necrobiotic tumor cells. Similarly, macrophages were re covered from pyran-treated animals, which potently ar rested DNA synthesis of M109 tumor cells in vitro.
[CANCER
RESEARCH
37, 3338-3343,
September
1977]
Association of Macrophage Activation with Antitumor Activity by Synthetic and Biological Agents Richard M. Schultz, Joseph D. Papamatheakis, Josef Luetzeler,' and Michael A. Chirigos Laboratory
of RNA Tumor Viruses. National
Cancer Institute,
NIH, Bethesda,
SUMMARY
Treatment of normal BALB/c mice i.p. with a number of adjuvants, including pyran copolymer, the copolymer of polyinosinic and polycytidylic acids, Bacillus CalmetteGuérin,glucan, and dextran sulfate, rendered macrophages nonspecifically cytostatic for syngeneic tumor cells. Macro phage activation was highly dose dependent. The validity of the inhibition of DMA synthesis assay for measuring macrophage-induced cytostasis of target cells was proven by demonstrating a concurrent decrease in RNA synthesis and a reduction in viable tumor cell number. Moreover, condi tioned supernatants from pyran-activated macrophages did not significantly decrease [3H]thymidine incorporation by freshly added leukemia cells. Biological or synthetic agents that activated macrophages were generally effective sys temic antitumor agents against the M109 lung carcinoma. Drugs that did not activate macrophages, such as typhoid vaccine, tilorone, levamisole, WY-13876, and thymosin, were ineffective in prolonging the life of tumor-bearing mice. Pyran treatment i.p. was the most effective antitumor adjuvant in two separate tumor models, and suppression of tumor growth appeared to be related not only to an increase in macrophage tumoricidal function, but also to a larger influx of macrophages responding at the tumor site. INTRODUCTION
A variety of biological and chemical adjuvants have the ability to enhance the metabolic and functional activity of mononuclear phagocytes. Although the mechanism of mac rophage activation by these agents is quite dissimilar, the end result is the same; these cells display heightened tu moricidal and microbicidal activity (7, 10, 15, 20, 22), en hanced phagocytosis, and the ability to amplify the afferent limb of immune responses (12). Macrophages may either be directly activated by Interferon inducers (e.g., lipid A, dou ble-stranded RNA, and pyran copolymer) (1, 19) or may require activating factors from sensitized T-derived lympho cytes in the presence of appropriate antigen (2, 6, 17). We have presented indirect evidence that the ability of agents to render macrophages cytotoxic for tumor cells correlates with their capacity to enhance antitumor resistance (22). Previous evidence showed that oyran copolymer mark ' Visiting
scientist
from the Immunopathology
Cologne, West Germany. Received April 7, 1977; accepted
3338
Department,
June 10, 1977.
University
of
Maryland
20014
edly increased resistance of BALB/c mice bearing the M109 lung carcinoma (18, 21). Systemic treatment with pyran resulted in a prominent histiocytic infiltrate invading the tumor in which macrophages were often associated with necrobiotic tumor cells. Similarly, macrophages were re covered from pyran-treated animals, which potently ar rested DNA synthesis of M109 tumor cells in vitro. The sensitivity of the M109 tumor to pyran copolymer and its lack of responsiveness to numerous cytoreductive agents (25) suggested that this tumor might have the potential to identify alleged immunopotentiating antitumor agents. The present study compares pyran to other adjuvants in regard to both systemic antitumor activity and macrophage activa tion. The IDS2 assay for measuring tumor cytostasis was found to be a sensitive and reliable index of macrophage function. The presence of growth-inhibitory macrophages following adjuvant therapy correlated with significant in creases in survival times of tumor-bearing mice.
