THE ROLE OF HOST DEFENSE EN 013~mCHLORGDEAMMINEPLATINUMGI) MEDEATED REGRESSWNS 0F SARC-OMA 180 m MViCE 'E‘hesisi far the Degree at 9:133 MECMGAE‘! STATE fiNEVERSiTY PHILEP B. coma 1973 I! 131: ml MIMI!!! in!!! ll in I ll Hfllfl ll - ~ ~--: L1” as v 7* 1.: I3... A _‘ '5 [Iv/A‘Cl‘zi"? '~s 1 5 O ‘ ‘ a 5 Um ' ,_ _ . 1‘ 3 ”1"”? ‘- «fun-Ill ‘n-p-nv a.“ This is to certify that the thesis entitled I THE ROLE OF HOST DEFENSBS IN ‘ ‘ C__I S-DICHLORODIAMMINEPLATINUM (II) MEDIATED REGRESSIONS OF SARCOMA 180 IN MICE [ presented by Philip B. Conran has been accepted towards fulfillment of the requirements for Ph .D . degree in PATFDLOGY fizzés/go G L Waxler,D Major professor Date July 17, 1973 0-7639 um?” sous' mm mm m: . L [39:19»! sworn; " FJRT lll‘u 3% ; 03,9 ‘ZW, ,. 7 35¢ 56.? £5 5 i997. A - ABSTRACT THE ROLE OF HOST DEFENSES IN CIS-DICHLORODIAMMINEPIATINUM(II) MEDIATED REGRESSIONS OF SARCOMA-IBO IN MICE By Philip B. Conran The role of host defenses in mediating the regression of Sarcoma 180 (8-180) tumors in mice treated with gig-DichlorodiammineplatinumuI) [c_i_§_-Pt(II)] was investigated. It was found that the marked antitmor efficacy of gig-Pun) against 8-180 implanted in Swiss mice was reduced when hydrocortisone (BC), an immnosuppressive drug, was administered 7 days before, 6 hours after or 7 days after the platinum compound. This reduction was most dramatic when BC was administered 7 days before or 6 hours after the platinum compound. In another series of experiments it was found that gig-Pram was ineffective in promoting regressions of 8-180 implanted in BALB/c mice. The administration of zymosan, a nonspecific iumme stimulant, in combination with gig-Pull) , however, promoted significant numbers of tumor regressions. This was particularly true if the zymosan was administered on day 1 of tumor growth followed in 7 days by a single injection of cis-Pt(II). Philip B. Conran From the results of the previously described experiments it was concluded that the antitumor efficacy of c_i__s_-Pt(II) is, at least in part, dependent on an active host response directed against the tumor. The immunologic integrity of BALB/ c mice treated with a combina- tion of zymosan and gig-Pan) was studied. Humoral antibody production was evaluated by the agarose slide technique using sheep red blood cells as antigen. There was virtually no difference in spleen cell plaque forming ability between control and treated animls when the antigen was administered on day 14 of the experiment. When the antigen was administered on day 21 of the experiment, however, those animals treated with a combination of zymosan and gig-Pt (II) had significantly higher numbers of plaque forming spleen cells than the saline treated animals. Similar studies. were performed on animals bearing 8-180. When antigen was injected 14 days after.- tumor implantation, plaque forming cells were found in significantly higher numbers in the animals treated with zymosan or saline than in those treated with gig-Pt (II) or gig-Pull) plus zymosan. When antigen was administered on day 21 of the experiment, however, those animals treated with gig-Pt (II) or gig-Pull) plus zymosan had significantly higher numbers of plaque forming cells than the other two groups. Thus it was concluded that, depending on the time of antigen administration, treatment with gig-Pun) or zymosan plus gi_s_-Pt(II) may have some stimulatory effect on humoral antibody responses. Cell mediated immune. responses were evaluated by skin allograft rejection. Allografts were rejected earlier in animals bearing 3-180 and treated with a combination of zymosan and cis-Pt(II). Consequently, Philip B. Conran it was concluded that combination therapy may stimulate cell mediated immune responses. Selected tissues from animals bearing 8-180 and treated‘with saline, zymosan, gig:Pt(II) or a combination of zymosan and Eingt(II) were evaluated histologically. Spleens and regional lymph nodes from all groups were markedly hyperplastic, particularly in the marginal zones of the lymphoid follicles. The thymuses of animals with regres- sing tumors had a normal appearing architecture with a somewhat hyper- plastic cortex. In contrast, the thymuses of those animals with non- regressing tumors were atrophic and the demarcation between cortex and medulla was obscured. Regressing tumors, regardless of treatment, were characterized by marked lymphocytic infiltration and were surrounded by a proliferating fibrous capsule similar to that seen in homograft rejection. This suggested that ultimate tumor regression may be mediated via immunologic mechanisms rather than a specific attack on tumor cells by the thera- peutic agents. THE ROLE OF HOST DEPENSES IN CIS-DICHLORODIAMMINEPLATINUM(II) MEDIATED REGRESSIONS OF SARCGMA 180 IN MICE By r {5 Philip B§\Conran A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1973 To all cancer patients 11 ACKNOWLEDGMENTS The author wishes to express his gratitude to Professor Barnett Rosenberg who afforded him the opportunity to pursue this investiga- tion and to Engelhard Industries and Matthey Bishop, Inc., for supporting it. Appreciation is given to my academic committee: Dr. Glenn Waxler, my major professor, for his untiring assistance; Dr. Robert Langham.for his invaluable training in pathology; Dr. Herbert Cox for stimulating my interest in immunology; and Dr. C. Cleon Morrill and Dr. Vance Sanger. Special thanks are extended to Mrs. Loretta VanCamp for her able technical assistance and provocative ideas, and Mr. Cliff Hale for his assistance. The author is especially indebted to Mr. George Moldovan for his tireless hours of assistance in computerizing the data reported in this investigation. Finally, the author wishes to express his sincere gratitude to his wife and children for their patience and understanding. iii TABLE OF CONTENTS INTRODUC HON O O O O O O O O O O O O O O O O O O O O O O LITERAME REVIEW. 0 I O O I O O O O O 0 O O O O 0 Host Defenses Against Neoplasia . . . . . . . . . Utilization of Sarcoma 180 in Cancer Chemotherapy md 1mm th‘tap y 0 O I O O I O O O O O O O O O O The Use of Platinum Compounds in Cancer Chemotherapy. . . The Properties of Zymosan and its Use as an mostmant O O O O O O O O O O O O O O O O O O O O O The Use of Steroid Hormones as Immunosuppressive Drugs. . MATERIAIIS AND mmons. O O O O O O O O O O O C O O O O O O O I 0 General Plan. . . . . . . . . . . . . . Source of Animals . . . . . . . . . . . Maintenance of Animals. . . . . . . . . Preparation of SigrPt(II) Compound. . . Preparation of Hydrocortisone . . . . . Preparation of Zymosan. . . . . . . . . Transplantation of Sarcoma 180. . . . . numeral Antibody Assay. . . . . . . .l. Cell Mediated Immunity Assay. . . . . . Treatment of Animals with Combined cis-Pt(II) Hydrocortisone (BC) . . . . . . . . . . . . . Treatment of Animals with Combined gigrPt(II) and Timing Experiment 1. . . . . . . . . . . . Timing Experiment 2. . . . . . . . . . . . Timing Experiment 3. . . . . . . . . . . . iv Page 12 16 20 24 24 24 25 25 26 26 26 27 30 31 32 34 34 37 Evaluation of the Integrity of Host Defenses. . . . Evaluation of Humoral Antibody Response. . . Evaluation of Cell Mediated Immune Responses Microscopic Examination of Tissues. . . . . . . . Statistical Analysis. . . RESULTS. 