg. Forum 27:105-106. 24. Yarkoni, E., L. Wang, and A. Bekierkunst. 1977. Stimulation of macrophages by cord factor and by heatkilled and living BCG. Infect. Immun. 16:1-8. 25. Zbar, B., 1. D. Bernstein, and H. J. Rapp. 1971. Suppression of tumor growth at the site of infection with living bacillus Calmette-Guerin. J. Natl. Cancer Inst. 46:831-839. 26. Zbar, B., and T. Tanaka. 1971. Immunotherapy of cancer: regression of tumors after intralesional injection of living Mycobacterium bovis. Science 172:271-273. 27. Zbar, B., H. T. Wepsic, H. J. Rapp, J. Whang-Peng, and T. Borsos. 1969. Transplantable hepatomas induced in strain-2 guinea pigs by diethylnitrosamine: characterization by histology, growth and chromosomes. J. Natl. Cancer Inst. 343:821-826.
INFECTION AND IMMUNITY, Sept. 1978, p. 914-917 0019-9567/78/0021-0914$0200/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Antitumor Activity of Mycobacterial Glycolipid Al ZULEMA REGGIARDO
Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland 21201 Received for publication 13 June 1978
Glycolipid Al isolated from Mycobacterium bovis BCG, when dissolved in olive oil and injected together with Line 10 transplantable hepatoma cells, is able to elicit a host response which results in the abrogation or retardation of tumor growth in syngeneic guinea pigs. Glycolipid Al does not have adjuvant activity for delayed type hypersensitivity, and antibodies to Al have not been detected in the sera of guinea pigs during or after the tumor abrogation induced by Al injection. Glycolipid Al does not share antigenic determinants with Line 10 cell lipid fractions. The possible role of the granuloma response elicited by Al in controlling tumor growth is discussed. Since the first observations (5, 15) that showed that living bacillus Calmette-Guerin (BCG) was active in inhibiting the growth of neoplastic cells in mice, many other studies have reported the antitumor effect of BCG in different animal species and in humans (1). The isolation and characterization of BCG components responsible for the antitumor effect are necessary not only to have a better understanding of the BCG tumor suppression-host response complex but also for the optimal use of these components in the therapy of cancer. Glycolipid Al is a serologically active lipid isolated and purified from Mycobacterium bovis BCG (17). Recently, it has been shown that glycolipid Al dissolved in olive oil is able to elicit a strong granulomatous response in the skin of guinea pigs (19). The inflammatory reaction appears early (24 h) and is long lasting (approximately 3 to 4 weeks), and it was thought that this property of Al might endow it with antitumor effect. This paper describes the effect of Al on the growth of hepatoma transplants in syngeneic guinea pigs.
MATERIALS AND METHODS Serologically active glycolipids. Glycolipids Al, Bi, and a mixture of Cl, C2, and C3 referred to as C were extracted from Mycobacterium bovis BCG (Research Foundation, University of Illinois, Chicago) and purified by silicic acid chromatography as previously described (17). The indicated amount of the glycolipids was dissolved in olive oil (highly refined, Sigma Chemical Co., St. Louis, Mo.) and heated for 15 min at 56"C before use. Line 10 tumor cells. Line 10 is a transplantable hepatocarcinoma originally produced by oral administration of diethylnitrosamine to guinea pigs. The ascites variant of Line 10 was used following the model developed by Rapp et al. (16, 27). In this model the