MATERIALS
AND METHODS
Mice. Male BALB/c and C57BL/6 mice, 6 to 8 weeks old, were obtained from the Mammalian Genetics and Animal Production Section, NIH, Bethesda, Md. All animals weighed at least 23 g before they were used for experimen tation. Drugs. The description and source of all adjuvants incor porated into this study are listed in Table 1. All drugs were made up in Dulbecco's phosphate-buffered saline, adjusted to pH 7.2, and given i.p. at 1% body weight. Target Cell Cultures. Cell strains derived from the M109 alveolar carcinoma and MBL-2 lymphoblastic leukemia have been established and maintained in RPMI-FCS. Both cell lines have been shown to be free of Mycoplasma con tamination (Microbiological Associates, Bethesda, Md.). Tumor Testing. The M109 lung carcinoma, which arose spontaneously in a BALB/c mouse in 1964, was received from Dr. Ruth I. Geran, Drug Research and Development, National Cancer Institute, NIH. The colon tumor 26, an undifferentiated colonie carcinoma originally induced in a BALB/c mouse by /V-nitroso-N-methyl-urethan (3), was gen-
2 The abbreviations used are: IDS, inhibition of DNA synthesis; RPMI-FCS, Roswell Park Memorial Institute Medium 1640 supplemented with 20% heatinactivated (56°for 30 min) fetal calf serum, gentamicin (100 M9/ml), 0.075% NaHCOj, and 10 mM W-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer; MST, median survival time; poly(l)-poly(C), copolymer of polyinosinic and polycytidylic acids; BCG, Bacillus Calmette-GuÉrin.
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VOL. 37
Macrophage Table 1 Agents tested for their antitumor activity and ability to produce growth-inhibitory macrophages and route of administration (mg/kg)25,
AgentChemicalDextran sulfateLevamisolePoly(l)-poly(C)Pyran FineChemicals, Upps SwedenJanssen ala, Pharma-ceutica, Beerse,BelgiumMiles
i.p.10,
i.p.10,
Laboratories,Inc., i.p.25, Ind.Hercules Elkhart, copolymer(NSC-46015)Tilorone(NSC-1 ResearchCenter, i.p.50, Wilming Del.Merrell-NationalLaboratories, ton, i.p.100, 43963)Wyeth-13,876SourcePharmacia Cin OhioWyeth cinnati, Laboratories,Philadelphia, i.p. Pa.Dose Biological BCG, Pasteur strain
Glucan
Thymosin (calf thymus extract Fraction 5)
Typhoid vaccine (U.S.P.)
Dr. S. D. Chaparas, Bureau of Biologics, Food and Drug Administra tion, Rockville, Md. Dr. N. R. DiLuzio, Tulane University School of Medi cine, New Or leans, La. Dr. A. L. Goldstein, University of Texas Medical Branch, Galveston, Texas Eli Lilly Company, Indianapolis, Ind.
107 organisms/ mouse, i.p.
25, i.p.
10, i.p.
-10e killed orga nisms/mouse, i.p.
were calculated. The percentage increase in life span of test groups (7) over control groups (C) was calculated by (7/C 1) x 100. The mean survivals of adjuvant-treated groups in comparison to those of groups receiving Dulbecco's phos phate-buffered saline were evaluated statistically with Stu dent's f test. Preparation of Macrophage Cultures. Noninduced BALB/ c or C57BL/6 mouse peritoneal macrophages were pre pared as described previously (22). Exúdales from 5 mice in each group were collected 6 days after drug treatment, pooled, washed once in 30 ml Hanks' balanced salt solu tion, and resuspended in 10 ml RPMI-FCS. Peritoneal mac rophages were purified by adherence on 100- x 20-mm
1977
Activation
and Ant/tumor
dishes. After 1 hr of incubation
Activity
at 37°,the