0 O I O O O O O C O I 0 Combined gigrPt(II) and Hydrocortisone Therapy. . . Combined Zymosan and cis-Pt(II) Therapy . . . . . . The Evaluation of Host Defenses in Animals Treated with Combinations of Zymosan and gig:Pt(II) Microscopic Evaluation of Spleen . . . Thymus . . . Lymph Nodes. Tumors . . . DISCUSSIW 0 O O O O O O O O O 0 WY. 0 O O O O O O O O O O O BmImwm O O O O O O O O O O VITA O I O O O O O O I O O O O 0 Selected Tissues. Page 37 37 4O 40 43 44 44 44 S7 61 61 64 64 64 75 91 94 105 Table 10 ll 12 14 LIST OF TABLES Treatment schedule for combined hydrocortisone and cis-Pt(II) therapy in Swiss mice bearing 8-180. . . . . . Treatment schedule for combined zymosan and cis-Pt(II) therapy in BALB/c mice bearing 8-180. . . . . .‘. . . . . Treatment schedule for combined zymosan and cis-Pt(II) therapy in BALB/c mice bearing 8-180. Timing study 1 . . Treatment schedule for combined zymosan and cis-Pt(II) therapy in BALB/c mice bearing 8-180. Timing study 2 . . Treatment schedule for combined zymosan and‘gig-Pt(II) therapy in BALB/c mice bearing 8-180. Timing study 3 . . Treatment schedule for evaluation of humoral antibody responses in BALB/c mice treated with zymosan and cis-Pt(II) but not hearing tumors . . . . . ... . . . . . Treatment schedule for evaluation of humoral antibody responses in BALB/c mice bearing 8-180 tumors and treated with zymosan and cis-Pt(II) . . . . . . . . . . . . . . . Treatment schedule for evaluation of cell-mediated immune responses in BALB/c mice treated with zymosan and cis-Pt(II) but not bearing tumors . . . . . . . . . . Treatment schedule for evaluation of cell-mediated immune responses in BALB/c mice bearing 8-180 tumors and treated with zymosan and cis-Pt(II) . . . . . . . . . Results of combined cis—Pt(II) and hydrocortisone therapy in Swiss mice bearing 8-180 . . . . . . . . . . . Results of combined zymosan and cis-Pt(II) therapy in Rule mice bearing 8-180 0 I O O C O O O O O O O O O O 0 Results of combined zymosan and cis-Pt(II) therapy in BALB/c mice bearing 8-180. Timing study 1. . . . . . . . Results of combined zymosan and cis-Pt(II) therapy in BALB/c mice bearing 8-180. Timing study 2. . . . . . . . Results of combined zymosan and cis-Pt(II) therapy in BALB/c mice bearing 8—180. Timing study 3. . . . . . . . vi Page 32 33 35 36 38 39 41 42 43 45 46 48 50 52 Table Page 15 Results of evaluation of humoral antibody responses in BALB/c mice treated with zymosan and cis-Pt(II), but not hearing tmrs. O O I O O Q C O C O O O I C O O O O O O 58 16 Results of evaluation of humoral antibody response in BALB/ c mice bearing 8-180 and treated with zymosan and Eg-Pt(II) O O O O O O O O O O O O O O O O O O O O O I O O O 59 17 Results of evaluation of cell-mediated imune responses in BALB/c mice treated with zymosan and cis-Pt(II), but mt baring wrei O O O O O O O O C C O O O O O O O C O O 62 18 Results of evaluation of cell-mediated immune responses in BALB/c mice bearing 8-180 and treated with zymosan deiB-Pt(II)seeeeeeseeeeoo000000ess 63 vii LIST OF FIGURES Figure Page 1 Growth of 8-180 in BALB/c mice treated with combinations of zymosan and cis-Pt(II). Numbers of surviving mice are indicated by the number at each point . . . . . . . . . 54 2 Changes in body weight in BALB/c mice bearing 8-180 and treated with combinations of zymosan and cis-Pt(II) . . . . 56 3 Thymus from BALB/c mouse treated with zymosan and hear- ing a regressing tumor. Note the wide cortical region (C) composed of lymphocytes and the more pale staining medulla CM) composed of epithelial cells. . . . . . . . . . 65 4 Thymus from BALB/c mouse treated with gig-Pt(II) and bearing a progressively growing tumor. The thymus is atrophic and has lost its normal architecture. Note absence of medulla. . . . . . . . . . . . . . . . . . . . . 66 S Axillary lymph node from BALB/c mouse treated with zymosan plus cis-Pt(II). There is marked hyperplasia of both the cortex and medulla. . . . . . . . . . . . . . . 67 6 Higher magnification of the medulla of the lymph node in Figure 5. Note macrophages (blunt arrow) and lymphocytes (arrow) in medullary sinuses. . . . . . . . . . 68 7 Axillary lymph node from BALB/c mouse treated with saline and containing metastatic tumor cells. Note sheets of tumor cells compressing cortical lymphocytes toward the periphery. . . . . . . . . . .... . . . . . . . 69 8 Higher magnification of a portion of the lymph node in Figure 7. Note tumor cells compressing and infiltrating cortical lymphocytes. . . . . . . . . . . . . . . . . . . . 70 9 Regressing tumor from BALB/c mouse treated with zymosan. There is proliferation of a fibrous capsule (F) on the periphery and marked lymphocytic infiltration (L) . . . . 71 11) Higher magnification of a portion of the tumor in Figure 9. Note fibrous tissue (F) on periphery and lymphocytes (L) infiltrating tumor cells. . . . . . . . . . . . . . . . 72 viii Figure Page 11 Progressively growing tumor from BALB/c mouse treated with gig-Pt(II). It is characterized by sheets of pro- liferating neoplastic cells, numerous mitotic figures (arrows) and multifocal areas of necrosis (N) . . . . . . . 73 12 Higher magnification of a portion of the tumor in Figure 11. Note area of necrosis (N) surrounded by pleomorphic, neoplastic cells . . . . . . . . . . . . . . . 