intradermal inoculation of 10" Line 10 cells results in progressive tumor growth, with death of the guinea pig in 7 to 8 weeks. Line 10 cells, passage 16, were originally supplied by B. Zbar. A fresh suspension of Line 10 cells was prepared as follows. Line 10 cells that had been stored in 10% dimethylsulfoxide at -70'C were thawed, washed once with saline, and resuspended in saline. Each guinea pig was injected intraperitoneally with 4 x 1I0 Line 10 cells. Approximately 2 weeks later, the cells were recovered by paracentesis, washed once with saline, and resuspended in saline to give a concentration of 2 107 viable cells per ml. Viability was determined by trypan blue exclusion. Animals. Sewall Wright inbred strain 2 male guinea pigs weighing 500 to 700 g, which have been bred in these laboratories from a breeding stock from the National Institutes of Health, were used. Inoculation of guinea pigs. Guinea pigs were shaved at least 2 h prior to the injections. All of the inoculations were done by the intradermal route. For the tumor studies, equal volumes of Line 10 cell suspensions, containing 2 x 107 cells per ml and the glycolipids in olive oil, were mixed well in a syringe with an 18 gauge needle. Each guinea pig received 0.1 ml of this mixture in the right flank. Guinea pigs received 10' Line 10 cells in combination with one of the following: 5 or 20 ,g of Al, 100 ,g of B1, or 100 jig of C. Control animals were injected with a mixture of X
Line 10 cells and olive oil. To find out whether Al was toxic for Line 10 cells, the following experiment was carried out. Equal volumes of Line 10 cells in saline and Al in olive oil were mixed. This mixture, containing 10' cells and 200 jig of Al per ml, was incubated at room temperature for 1 h, then centrifuged, and the oil phase containing Al was removed. An approximately equal volume of olive oil was added to the cell suspension, mixed, and centrifuged, and the oil phase was discarded. This process was repeated two times more to remove Al and finally the suspension was adjusted to contain 107 Line 10 cells per ml of saline. A control suspension of Line 10 cells was prepared in similar fashion with Line 10 cells and olive oil alone. Six guinea pigs were injected intradermally with 0.1 914
VOL. 21, 1978
ANTITUMOR ACTIVITY OF GLYCOLIPID Al
ml of the Line 10 suspension that had been treated with Al, and six guinea pigs received the control suspension. Line 10-Al-inoculated animals that were capable of abrogating tumor growth were rechallenged intradermally on the left flank with 10 Line 10 cells 20 or 24 weeks after the first inoculation. For the adjuvant studies, one group of 10 guinea pigs was inoculated with 0.1 ml of a mixture of equal volumes of Freund complete adjuvant (Difco Laboratories, Detroit, Mich.) and bovine serum albumin (BSA) (Fraction V, Armour Pharmaceutical Co., Phoenix, Ariz.) in saline. Another group of 10 guinea pigs was injected with 0.1 ml of a mixture of equal volumes of 400 ,g of Al per ml of olive oil and 10 mg of BSA per ml of saline. Skin testing. Half of the guinea pigs in each group of the adjuvant studies were skin tested 15 days after inoculation and the other half at 30 days. The skin tests were done with 10 and 50Mg of BSA; the reactions were read at 24 and 48 h, and the size (millimeters) of erythema and induration were recorded. As a control, 0.1 ml of saline was used. All injections were done in
duplicate. Preparation of lipids from Line 10 cells. Six guinea pigs were injected intraperitoneally with 4 x 10" Line 10 cells. Approximately 2 weeks later, the Line 10 cells were recovered by paracentesis and washed two times with saline. A sample was suspended at 10' cells per ml in saline containing 0.5% phenol, and the rest of the cells were washed with acetone. The acetone-washed cells were then extracted, first three times with methanol at 560C and then three times with chloroform at 560C. These extracts were specially treated as previously described (17) to eliminate the nonlipidic contaminants and cell debris. The methanol extract was chromatographed on silicic acid columns as described (17). The chloroform extract was not chromatographed and tested as such. Serological procedures. Antibodies against Al were investigated in the sera of guinea pigs by indirect hemagglutination (17). Guinea pigs were bled 3, 6, and 10 weeks after the injection of the Line 10-Al mixture. To investigate whether Line 10 and Al share antigenic determinants, the Line 10 lipid fractions, suspended at 1,000 and 100 /g/ml, and the Line 10 cell suspension were tested by hemagglutination inhibition (17).