adhering cells were gently scraped with a soft rubber policeman into RPMI-FCS, adjusted to 1 x 106 viable cells per ml RPMI-FCS, and kept in an ice bath before use to prevent adherence. Greater than 95% of the cells prepared in this manner had morphological characteristics of macrophages and the capability to ingest latex beads. Assay for Macrophage-mediated Cytostasis. The ability of activated macrophages to diminish target cell prolifera tion was measured by the IDS assay previously described (21). This assay is advantageous for measuring macrophage-mediated cytostasis since macrophages do not pro liferate and incorporate significant thymidine under the cul ture conditions used. Briefly, target cells were trypsinized from exponentially growing cultures and resuspended at 5.0 x 104 cells per ml RPMI-FCS, and 2-ml aliquots were placed in 30-mm tissue culture dishes. Purified peritoneal macrophages were adjusted to 1.0 x 10" cells per ml RPMIFCS, and 1 ml was added to the target cell cultures; the effectortarget cell ratio was, therefore, 10:1. DMA synthesis of the target cells was assessed after 20 hr of incubation at 37°. Triplicate cultures of each set of dishes as well as cultures consisting of tumor cells alone and macrophages alone were pulsed with 2.0 /xCi of [3H]thymidine (specific activity, 10 Ci/mmole) for 2 hr at 37°.At the end of the incubation, the cells were detached with trypsin, centrifuged at 600 x g for 10 min, and resuspended in 0.5 ml Dulbecco's phosphate-buffered saline. Four ml of chilled 10% trichloroacetic acid were added to each tube, and precipitation was allowed to go on for 30 min at 4°.The
erously supplied by Dr. F. Schabel from Southern Research Institute, Birmingham, Ala. Single-cell suspensions were prepared from the tumors by enzymatic digestion of minced tissues with 0.25% trypsin. For adjuvant studies, 5 x 10s viable tumor cells, suspended in serum-free Roswell Park Memorial Institute Medium 1640, were injected s.c. into the inguinal region of each BALB/c mouse. Selected adjuvants or placebo were administered i.p. on Day 7 after tumor inoculation. Deaths of mice were recorded daily, and MST's
SEPTEMBER
tissue culture
resultant precipitate was collected on glass-fiber paper (Millipore Corp., Bedford, Mass.) and washed with cold 10% trichloroacetic acid. The filters were then air-dried and as sayed for radioactivity in a Packard Tri-Carb scintillation counter with Aguasol solubilizer (New England Nuclear, Boston, Mass). The percentage of specific inhibition of DNA synthesis was calculated by the formula: . Specific inhibition = I
- cpmf; cpm.v
100
where cpmv = mean cpm in cultures containing effector cells from normal control mice, and cpmf = mean cpm in cultures containing test effector cells. Histopathology. Whole tumors were excised at different time intervals after tumor inoculation and fixed in 10% neutral formalin. These tissues were sectioned and rou tinely stained with hematoxylin and eosin. RESULTS Association of Antitumor Activity of Biological and Syn thetic Agents with Macrophage Activation. The biological and synthetic adjuvants were tested for systemic antitumor activity against the syngeneic metastasizing M109 lung car cinoma. The MST of tumor-bearing mice that received pla cebo was 35.5 days. Pyran copolymer at 25 mg/kg on Day 7 after tumor inoculation was the most significant (p < 0.001) at prolonging survival times (MST = 55 days) of tumorbearing mice (Chart 1a). Poly(l)-poly(C), BCG, and glucan
3339
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Chart 1.a, effect of synthetic and biological agents on life span of mice bearing the M109 lung carcinoma. Tumor cells (5 x 10s)were inoculated s.c. into BALB/c mice on Day 0. Drugs were given i.p. on Day 7. Experiments involved 10 mice/group. Probabilities that the differences are statistically significant from tumor-bearing controls receiving phosphate-buffered saline are: *,p < 0.05; **,p , effect of various agents on the cytostatic activity of BALB/c peritoneal macrophages. Drugs at the same dose levels as in a were inoculated i.p. on Day 0. Macrophages were har vested 6 days later and tested for their ability to inhibit M109 cell DMA synthesis. The values represent the means of 2 experiments, each involving triplicate samples. S.E. of individual experiments never exceeded ±8%.