74 INTRODUCTION The role of host defense mechanisms in promoting the regression of tumors in animals treated with gig-Dichlorodiammineplatinum(II) [gig-Pt(II)] has recently become of great interest. Rosenberg and vanCamp (1970) reported that treatment with gig-Pt(II) caused regres- 7 sions in 63-100! of Swiss mice bearing advanced Sarcoma 180 (3-180). Generally, 8-180 will regress spontaneously in 0-252 of the Swiss mice in which it is implanted (Mihich, 1969). This suggests that there are marked histocompatibility differences between the tumor and the host and that host responses against the tumor are vigorous. It has been observed that various cancer chemotherapeutic agents are less effective when the tumor and host are histocompatible (Bradner and Pindell, 1965). In addition it has also been noted that there is a reduction in chemotherapeutic efficacy if host responses are purposely suppressed (Ferrer and Mihich, 1967; Tarnowski and Stock, 1957; Mihich and Nichol, 1959; Martin at aZ., 1962). On the other hand, numerous investigators have reported that the efficacy of cancer chemotherapeutic agents can be enhanced by using specific or nonspecific immunostimulation in combination with the chemical agents (Martin et al., 1962; Martin et al., 1964; Mathé, 1970; Kalpaktsoglou and Good, 1970; Martin et al., 1970; Fugmann at al., 1970; Esber at al., 1972; Pass and Fefer, 1972; Pearson at al., 1972). 2 Since-gig-Pt(II) is a relatively new drug and not related chemically to any cancer chemotherapeutic agents presently in use, a series of experiments was performed to ascertain what effect suppression or stimulation of host defenses would have on its efficacy as a cancer chemotherapeutic agent. LITERATURE REVIEW Host Defenses Against Neoplasia It has been postulated that the development of neoplasms is due to a breakdown in immunologic surveillance (React, 1970). Although this hypothesis must be kept in perspective because of the many factors involved in the development of neoplasia, it is of interest to note that cancer in man and animals develops in the extremes of life when the immune system is maturing or when it is weakened by thymic atrophy. In concert with this observation is the fact that the incidence of neoplastic diseases is documented as being 100 times greater in immuno- logically deficient or immunosuppressed indivuals than in the normal population (Fahey, 1971). Data from animal studies are equally convincing since it has been shown that animals which have undergone neonatal thymectomy, radiation treatment, treatment with antilymphocyte serum or treatment with immunosuppressive drugs have an increased incidence of neoplastic diseases and are more susceptible to the implantation of tumors (McMichael, 1967; Allison, 1970; 13911 and Kinlen, 1970; Klein, 1970; Law, 1970; Fahey, 1971; Kreider at al., 1971). Contrasted to this, both specific and nonspecific stimulation of host defenses have, in some instances, had a marked influence on the induction time, growth and persistence of experimental tumors as well as promoting reduction of tumor mass and prolongation of remission time in spontaneous neoplasms (Klein, 1969; Alexander, 1970; Maths, 4 1970; Bernstein at al., 1971; Humphrey et aZ., 1971a; Humphrey at al., 1971b; Morton at al., 1971). One could also assume that the reported spontaneous regression of autochthonous tumors in man and animals may also be immunologically mediated (Sumner and Foraker, 1960; Everson and Cole, 1966; Refer et aZ., 1968; Bell, 1970; Kreider et al., 1971). In accordance with the hypothesis that imunologic factors play a role in mediating the regression of neoplasms. Doniach et al. (1958) and Klein (1969) reported that two of the most antigenic tumors known. Burkett's lymphoma and choriocarcinoma, are also extremely responsive to chemotherapy and immunotherapy. The revitalization of interest in tumor immunology came in 1953 when the results of a series of experiments were reported by Foley. Prior to this time the lack of successful immunization attempts and the paucity of standardized laboratory animals led manyinvestigators to believe that nothing fruitful could be gained from studying immunologic responses to tumors. Foley, however, found that if he ligated iso- grafted, methylcholanthrene-induced sarcomas in C3meice he could induce necrosis in the tumors. More importantly, subsequent rechallenge of the mice with live pieces of the same tumor resulted in rejection of the implants. Since these animals did not reject implants of other tumors, it was apparent that a tumor specific immunity had developed. In recent years tumor specific transplantation antigens (TSIA) have been demonstrated in a wide array of neoplasms including those induced by chemicals, physical agents and both DNA and RNA oncogenic viruses. These TSIA are antigens which are capable of inducing rejection responses in syngeneic hosts in a preimmunizationrviable cell challenge type of experiment (Klein, 1969). For the most part S virally induced TSTA are common to all tumors produced by the same virus regardless of morphological appearance or strain of animal in which it arises. With DNA viruses, e.g., Polyoma, SV40 and Shope papilloma, the TSTA are virus related but distinct from virus specific antigens since they can be demonstrated after the infecting virus is no longer present. Evidence for separate virus related TSTA versus virally specific antigens is lacking in RNA viruses, however, since tumor cells continue to shed viral particles (Pessens, 1970; Law, 1969). The tumors induced by chemical or physical agents have TSTA which are tumor specific but not carcinogen specific. Thus there is virtually no cross-reactivity even when tumors are morphologically identical and arise in a single animal. This difference in specificity between the TSTA of chemically induced and virally induced tumors may, however, be more spurious than real (Klein, 1969). Recently Horton et a2. (1969) demonstrated antigens which were specific for individual spontaneous mammary tumors. These tumors arose in mice which carried memory tumor virus and were presumed to be virally induced. Their antigenicity and growth behavior patterns, however, mimicked chemically induced tumors. 0n the other hand, G antigen (Gross virus) has been detected in methyl- cholanthrene induced sarcomas which, as Old and Boyse (1965) pointed out, considerably weakens the argument that viruses are not involved in chemically induced tumor systems. Although these TSTA are capable of immunizing hosts against subse- quent implantations of the same or similar tumors, their overall ability to promote effective immunologic responses against these tumors is questionable. Law (1969) summarized their biological activities by declaring that those antigens which are located on the surface of tumor cells, e.g., mammary tumor virus and murine sarcoma 6 virus, are of the histocompatibility type and are ostensibly of potential importance in relation to contact inhibition, cell division and immuno- logic reactions. Those antigens which are located intracellularly and which have been demonstrated by complement fixation or immunofluorescence, however, are of questionable significance as defense mechanisms. Many neoplasms of man have also been shown to have what appears to