RESULTS Effect of glycolipid Al on tumor growth. (i) In the group injected with 106 Line 10 cells and 20 ,tg of Al in olive oil, 50% (13) of the animals were able to abrogate tumor growth, and in the remaining guinea pigs tumor growth was delayed. In four guinea pigs the tumor began to grow in week 2 to 3 postinoculation, and the animals died between weeks 11 and 12. In four other guinea pigs, the tumor began to grow between weeks 4 and 6 after injection, and the animals died between weeks 12 and 15 postinoculation. In three guinea pigs, the tumor began to grow between weeks 7 and 11 postinoculation. Metastases were palpable in the axillary regional nodes when the tumor nodules were approxi-
915
mately 12 to 15 mm in diameter. In two guinea pigs, there was no tumor growth at the site of injection, but a regional lymph node metastasis was palpable at week 9 to 10 postinoculation and the guinea pigs died between weeks 15 and 16. The 13 surviving guinea pigs remained tumor free and well 5 and 6 months after the inoculation, at which time 10 of them were rechallenged with 10" Line 10 cells. No observable tumor appeared in the skin, no palpable regional metastases were present, and these animals are living and healthy 3 months after the rechallenging. (ii) In the group injected with 10" Line 10 cells and 5 Iug of Al in olive oil, four guinea pigs were used, and only one was able to abrogate tumor growth. In the other three animals, tumor growth was retarded and death occurred in week 10 postinoculation. (iii) In the group injected with 10" Line 10 cells and olive oil, all the guinea pigs showed evidence of tumor growth during the first week after inoculation, and the animals died in week 7 to 8 postinoculation. Effect of glycolipids Bi and C on tumor growth. Guinea pigs injected with mixtures of Line 10 cells with B1 or C failed to abrogate tumor growth. The animals died in week 7 to 8 postinoculation. Effect of glycolipid Al on Line 10 cells. Glycolipid Al was not toxic for Line 10 cells. In the guinea pigs that were inoculated with Line 10 cells preincubated with Al (which was subsequently removed), the tumor grew as in the control guinea pigs that had received Line 10 cells preincubated with olive oil alone. All the animals died in week 7 to 8 postinoculation. Adjuvant studies. Glycolipid Al in olive oil failed to act as an adjuvant for delayed type hypersensitivity to BSA. The guinea pigs inoculated with the mixture of BSA and Al in olive oil did not show delayed type hypersensitivity to BSA when skin tested 15 and 30 days after inoculation. Guinea pigs injected with BSA in Freund complete adjuvant showed hypersensitivity of 11- to 12-mm induration with central necrosis when skin tested with 10 jig of BSA at 30 days but not at 15 days. Studies on common antigenic determinants between Line 10 lipids and glycolipid Al. No antigenic cross-reactions were observed between glycolipid Al and Line 10 cells or Line 10 lipid extracts and fractions. None of the Line 10 preparations was capable of inhibiting the hemagglutination reactions between Al and human serum antibodies. Studies on antibody response to Al. Antibodies against glycolipid Al were not detected in the sera of guinea pigs during or after tumor
916
REGGIARDO
abrogation as a consequence of Al injection. No Al antibodies were detected in the sera of those guinea pigs which failed to abrogate tumor growth. DISCUSSION Living BCG injected together with Line 10 cells or into 7- to 8-day established tumors is able to suppress or regress hepatoma transplants in strain 2 guinea pigs (25, 26). The studies presented in this paper show that mycobacterial glycolipid Al, when dissolved in olive oil and injected together with Line 10 cells, is able to elicit a host response which results in the abrogation or retardation of tumor growth in guinea pigs. Al was not toxic for Line 10 cells. In some guinea pigs there was no evidence of tumor growth for several weeks, although eventually tumors began to grow 7 to 11 weeks postinoculation. It is reasonable to speculate that the majority of Line 10 cells were killed at the beginning and later on the few surviving cells began to multiply. Why the host was unable to control the growth of these remaining cells after the initial killing is not understood. It is possible that some Line 10 cells were in a dormant state and could not be killed by the host. Glycolipids B1 and C did not have any effect on tumor growth. Other purified mycobacterial lipid fractions have been shown to control Line 10 growth in syngeneic guinea pigs. Mycobacterial trehalose 6,6'-dimycolate (TDM), so-called "cord factor," has been reported effective when combined with cell walls or dead BCG and injected into 6- to 10-mm tumors (2). P3, a highly purified TDM preparation, has been shown to be active when mixed with mycobacterial cell walls or bacterial endotoxins and injected on mineral oil droplets either together with Line 10 or into 7- to 8-day tumors (13, 20, 21). Neither of these trehalose mycolate preparations had an antitumor effect in guinea pigs when administered alone. Both of them have been reported to have adjuvant effects, TDM for antibody formation in mice and rats (4) and P3 for delayed-type hypersensitivity in guinea pigs (8). Also P3 is reported to be a mild granulomagenic agent in guinea pigs (12). TDM is granulomagenic in mice but not in guinea pigs when given intradermally (2, 22). Recently, using guinea pig macrophages, Kelly has shown that glycolipid P3 is able to generate chemotactic activity from plasma (11). TDM is said to be directly chemotactic for mouse leukocytes and macrophages and able to stimulate mouse macrophages (14, 24). All of the abovementioned properties could contribute to the antitumor effect(s) of P3 and TDM.
INFECT. IMMUN.