also exhibited significant antitumor activity (MST of 44.0, 42.5, and 42.0 days, respectively), whereas dextran sulfate, typhoid vaccine, tilorone, levamisole, WY-13876, and thymosin were without effect under the experimental condi tions used. These agents were tested under similar conditions for the ability to produce growth-inhibitory macrophages. Perito neal macrophages were collected 6 days after i.p. adminis tration of drugs to normal, non-tumor-bearing mice and tested against syngeneic M109 target cells (Chart 1t>). All polyanions [pyran, poly(l)-poly(C), and dextran sulfate] markedly stimulated BALB/c macrophages to inhibit M109 DNA synthesis (p < 0.001). In addition, the biological agents BCG and glucan rendered macrophages cytostatic, whereas typhoid vaccine, tilorone, levamisole, WY-13876, and thymosin were without significant effect. These agents, which failed to activate macrophages, were similarly inef fective in prolonging the life span of tumor-bearing mice. Biological or synthetic agents that activated macrophages, such as pyran, poly(l)-poly(C), BCG, and glucan, were gen
3340
erally effective systemic antitumor agents against the M109 lung carcinoma. To determine whether a positive correlation existed be tween macrophage activation and antitumor activity in a different tumor system, the various agents were tested against the colon tumor 26. Only pyran copolymer was found to significantly (p < 0.01) enhance BALB/c resist ance in 2 separate screens. The MST's were increased 28.2 and 46.0%, respectively, by pyran treatment. Since pyran was the only effective adjuvant against the colon tumor 26, the histopathology of the host response against the primary tumor was studied. Histologically, the colon tumor 26 represents a poorly differentiated carci noma interspersed with spindle cell-like areas (Fig. 1). As early as 2 days after pyran therapy (Day 9), the host tissue showed signs of cellular activity with increasing numbers of histiocytes, lymphocytes, and some granulocytes at later intervals (Days 11 and 13). At Day 18, histiocytes and macro phages predominated around and within the tumor tissue. The tumor was clearly demarcated from the surrounding fibrofatty tissue which showed a heavy infiltration of histio cytes, numerous macrophages with foamy appearance of the cytoplasm, as well as some lymphocytes (Fig. 2). These cells were observed infiltrating tumor tissue in groups from the periphery, encircling viable-looking tumor cells, and separating the tumor tissue toward the center. Although the migration of granulation tissue due to pyran therapy was less developed than previously reported in the M109 lung tumor (21), the attack of histiocytes and macrophages against the colon carcinoma cells was more obvious. In contrast, the host reaction in Dulbecco's phosphatebuffered saline-treated animals bearing the colon tumor 26 was nearly absent. The growth was more progressive with infiltration of the tumor up to the deeper layers of the skin (Fig. 3). Tumor cell necrosis was only moderate in compari son to the tumors of pyran-treated animals taken at similar intervals. Dose Response of Macrophage Activation in Vivo. Fur ther experiments were performed to test the dose depend ency of adjuvant-induced macrophage activation. Drugs were administered i.p. at doses ranging from 100 to 0.1 mg/ kg. Macrophages were harvested on Day 6. Activation by polyanions was sharply dose dependent (Chart 2). Pyran copolymer and poly(l)-poly(C) treatment produced optimal macrophage stimulation at 10 mg/kg, whereas dextran sul fate required 25 mg/kg. Antitumor activity by pyran is simi larly dose dependent, although a single i.p. treatment of pyran on Day 7 after M109 tumor implantation is effective over a dose range of 1 to 100 mg/kg (18). Macrophage activation by glucan was active over a broader range from 1 to 25 mg/kg. Higher levels of drug were not active. Validity of IDS Assay. Since macrophages have been reported to secrete thymidine or an analog thereof (24), we tested whether our IDS assay actually reflected target cell proliferation or merely competition of macrophage secre tions for radioactive thymidine used for pulsing the cells. MBL-2 lymphoblastic leukemia cells were used as targets. Due to their nonadherent nature in cell culture, these cells have the advantage of readily being distinguished from macrophage effectors and are sensitive to the cytotoxic effects of activated macrophages (19). Macrophages actiCANCER
RESEARCH
VOL. 37
Macrophage Activation and Antitumor Activity Drugs •Pycan
A PrtyII). PolyID O Oeitian
Sudale
A Glucan
1.0
100
2SO
1WD
LOG DOSE Img/kgl
Chart 2. Dose dependency of macrophage activation in vivo. Macro phages were harvested 6 days after ¡.p.drug administration at the concentra tions shown and tested for their ability to inhibit M109 cell proliferation. PolyO)-poly(C) was lethal for mice at 100 mg/kg. Values represent the means of triplicate samples.