be tumor-related antigens. However, some of these antigens can be associated with nonrneoplastic fetal cells as well. Tumors in which antigenicity can be demonstrated are: Burkett's lymphoma, choriocarcinoma, nasopharyngeal carcinoma, melanoma, neuroblastoma, colonic carcinomas and osteogenic sarcoma. Some of these antigens are intracytoplasmic, while others are bound to cell membranes (Klein, 1969; Fairly, 1970; Pessens, 1970; Thompson et aZ., 1969; Morton et al., 1971; Oettgen at al., 1971). In most of these cases cytotoxic antibodies are found in the sera of patients bearing these tumors. In many instances the antibodies cross-react with similar tumor cells from other patients indicating that there may be common antigenicity between certain tumor types (Hellstrom et aZ., 1968; Pessens, 1970; Morton et aZ., 1971). In addition, lymphocytes from patients bearing these tumors have been shown to be cytotoxic for their tumors as well as for neoplastic cells of patients bearing tumors of the same class (Hellstrom at al., 1968; Pessens, 1970; Hellstrom, 1971; Gettgen at al., 1971). Aside from tumor—specific antigens, there are other indications «of active host responses against tumors. One of the most obvious is the fact that regional lymph nodes draining neoplastic sites become hyperplastic resembling the reactions seen in nodes responding to non- ne0p1astic antigenic stimuli. This hyperplastic response is found in Patients bearing primary tumors without metastatic lesions. It has 7 also been observed that the presence of lymphocytic infiltrates in neoplasms is associated with a more favorable prognosis (Alexander et aZ., 1966). Another indication of host reactivity is the development of cone comitant immunity. This type of immunity is defined as the ability of the immunologic resistance of the host to destroy small implants of tumors even though large tumors, and in some cases metastatic lesions, are growing progressively in the host (Klein, 1969). The importance of concomitant immunity was emphasized by the independent experiments of Gershon et a1. (1968) and Crile and Deodhar (1971). Gershon and his co~workers found that a normally nonmetastasizing lymphoma of hamsters did metastasize if the primary tumor was removed. Their experiment indicated that removal of the primary tumor 7 days after transplantation led to a rapid decrease in immunity, the pro- duction of enhancing antibodies and the appearance of metastatic nodules. The metastatic lesions were thought to be derived from pre- existing tumor cells in the blood and lymphoid tissues. Crile and Deodhar (1971) utilized two different tumor systems in their experiments studying concomitant immunity. In one experiment, 8-180 was implanted in the legs of mice. They found that if the tumor bearing leg was amputated 10 days after implantation, 60% of the animals veers susceptible to tumor challenge 4 days later. If, on the other hand, the tumors were irradiated but not removed, the animals were immune to challenge for 3 weeks. The investigators suggested that the irradiated tumor cells were capable of absorbing enhancement antibody, thus maintaining the state of concomitant immunity. They also suggested that the continued antigenic stimulation produced by the irradiated tumor cells may have helped to sustain cell-mediated immunity. 8 In a second experiment, the same workers found that the incidence of pulmonary metastasis of Lewis T241 fibrosarcoma in mice was higher when the foot bearing the primary tumor was amputated. The incidence of metastasis was reduced when the tumor bearing foot was irradiated but not amputated. It was suggested that the release of tumor antigens from the irradiated tumor may have increased immunity which in turn was responsible for destroying metastatic progenitors. It was previously mentioned that both humoral and cell mediated immune responses have been observed in a vast array of neoplastic diseases of both animals and man. Fairly (1970) suggested that circu- lating antibodies, even if cytotoxic, have little effect on solid tumors but may prevent blood borne metastasis. This postulation is suggested by the fact that many neoplasms metastasize to local lymph nodes where they remain for some time before spreading via the circula- tory system. This postulate might also explain why neoplasms metastasize by local lymphatics when antibodies are present but via the blood when they are absent. Alexander (1970) summarized the effector mechanisms which are recognized in immunological reactions against neoplasms in viva. He suggested that humoral antibodies, probably in conjunction with complement, are capable of destroying tumor cells, assuming they have adequate access to them. In concert with these are cytotoxic lympho- cytes which infiltrate the neoplasms like a homograft and macrophages which may be coated with cytophilic antibody which destroy tumor cells by direct contact. 9 Utilization of Sarcoma 180 in Cancer Chemotherapy and Immunotherapy Sarcoma 180 (Crocker Sarcoma-180, Crocker tumor 180, Mouse Sarcoma 180) arose spontaneously in the right axillary region of a white male mouse necropsied in 1914. It was initially described as a carcinoma. However, the tissue of origin was not cited. With serial transplanta- tion, the histologic pattern was altered so that, by 1919, the tumor was described as a sarcoma. Stewart et al. (1956) suggested that the tumor be classified as undifferentiated because of the anaplastic appearance of the cells and lack of any specific histologic pattern. Sarcoma 180 has little strain specificity and, for the most part, grows well in a number of mouse strains. For this reason it is often referred to as a "nonspecific" tumor. The untreated tumor kills its host in 4 to 5 weeks. Generally the rate of spontaneous regression varies from 5-25Z (Stewart at al., 1956; Mihich, 1969; Sellei et al., 1970). Snell et al. (1953) reported that inbred strains of mice carrying the H-Zd histocompatibility locus, e.g., BALB, BALB/c, DEA/2, had fewer spontaneous regressions than those carrying other H92 genes. They explained this on the basis that the tumor may have arisen in mice of the genotype H-Zd/H-Zd. The 3-2 locus is one of a number of loci determining suscepti- bility or resistance to transplants. It is a particularly strong locus, however, since when tumor and host differ at this locus a powerful deterrent against progressive tumor growth is evoked. The fact that the proposed H—Zd origin still prevails in 8—180 after many years of transplantation in innumerable host suggests that the "non- specific" nature of the tumor may have occurred