Several different mechanisms may be involved in the antitumor activity of glycolipid Al. It is possible to assume that antibodies against Al could contribute to the killing of Line 10 cells. Antibodies against Al have been found in the sera of tuberculous and leprous patients (18) and in the sera of patients undergoing BCG immunotherapy (23). However, Al antibodies have not been detected in the sera of strain 2 guinea pigs during or after tumor abrogation as a consequence of Al injection. It is possible that Al antibodies are cytophilic and could specifically direct the macrophages toward the tumor target cell if Al and Line 10 cells have a common antigenic determinant. It has been reported that BCG shares antigenic determinants with Line 10 cells that are tumor specific and could provide one possible mechanism for the antitumor effect of living BCG (6). In the experiments reported in this paper, glycolipid Al did not antigenically cross-react with lipid fractions and extracts from Line 10 cells, providing further evidence for a nonspecific effect of Al and ruling out a role for Al antibodies in this model. Numerous observations indicate that macrophages may play an important role in controlling tumor growth (7). The granuloma elicited by Al consists of an extensive accumulation of histiocytes (19), and it is reasonable to speculate that these macrophages are activated in some fashion and capable of killing the neoplastic cells, allowing the development of tumor-specific immunity and enabling the guinea pig to resist subsequent tumor challenge. Histological studies at the skin site of Line 10 and living BCG injection suggested that the granulomatous response may be important for the regression of tumor growth (10). BCG-activated macrophages may nonspecifically kill tumor cells by a different mechanism(s) than Al-activated macrophages. In the former, sensitized lymphocytes may play a crucial role. For instance, immunosuppressed guinea pigs which were unable to develop tuberculin sensitivity failed to control tumor growth at the site of BCG infection (9). On the other hand, the granuloma elicited by Al seems to be of the nonallergic type, with lymphocytes being rare (19). Furthermore, Al when injected in olive oil does not have adjuvant activity for delayed type hypersensitivity to an unrelated antigen, which also suggests that T lymphocytes or "specific arming" of macrophages by immune T cells may not be involved in this model. Whether some other components of the inflammatory reaction induced by Al besides granuloma formation may participate in controlling tumor growth remains to be studied. Finally, if Al activates macrophages by a nonimmunolog-
VOL. 21, 1978
ANTITUMOR ACTIVITY OF GLYCOLIPID Al
ical pathway, it would be expected that immunosuppressed guinea pigs would be able to control tumor growth after Al injection. This hypothesis and studies on regression of established tumors are under investigation. ACKNOWLEDGMENTS This work was supported in part by research grants from the American Cancer Society, Maryland Division, Inc. LITERATURE CITED 1. Bast, R. C., Jr., B. Zbar, T. Borsos, and H. J. Rapp. 1974. BCG and cancer. N. Engl. J. Med. 290:1413-1420. 2. Bekierkunst, A. 1968. Acute granulomatous response produced in mice by trehalose-6,6'-dimycolate. J. Bacteriol. 96:958-961. 3. Bekierkunst, A., L. Wang, R. Toubiana, and E. Lederer. 1974. Immunotherapy of cancer with nonliving BCG and fractions derived from mycobacteria. Role of cord factor (trehalose-6,6'-dimycolate) in tumor regression. Infect. Immun. 10:1044-1050. 4. Bekierkunst, A., E. Yarkoni, I. Flechner, S. Morecki, E. Vilkas, and E. Lederer. 1971. Immune response to sheep red blood cells in mice pretreated with mycobacterial fractions. Infect. Immun. 4:256-263. 5. Biozzi, G., C. Stiffel, B. N. Halpern, and D. Mouton. 1959. Effect de inoculation du bacille de CalmetteGuerin sur le developpement de la tumeur ascitique d'Ehrlich chez la souris. C. R. Soc. Biol. (Paris) 153:987-989. 6. Borsos, T., and H. J. Rapp. 1973. Antigenic relationship between Mycobacterium bovis (BCG) and a guinea pig hepatoma. J. Natl. Cancer Inst. 51:1085-1086. 7. Evans, R., and P. Alexander. 1976. Mechanisms of extracellular killing of nucleated mammalian cells by macrophages, p. 535-575. In D. S. Nelson (ed.), Immunobiology of the macrophage. Academic Press, Inc., New York. 8. Granger, D. L, K. Yamamoto, and E. Ribi. 1976. Delayed hypersensitivity and granulomatous response after immunization with protein antigens associated with a mycobacterial glycolipid and oil droplets. J. Immunol. 116:482-488. 9. Hanna, M. G., Jr., M. J. Snodgrass, B. Zbar, and H. J. Rapp. 1973. Histopathology of tumor regression after intralesional injection of Mycobacterium bovis. IV. Development of immunity to tumor cells and BCG. J. Natl. Cancer Inst. 51:1897-1908. 10. Hanna, M. G., Jr., B. Zbar, and H. J. Rapp. 1972. Histopathology of tumor regression after intralesional injection of Mycobacterium bovts. I. Tumor growth and metastasis. J. Natl. Cancer Inst. 48:1441-1455. 11. Kelly, M. T. 1977. Plasma-dependent chemotaxis of macrophages toward BCG cell walls and the mycobacterial glycolipid P3. Infect. Immun. 15:180-183. 12. Meyer, T. J., E. Ribi, and I. Azuma. 1975. Biologically active components from mycobacterial cell walls. V. Granuloma formation in mouse lungs and guinea pig skin. Cell. Immunol. 16:11-24.