vated by a peak concentration (20 mg/kg) of pyran were used as effector cells. After 18 hr of coincubation with MBL2 cells, the cultures were either pulsed with [3H]thymidine or [3H]uridine, or the actual number of surviving leukemia cells was quantitated with a hemacytometer. Cultures that showed a 46% inhibition of DMA synthesis were accompa nied by a concurrent 65% inhibition of RNA synthesis and 84% inhibition of MBL-2 cell growth. Moreover, conditioned supernatants from pyran-activated cultures did not signifi cantly decrease [3H]thymidine incorporation of freshly added log-phase cultures of leukemia cells. Accordingly, in the present system, it seems reasonably certain that thymidine incorporation actually reflects target cell proliferation. DISCUSSION
The vital role that mononuclear phagocytes play in con trol of tumor growth and its dissemination are now being justly recognized. The conversion of the resting macro phage to an activated state by polyanions or soluble media tors constitutes a primitive yet highly effective surveillance and potent antitumor mechanism. Once activated, macro phages can regulate cell proliferation (10) and selectively destroy cells with abnormal growth properties (9). The studies presented here show that the ability of biolog ical and synthetic agents to render macrophages cytostatic for tumor cells correlated with their capacity to increase life span of mice bearing the syngeneic M109 lung carcinoma. Macrophage activation by pyran, poly(l)-poly(C), glucan, and dextran sulfate was sharply dose dependent, with su praoptimal levels of drug having lost their activity. The reason for this dose dependency is unknown, although direct macrophage activation by polyanions in vitro shows similar kinetics (19). Pyran was the most effective adjuvant in regard to both macrophage activation and antitumor activity in 2 tumor models. Since pyran is not directly toxic for tumor cells in vitro, modulation of macrophage activity has been implicated in the antitumor activity (21). Pyran has previously been shown to suppress the growth of numerous SEPTEMBER 1977
solid tumors (15,18, 21, 23, 25) and is capable of producing a significant number of "cures" when combined with remis sion-inducing chemotherapy against the Lewis lung carci noma and LSTRA murine leukemia (13). The antitumor ac tivity of pyran has been studied in depth against the primary M109 lung carcinoma and its métastases(18, 21). Systemic pyran therapy was active over a wide range of doses from 1 to 100 mg/kg/day, and multiple doses were not significantly better than single treatments. A single treatment was effec tive even relatively late in the course of neoplasia (Day 14) in a system where the MST of control mice is about 30 days. Pyran copolymer was previously found to be toxic in Phase 1 clinical trials involving advanced cancer patients (16); toxic side effects, including thrombocytopenia and hypotension, precluded further study. However, at that time, the ability of pyran to enhance host survival against neopla sia was believed to result from direct antimitotic effects. Of particular significance are our findings that pyran is effec tive over a large range of doses and that multiple treatments do not give additional protective effects (21). Moreover, we present evidence that pyran dosage at high levels inhibits macrophage function. It now appears that pyran therapy can be tailored to the individual tumor system to minimize patient toxicity and retain good activity. Although it is possible that pyran and the biological adju vants stimulate other cell types that both inhibit tumor growth and, in turn, activate macrophages, in vivo studies have further implicated activated macrophages as effectors of tumor resistance. Histopathological studies have re vealed an intense histiocytic reaction in the connective tis sue surrounding the s.c. primary tumor in mice treated intralesionally with BCG (8) or glucan (11). Intimate contact between macrophage and target cell was a requirement for tumor cell destruction. The presence of large numbers of histiocytes in several untreated animal tumors was similarly associated with tumor rejection and the lessened likelihood of metastasis (4). Thus, the major limitation of the macro phage effector arm of the immune response appears to be cell concentration at the primary or disseminated tumor site. It is tempting to compare pyran copolymer with BCG, since pyran copolymer therapy provoked histiocytosis around the s.c.-transplanted M109 lung carcinoma (21) and colonie carcinoma 26 in BALB/c mice, and since both agents activate macrophages in vivo. However, 3 distinct differences are involved: (a) there is no granuloma develop ment as would be seen with BCG; (b) macrophage activa tion and mobilization by BCG requires functional T-cells (17), whereas activation by pyran is direct (19) and does not require an immunocompetent host; and (c) BCG requires intralesional therapy to produce a migration of histiocytes to the tumor (8), whereas i.p. pyran treatment produces a histiocytic reaction at the s.c. tumor site (21, 23). Pyran therapy also reduces the number of pulmonary lesions that develop after the i.v. inoculation of M109 tumor cells (18). Snodgrass ef al. (23) have shown that small numbers of macrophages, presumably of hematogenous origin, accumulated in the pulmonary interstitium of pyrantreated animals. There were regions where macrophage accumulations formed nodules that disrupted much of the normal architecture. It is tempting to speculate that these pyran-activated macrophages have a surveillance function 3341
R. M. Schultz et al. in inhibiting or controlling metastatic cell growth. Similarly, Fidler (5) has reported that activated macrophages injected i.v. inhibit pulmonary metastasis of B-16 melanoma cells. The data presented are consistent with the hypothesis that nonspecifically activated macrophages are major effec tors of the antitumor resistance induced by both synthetic and biological immunopotentiators. The effect of pyran on modulating host immunological factors, even in an ad vanced tumor system (21), its ability to augment specific immune responses (14), its compatibility with cytoreductive chemotherapy (13), and its effect on inhibiting distant me tastasis (18) strongly support the potential use of pyran copolymer as an adjuvant to conventional tumor treatment modalities.