by an increase in virulence which allowed it to overpower histocompatibility differences 10 rather than by attenuation or modulation of histocompatibility genes (Snell at al., 1953). Due to its lack of specificity, 8-180 has been used as a cancer chemotherapy screening tumor for many years. It has the disadvantage of being only moderately sensitive to chemotherapeutic agents. Complete regressions are usually attained in a low percentage of cases and mostly by using doses very close to toxic levels (Issekutz, 1969). Sellei et a1. (1970) reported that nitrogen mustard, Degranol, Mitomen and Sarcolysin, which are normally high in effectiveness in other tumors, had only moderate effectiveness against 8-180 and that they produced excessive weight loss, indicating toxicity. Iuyleran, Mannagranol and Colchicine were ineffective against the tumor. Azaserine, which was very effective in inhibiting this tumor, failed to have similar favor- able effects on other experimental tumors and was found to be of no value in clinical practice. Although Sellei stated that an effect by Mercaptopurine cannot be detected against 8-180, Ferrer and Mihich (1967) found that 6~mercaptopurine (6-H?) could promote cures in 42-52% of animals. It appears that a crucial issue in the success or failure of chemotherapy against 8-180 is the strain of mice in which it is implanted. Bradner and Pindell (1965) found that, by implanting the tumor in DEA/2 mice, which are compatible with 8-180 at the npzd locus, they could reduce the chemotherapeutic efficacy of 6-HT, Actinogan and Phleomycin. The same drugs were effective in promoting regressions of 8-180 when it was implanted in Swiss Ha/ICR.mdce, a noncompetible host. Sarcoma 180 does not lend itself to sophisticated.immunologic study because of its lack of specificity. It does appear that its growth and ultimate disposition, however, are regulated by host 11 responses. 01d at al. (1961) found that phagocytic activity increased and splenomegaly occurred in Swiss mice in which 8-180 was implanted. These changes occurred relatively early but disappeared prior to the death of the animal. The same authors noted similar enhanced phagocytic activity and splenomegaly when they injected the supernatant from spleen homogenates taken from animals bearing 8-180. Although they could not induce tumors with this material, the effect on the reticula- endothelial system (RES) suggested that a transmissible agent might be associated with this tumor. Mihich and Nichol (1959) and.Mihich (1962) found that spontaneous regression rates of 8-180 increased markedly.in mice fed a diet deficient in vitamin B6. They attributed this effect to an altera- tion in the production of humoral enhancement antibodies. Similar increases in spontaneous regressions were found by Old at al. (1962), Ferrer (1968) and Ferrer.and Mihich (1968) when mice were splenectomized prior to tumor implantation. Since the spleen is the major contributor of humoral antibody in the rodent, the authors concluded that splenectomy served to decrease the quantity of enhancement antibody formed. These same investigators noted that the efficacy of chemotherapeutic agents and nonspecific immunostimulants was enhanced when prior splenectomies were performed. Ferrer and Mihich (1968) found that the incidence of tumor regressions in animals fed vitamin B6 deficient diets or treated with 6-MP or Kethoxal-Bis (Thiosemicarbazone) (KTS) was greatly reduced by active immunization with frozen-thawed tumor cells or by passive transfer of specific hyperimmune serum. They attributed this to the development of an enhancement phenomenon. While splenectomies enhanced the efficiency of various chemothera- peutic agents, neonatal thymectomy was found to reduce the 12 chemotherapeutic effects of 6-H? and KTS (Ferrer and Mihich, 1967). It was concluded that removing the thymus caused cellemediated immune responses to be greatly impaired. Besides the use of chemotherapeutic agents, numerous investigators have studied the effects of nonspecific immunostimulation in promoting the regression of 8-180. Certain agents capable of stimulating the RES, such as Bacillus Calmette Guerin (01d at aZ., 1962), Zymosan (Bradner and Pindell, 1965), Bacillus pertussis lipopolysaccharide CMalkiel and Hargis, 1961), lipopolysaccharide from.Proteus vulgaris (Mizuno at al., 1963), Lentinan (Needs and Chihara, 1971) and various other polysaccharides from plants and yeasts (Fukuoka at aZ., 1968; Kamasuka st aZ., 1968; Suzuki et al., 1969; Komatsu at al., 1969; Tanaka, 1967) were either capable of inhibiting the growth of 8-180 or were capable of promoting tumor regressions. Lemperle (1966) demonstrated that the combination of imunizing mice with killed 8-180 tumor cells and stimulating the RES with restin, a crystalized lipid fraction of shark 1iver,or glucan, the active polysaccharide fraction of zymosan, rendered mice significantly resistant to the growth of 8-180. The Use of Platinum Compounds in Cancer Chemotherapy_ In 1965, Rosenberg et al. reported that Escherichia coli underwent filamentous growth (elongation) but would not divide when under the influence of electric current delivered by platinum electrodes. After a number of experiments, it was disclosed that several new species of platinum salts were generated by the electrical current. Further investigation indicated that many of these salts were capable of inducing bacterial elongation when added to their growth medium 13 (Rosenberg at al., 1967). In 1969, Rosenberg et al. reported that one of these salts, gigrDichlorodiammineplatinum(II) [gingt(II)] was capable of retarding the growth of 8-180 and mouse leukemia L1210. An additional report by Rosenberg and VanCamp (1970) indicated that delayed gigrPt(II) treatment was capable of promoting regressions of 8-180 in 63-100! of the animals treated. Since the original reports of Rosenberg, oncostatic properties of Singt(II) have been demonstrated against the Ehrlich's Ascites tumor (Howle and Gale, 1970), L1210 leukemia when combined with other drugs (Speer st aZ., 1971; Vanditti, 1971; Sirica et al., 1971; Woodman at al., 1971), Rous Sarcoma (Hinz, 1970), Dunning ascitic leukemia and walker 256 carcinosarcoma (Kociba et al., 1970), chemi- cally induced myeloid and lymphatic leukemias (Leonard et aZ., 1971), mouse reticulum cell sarcoma (Talley, 1970), chemically induced rat mammary carcinoma (welsch, 1971) and a variety of other tumor systems such as Lewis Lung carcinoma, B-l6 melanocarcinoma, P388 leukemia and ADJ-PC6A plasma cell tumor (Rosenberg, 1971). The drug is presently in the early phases of testing