917
1;3. Meyer, T. J., E. Ribi, 1. Azuma, and B. Zbar. 1974. Biologically active components from mycobacterial cell walls. II. Suppression and regression of strain-2 guinea pig hepatoma. J. Natl. Cancer Inst. 52:103-111. 14. Ofek, I., and A. Bekierkunst. 1976. Chemotactic response of leukocytes to cord factor (trehalose-6,6'-dimycolate). J. Natl. Cancer Inst. 57:1379-1380. 15. Old, L. J., D. A. Clark, and B. Benacerraf. 1959. Effect of bacillus Calmette-Guerin infection on transplanted tumours in the mouse. Nature (London) 184:291-292. 16. Rapp. H. J., W. H. Churchill, Jr., B. S. Kronman, R. T. Rolley, W. G. Hammond, and T. Borsos. 1968. Antigenicity of a new diethylnitrosamine-induced transplantable guinea pig hepatoma: pathology and formation of ascites variant. J. Natl. Cancer Inst. 41:1-7. 17. Reggiardo, Z., and G. Middlebrook. 1974. Serologically active glycolipid families from Mycobacterium bovis BCG. I. Extraction, purification and immunological studies. Am. J. Epidemiol. 100:469-476. 18. Reggiardo, Z., and G. Middlebrook. 1974. Serologically active glycolipid families from Mycobacterium bovis BCG. II. Serological studies on human sera. Am. J. Epidemiol. 100:477-486. 19. Reggiardo, Z., and A. K. M. Shamsuddin. 1976. Granulomagenic activity of serologically active glycolipids from Mycobacterium bovis BCG. Infect. Immun. 14:1369-1374. 20. Ribi, E., R. Toubiana, S. M. Strain, K. C. Milner, C. McLaughlin, J. Cantrell, I. Azuma, B. C. Das, and R. Parker. 1978. Further studies on the structural requirements of agents for immunotherapy of the guinea pig Line-10 tumor. Cancer Immunol. Immunother. 3:171-177. 21. Ribi, E. E., D. L. Granger, K. C. Milner, and S. M. Strain. 1975. Tumor regression caused by endotoxins and mycobacterial fractions. J. Natl. Cancer Inst. 55:1253-1257. 22. Saito, R., A. Tanaka, K. Sugiyama, I. Azuma, Y. Yamamura, M. Kato, and M. Goren. 1976. Adjuvant effect of cord factor, a mycobacterial lipid. Infect. Immun. 13:776-781. 23. Wile, A., Z. Reggiardo, F. C. Sparks, and D. L. Morton. 1976. Monitoring BCG immunotherapy with serologically active mycobacterial glycolipids. Surg. Forum 27:105-106. 24. Yarkoni, E., L. Wang, and A. Bekierkunst. 1977. Stimulation of macrophages by cord factor and by heatkilled and living BCG. Infect. Immun. 16:1-8. 25. Zbar, B., 1. D. Bernstein, and H. J. Rapp. 1971. Suppression of tumor growth at the site of infection with living bacillus Calmette-Guerin. J. Natl. Cancer Inst. 46:831-839. 26. Zbar, B., and T. Tanaka. 1971. Immunotherapy of cancer: regression of tumors after intralesional injection of living Mycobacterium bovis. Science 172:271-273. 27. Zbar, B., H. T. Wepsic, H. J. Rapp, J. Whang-Peng, and T. Borsos. 1969. Transplantable hepatomas induced in strain-2 guinea pigs by diethylnitrosamine: characterization by histology, growth and chromosomes. J. Natl. Cancer Inst. 343:821-826.