ACKNOWLEDGMENTS The authors wish to thank Douglas C. Jones, Virus Studies Section, Office of Coordinator of Ultrastructure Studies, National Cancer Institute, for pho tographic assistance.
REFERENCES 1. Alexander, P., and Evans, R. Endotoxin and Double Stranded RNA Render Macrophages Cytotoxic. Nature New Biol., 232. 76-78, 1971. 2. Churchill, W. H., Piessens, W. F., Sulis, C. A., and David, J. R. Macro phages Activated as Suspension Cultures with Lymphocyte Mediators Devoid of Antigen Become Cytotoxic for Tumor Cells. J. Immunol., 775: 781-786, 1975. 3. Corbett.T. H., Griswold, D. P., Roberts, B.J., Peckham, J., andSchabel, F. M. A Mouse Colon-Tumor Model for Experimental Therapy. Cancer Chemotherapy Rept. Part 2, 5: 169-186, 1975. 4. Eccles, S. A., and Alexander, P. Macrophage Content of Tumours in Relation to Metastatic Spread and Host Immune Reaction. Nature, 250: 667-669, 1974. 5. Fidler, I. J. Inhibition of Pulmonary Metastasis by Intravenous Injection of Specifically Activated Macrophages. Cancer Res., 34: 1074-1078, 1974. 6. Fidler, I. J., Darnell, J. H., and Budmen, M. B. In Vitro Activation of Mouse Macrophages by Rat Lymphocyte Mediators. J. Immunol., 117: 666-673, 1976. 7. Gottlieb, A. A., and Waldman, S. R. The Multiple Functions of Macro phages in Immunity. In: A. I. Laskin and H. Lechevalier (eds.), Macro phages and Cellular Immunity, pp. 13-38. Cleveland: Chemical Rubber Co. Press, 1972. 8. Manna, M. G., Zbar, B., and Rapp, H. J. Histopathology of Tumor Regression after Intralesional Injection of Mycobacterium bovis. 1. Tu
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mor Growth and Metastasis. J. Nati. Cancer Inst., 48:1441-1455,1972. 9. Hibbs, J. B. Discrimination between Neoplasia and Non-neoplastic Cells in Vitro by Activated Macrophages. J. Nati. Cancer Inst., 53: 1487-1492, 1974. 10. Keller, R. Cytostatic and Cytocidal Effects of Activated Macrophages. In: D. S. Nelson (ed.), Immunobiology of the Macrophage, pp. 487-508. New York: Academic Press, Inc., 1976. 11. Mansell, P. W. A., Ichinose, H., Reed, R. J., Krementz, E. T., McNamee, R.. and DiLuzio, N. R. Macrophage-mediated Destruction of Human Malignant Cells in Vivo. J. Nati. Cancer Inst., 54: 571-580, 1975. 12. Meltzer, M. S., and Oppenheim, J. J. Bidirectional Amplification of Macrophage-Lymphocyte Interactions: Enhanced Lymphocyte Activa tion Factor Production by Activated Adherent Mouse Peritoneal Cells. J. Immunol., 778: 77-82, 1977. 13. Mohr, S. J., Chirigos, M. A., Fuhrman, F. S., and Pryor, J. W. Pyran Copolymer as an Effective Adjuvant to Chemotherapy against a Murine Leukemia and Solid Tumor. Cancer Res., 35: 3750-3754, 1975. 14. Mohr, S. J., Chirigos, M. A., Smith, G. T., and Fuhrman, F. S. Specific Potentiation of L1210 Vaccine by Pyran Copolymer. Cancer Res., 36: 2035-2039, 1976. 15. Morahan, P. S., and Kaplan, A. M. Macrophage Activation and Antitumor Activity of Biologic and Synthetic Agents. Intern. J. Cancer, 17: 82-89, 1976. 