against human neoplasms, and its activities against some of these have been reported (Speer at aZ., 1971; Talley et al., 1972). The toxic properties associated with gig:Pt(II) are particularly pronounced in tissues having rapidly dividing cellular constituents, e.g., gastrointestinal tract, lymphoid organs, bone marrow. Kociba and Sleight (1971) observed that, although erythrocyte numbers, packed cell volume and hemoglobin concentration remained normal, there was panleukocytopenia, reticulocytopenia and depressed numbers of platelets in rats 2 to 4 days after treatment. There were also decreases in the levels of serum proteins. Thompson and Gale (1970, 1971) reported 14 depression in hematopoiesis as well as a reticulocytopenia and lympho- penia in rats. Histologically, rats and mice treated with gigrPt(II) were characterized by thymic and splenic atrophy, denudation of intestinal epithelium and acute nephrosis (Kociba et al., 1970; Thompson and Gale, 1970, 1971; Toth-Allen, 1970; Leonard et aZ., 1971). Since the organs most sensitive to the cytotoxic properties of EggrPt(II) are those with rapidly dividing cells, the ability of the drug to suppress immune responses has been studied. Richardson (1969) observed that gingt(II) suppressed the formation of antibodies in dispersed spleen cells stimulated in vitro with Brucella abortus antigen. In comparing it to 4 commonly used immunosuppressive drugs, cyclo- phosphamide, metotrexate, 6-H? and puromycin, she found that the con! centrations of gig:Pt(II) required to suppress antibody formation were lower than any of the latter drugs. Khan and Hill (1971) and Howle et a1. (1971) found it to be a potent inhibitor of phytohemagglutinin- induced mitogenesis of human lymphocytes. Berenbaum (1971) and Khan and Hill (1971) reported that 10 mg per Kg doses of gigfrt(II) reduced the number of antibody forming cells in the spleens of mice immunized with sheep erythrocytes. Khan and Hill observed significant reduction when the drug was administered at any time between 2 days before and 2 days after antigenic stimulation. They considered the drug to be a potent immunosuppressive. Berenbaum, on the other hand, noted that maximum immunosuppression was attained 2 days after antigenic stimula- tion and that administration of the drug prior to or concurrent with the antigen.was ineffective. In comparing sis-Pt(II) to cyclophosphamide, he concluded that gigrPt(II) was a weak.immunosuppressor at thera- peutic levels. 15 The platinum compound has also been shown to have an ability to suppress cell-mediated immunity. Khan and Hill (1971, 1972) reported that it inhibited graft versus host responses and prolonged the life of skin allografts in mice. It has been proposed that gigrPt(II) mimicks the alkalating agents in regard to its antitumor activities. Connors (1971) found that it was active against 2 tumors which are sensitive to an alkylating agent. melphalan. 0n the other hand, its antitumor activity was markedly diminished against a tumor resistant to alkylating agents, e.g., Walker R. tumor. Similarities were also seen in the ability of cysteine and other nucleOphiles to reduce the toxicity of both alkylating agents and the platinum compound. Harder and Rosenberg (1970) and Howls and Gale (1970) found that Eingt(II) inhibited DNA.synthesis primarily and RNA and protein synthe- sis secondarily. The effects on DNA were considered to be direct since the synthesis of DNA precursors and the ability of the precursors to enter cells were not impaired (Harder and Rosenberg, 1970). Roberts and Pascoe (1972) compared the mechanisms of action of EggrPt(II) and mustard gas. The results of their experiments indicated that both compounds selectively inhibited DNA synthesis while showing no effects on gross RNA and protein synthesis. They also indicated that the effect on DNA.was due to a direct reaction rather than inter- ference with the enzymes involved in DNA synthesis. The authors pro- posed that, like mustard gas, gingt(II) produced an inter-strand cross-linking reaction in the DNA molecule. They suggested that likely regions for this cross-linking to occur would be between the 6-amino groups of adenosine in an adenosine phosphate thymidine sequence, the l6 2~amino groups of guanine in the narrow groove and the 6-amino groups of cytosine in the wide groove of DNA in a cytidine phosphate guanosine sequence. Although the available evidence suggests that cross-linking of complimentary strands of DNA is a mechanism.by which gigrPt(II) exerts its biological activity, it still remains to be proven that this is the principal way in.which it produces cytotoxicity. The Properties of_gymosan and its Use as an Immunostimulant Zymosan is an insoluble polysaccharide of yeast cell walls. Chemically it is composed of approximately 50-602 glucan, 16-202 mannan, 13-171 protein, 6-71 lipid, 3.0-3.5! ash and less than 11 chitin (Fitzpatrick and DiCarlo, 1964). It was concluded that the active component of zymosan is glucan and that its functions may depend upon the specific configurations and spatial arrangements of its sugar residues (Fitzpatrick and DeCarlo, 1964). In concert with this hypothe- sis were the findings of Sakai et a1. (1968), who found that glucans composed of beta-(1-3)-linked D-glucose residues were more active against neoplasms than those with alpha linkages. Diller et a1. (1963) found hydroglucan superior to zymosan in promoting regressions of 8-180 and 8-37. Suzuki et al. (1969), on the other hand, demonstrated that the oncostatic properties of mannan fractions from Saccharamyces cerevisiae were superior to the glucan fractions. The administration of zymosan causes marked reticuloendothelial (R.E.) activity. ‘Machado et a1. (1968) observed that low doses, i.e., 2 to 4 mg per Kg per day for 6 days, caused marked Kupffer cell and reticuloendothelial cell hyperplasia in the liver. The R.E. cells proliferated and formed nodules. There were also increases in the 17 numbers of lymphocytes, neutrophils and monocytes. The spleen was characterized by marked proliferation of R.E. cells and macrophages in the red pulp and marginal zones of the lymphoid follicles. 0ccasionally, multinucleated giant cells were also observed. Zymosan inactivates the third component of complement (C'3) and in so doing causes reduction in the levels of properdin. Properdin is a heat labile beta globulin in serum which is important in natural resistance. In combination with complement it enhanced phagocytosis, inactivates certain viruses, e.g., Newcastle virus, is bactericidal to some gram negative bacteria, e.g., Shigella dysenteriae and Bacillus subtilis; lyses certain unsensitized but abnormal erythrocytes, e.g., in paroxysmal nocturnal hemoglobinuria; and protects against the lethal effects of whole body irradiation (wardlaw and Pillemer, 1956; Fitz- patrick and DeCarlo, 1964). The reduction of properdin levels by zymosan are dose dependent and apparently transient in nature. Pillemer and Ross (1955) found that the injection of low doses of zymosan, i.e., 5 mg per Kg, produced a precipitous drop in properdin levels within 1 to 2 hours after injection followed in 2 to 14 days by a marked rise which increased to 200 to 300% above that of control levels. At high doses, i.e., 25 to 125 mg per Kg, the properdin levels decreased to levels lower than that found with the 5 mg per Kg dose and within 6 to 10 days were 25% lower than controls. Zymosan was reported to enhance hemolysin titers in rats when given 48 hours before, at the same time as or 48 hours after antigen administration (Cutler, 1959). This adjuvant effect was most pronounced when the zymosan was administered in low doses and 48 hours following antigen administration. 18 Blattberg (1957) reported the production of heat stable agglutinins in response to zymosan in rabbits. The best results were obtained when it was combined with adjuvants since zymosan alone elicited very little antibody response. He also observed that it was capable of increasing the bactericidal activity of rabbit ears for Escherichia coli B. It was proposed that zymosan and E. coli B were related antigenically and that antibodies produced against zymosan might cross-react with similar antigenic determinants found on the bacteria. In light of the experi- ments cited by Pillemer and Ross (1955), however, Blattberg may have been observing an effect due to enhanced properdin levels rather than a cross-reacting antibody response. Zymosan has a pronounced stimulatory effect on the reticuloendo- thelial system (RES). Cutler (1960) demonstrated that, within 48 hours after an intravenous injection of 3 mg, the phagocytic index in rats rose to 6 times that of normal values. This stimulation persisted for 6 weeks. Shinichiro and Shinoki (1968) observed that zymosan was not only capable of causing increases in the phagocytic index, but also in the levels of subcutaneous pre-histiocytes. The activity of zymosan against neoplasms, both by itself and in combination with chemotherapeutic agents, has been studied by numerous investigators. Modica (1958) observed that 1 mg administered before, during or after the application of 3,4-benzopyrene to rats prolonged the latent period of tumor growth and the survival time of the host. Larger doses, however, hastened tumor appearance and shortened survival time. Bradner et al. (1958) studied the effects of low and high doses of zymosan against 8-180 in Swiss mice. At doses between 10 and 160 mg per Kg given on day 1 of tumor growth, they found an average survival rate of 62 over the controls. Doses below and above these levels 19 yielded survival rates comparable to untreated controls. If 20 mg per Kg were administered on the first day of tumor growth, 50% regressions were obtained. Multiple dose schedules were most satisfactory when low doses were administered, i.e., 5 mg or 20 mg per Kg. These multiple doses, however, were no more effective than equivalent amounts of zymosan given in a single dose. Generally, large amounts, i.e., 320 mg per Kg, appeared to be deleterious, and large doses following small doses abrogated antitumor activity. 01d at al. (1960) found that 1 mg of zymosan injected intravenously in Swiss mice 1 week prior to implantation of 8-180 yielded better protection than zymosan injected on the day of tumor challenge. Diller et‘al. (1963) compared zymosan and hydroglucan. ‘with these products there were regressions in 90-95% of animals in which Sarcoma-37 or 8-180 had been implanted and 832 of animals in which Krebs-2 carcinoma had been implanted. They found hydroglucan to be superior to zymosan and suggested that the intravenous route of administration was superior to any others. Shinichiro and Shinoki (1968) observed that zymosan.was effective in promoting inhibition of rat Ascites Hepatoma (AH-130). Since the most effective oncostatic results were found when it was administered 3 days prior to tumor implantation, they concluded that the effect of zymosan was mediated via enhanced host responses rather than a direct effect on the tumor. Bradner and Pindell (1965) compared the ability of zymosan, 6-HT, actinogan, phleomycin and a pyridoxine-deficient diet to promote regression in 8-180 in Swiss Ha/ICR or DEA/2 mice. They found that all treatments were capable of increasing the incidence of recovery in the Swiss mice. However, only zymosan given intravenously at 50 mg 20 per Kg increased recovery from 8-180 when the tumor was grown in DEA/2 mice. Zymosan has also been shown to potentiate the antitumor effects of various chemotherapeutic agents and/or surgery. Martin et a1. (1962) reported that the efficacy of both 64M? and cyclophosphamide against Adenocarcinoma R.C. was enhanced when zymosan was used in combination with the two drugs. It was also observed that a combina- tion of zymosan and 6-H? was far superior to either compound alone in promoting regressions of 8-180. Again the reduction of tumor size by surgery potentiated the success of combination therapy even more. Martin et al. (1964) found that the combination of zymosan, surgery and cyclophosphamide was significantly more effective in reducing the recurrence of spontaneous mammary tumors than surgery or cyclophosphamide alone or in combination. Martin et al. (1970) observed that zymosan in combination with 4 chemotherapeutic agents, i.e., Streptomigran, cyclophosphamide and Mitomycin C, effected local cures in 541 of the animals treated. Local cures were defined as a lack of tumor recur- rence. The 4 chemotherapeutic agents plus surgery, on the other hand, effected local cures in only 15-39%. The Use of Steroid Hormones as Immunosgppressive Bragg Hydrocortisone (HC) is a steroid hormone with glucocorticoid activity. The effects of this drug are basically the same as cortisone, but HC is more potent. There are numerous physiological activities attributed to HC. However, for the purpose of this manuscript, the immunosuppressive and anti-inflammatory effects will be emphasized. Kass and Finland (1953) reported atrophy of lymphoid tissues and a reduced concentration of pentose nucleic acids after the 21 administration of cortisone and HC. They also reported that, after allotypic erythrocytes were injected into rabbits treated