16. Morahan, P. S., Munson, J. A., Baird, L. G., Kaplan, A. M., and Regelson, W. Antitumor Action of Pyran Copolymer and Tilorone against Lewis Lung Carcinoma and B-16 Melanoma. Cancer Res., 34: 506-511, 1974. 17. North, R. J. T-cell Dependence of Macrophage Activation and Mobiliza tion during Infection with Mycobacterium tuberculosis. Infect. Immun., 70: 66-71, 1974. 18. Papamatheakis, J. D.. Chirigos, M. A., and Schultz, R. S. Effect of Dose, Route, and Timing of Pyran Copolymer Therapy against the Madison Lung Carcinoma. In: M. A. Chirigos (ed.), Control of Neoplasia by Modu lation of the Immune System, Vol. 3. New York: Raven Press, in press. 19. Schultz, R. M., Papamatheakis, J. D., and Chirigos. M. A. Direct Activa tion in Vitro of Mouse Peritoneal Macrophages by Pyran Copolymer (NSC 46015). Cellular Immunol., 29: 55-61, 1977. 20. Schultz, R. M., Papamatheakis, J. D., and Chirigos, M. A. Tumoricidal Effect in Vitro of Mouse Peritoneal Macrophages from Mice Treated with Glucan. In: M. A. Chirigos (ed.). Control of Neoplasia by Modulation of the Immune System, Vol. 3. New York: Raven Press, in press. 21. Schultz, R. M., Papamatheakis, J. D., Luetzeler, J., Ruiz, P., and Chiri gos, M. A. Macrophage Involvement in the Protective Effect of Pyran Copolymer against the Madison Lung Carcinoma (M109). Cancer Res., 37: 358-364, 1977. 22. Schultz, R. M., Papamatheakis, J. D., Stylos, W. A., and Chirigos, M. A. Augmentation of Specific Macrophage-Mediated Cytotoxicity: Correla tion with Agents which Enhance Antitumor Resistance. Cellular Immu nol. ,25. 309-316, 1976. 23. Snodgrass, M. J., Morahan, P. S., and Kaplan, A. M. Histopathology of the Host Response to Lewis Lung Carcinoma: Modulation by Pyran. J. Nati. Cancer Inst., 55: 455-462, 1975. 24. Unanue, E. R. Secretory Function of Mononuclear Phagocytes. Am. J. Pathol., 83: 393-417, 1976. 25. Woodman, R. J., and Gang, M. Unique Therapeutic Activity of Pyran Polymer against a New Mouse Lung Tumor. Proc. Am. Assoc. Cancer Res., 75: 109, 1974.
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Macrophage Activation and Antitumor Activity
\
*
.'
'>&P/¿™3*?*^
+9
' .•&*•, ^ÌÌrST* * '•••-, ^> kSiHVWjfeS
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,' •'' . rv ' ^-»« m. V
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1. Colon tumor 26. Poorly differentiated carcinoma with intertwining arrangement of tumor cells. Mitotic figures are abundant, x 250. 2. Numerous macrophages (short arrows) with typical foamy cytoplasm together with some clusters of lymphocytes (long arrow) invading tumor 18 days after pyran treatment, x 250. 3. Tumor surrounded by connective tissue without host reaction. Expansion of the periphery of the tumor up to the deeper layers of the skin (hair and sebaceous gland in right upper corner), 18 days after placebo treatment, x 250.
SEPTEMBER 1977
3343