with HC, they were observed in.lymph node macrophages for as long as 10 days. In untreated rabbits the erythrocytes were generally eliminated within 48 hours. Thus, it was suggested that the immunosuppressive effects of cortisone and HC may be due to an interference with lymphocyte nucleic acid metabolism as well as interference with the mechanism by which cells dispose of phagocytized material. Snell (1962) reported that low doses of HC stimulated the phago- cytic index but that high doses blocked phagocytosis by interfering with the mechanism by which phagocytized material is eliminated. Lurie (1962) also reported depressed RES activity after treatment with H6. The ability of HC to suppress antibody formation mimics irradia- tion (Taliaferro, 1957; Berenbaum, 1967). It was found that maximum antibody suppression could be obtained in the rat when the drug was administered prior to and/or on the same day as antigen administration. It was also reported that the drug suppressed anamnestic responses but had no effect on the biologic half life of antibodies in passively immunized animals. Finally, hemolysin production could be restored after cortisone treatment by the administration of thymus or spleen cells. Schlesinger (1967) reviewed the effects of corticosteroid hormones on the antigenicity of tissue. Allogeneic skin grafts of mice and rats treated with HC were rejected much later than those from untreated controls. It was also reported that the involution of the thymus in HC treated mice was accompanied by loss of thymus-distinctive serological properties. This loss is similar to that observed in the involuted thymus of tumor-bearing mice. As an example, the reactivity of thymus 22 cells of A and SJL/J strains of mice to TL antibody disappeared within a day after the subcutaneous administration of 1 mg of HC, and no anti- body was detected for 6 days. Concomitantly, the thymus cells also lost their sensitivity to the cytotoxic effects of guinea pig serum, but their reactivity to H92 antibodies was unaltered. After the 6-day period, the thymus cells returned to normal. It was suggested that these antigenic changes observed in the thymus of tumor-bearing and HC—treated animals might be due to: 1) a selective detrimental effect on thymus cells possessing distinctive serological properties, leaving a cell population devoid of these properties, 2) inhibition of the induction of thymus-distinctive properties in stem cells entering the thymus, or 3) an effect on the genetic regulatory mechanism of thymus cells. This latter possibility is based on the fact that HC inhibits purine nucleotide and protein synthesis in the thymus. Corticosteroids have been reported to render animals more susceptible to oncogenesis. This is believed to be due to their ability to depress host responses. The onset of regressions of tumors initiated with fibroma virus in rabbits was delayed in animals treated with prednisone, and antibodies to fibroma virus were delayed in appearance and at reduced titers (Bergman at al., 1962). Hurst (1964) reported that cortisone enhanced the growth of Shope fibroma but did not greatly inhibit the development of antibodies to the virus. The tumors in treated animals grew one week longer and were twice the size of the tumors in untreated animals. Methylprednisolone has been shown to reduce the regression rates of Shope papilloma in rabbits and prolong the period of tumor growth in rats (McMichael, 1967; Kreider at aZ., 1971). Shachat et al. (1968) observed that mice pretreated with 23 cortisone had an increased susceptibility to oncogenesis with‘Maloney murine sarcoma virus. The administration of corticosteroids in conjunction with cancer chemotherapeutic agents and/or nonspecific immunostimulants has been reported to reduce the therapeutic efficacy of the latter. Hydro- cortisone blocked the anti-tumor activity of zymosan in promoting regressions of S-180 in Swiss mice (Bradner and Clarke, 1959). Corti- sone has been reported to reduce the number of regressions of 5-180 in animals fed a diet deficient in vitamin 36 (Stoerck, 1954; Mihich and Nichol, 1959), reduce the efficacy of 6-MP, Puromycin, cyclophosphamide, surgery and zymosan against various transplantable tumors (Tarnowski and Stock, 1957; martin et al., 1962) and decrease the number of local cures of spontaneous murine mammary carcinomas when used in conjunc- tionuwith Streptonigrin, Thioguanine, cyclophosphamide, Mitomycin C and Actinomycin D (Fugmann et aZ., 1970). MATERIALS AND METHODS General Plan A three-phase study was undertaken to investigate the role of host defenses in promoting tumor regression in animals treated with gingt(II). Phase one consisted of treating Swiss mice bearing 8-180 with SiEfPt(II) and hydrocortisone acetate, an immunosuppressive drug. Phase two consisted of treating BALB/c mice with gigrPt(II) and zymosan, an immunostimulant. Phase three consisted of evaluating the integrity of host defenses in BALB/c mice treated with a combination of Egngt(II) and zymosan. The agarose—slide technique was used to evaluate humoral antibody production, and the ability to reject skin allografts was used to evaluate cell-mediated immune responses. Some of these animals were killed on day 21 of the experiment, and selected tissues were evaluated histologically. Source of Animals Female Swiss mice, 8 weeks of age, were procured from a commercial supplier.* Female BALB/c mice, 8 weeks of age, and EBA/2 female mice, 8 weeks of age, were obtained from a second commercial supplier. The latter 2 strains of mice are inbred and bear the H-2d histo- compatibility locus. e Spartan Research Animals, Haslett, Michigan. ** Simonsen Laboratories, Gilroy, California. 24 25 Maintenance of Animals All mice, with the exception of those used in the allograft experiments, were maintained in the Biophysics Department animal quarters. The animals were maintained on a commercial diet* and water. ad Zibitum. Animal care was provided by Biophysics Department personnel. The animals utilized for skin allograft rejection experiments were maintained in quarters provided by the Michigan State University Center for Laboratory Animal Resources (CLAR) and cared for by CLAR personnel. A.Montadale wether lamb was maintained at Michigan State University's Veterinary Research Barn No. 1 as a source for sheep erythrocytes (SRBC) and was cared for by the Department of Large Animal Surgery and Medicine personnel. The housing and care of animals was performedin accordance with the standards promulgated by the National Society of Medical Research. Preparation of cis-Pt(II) Compound The compound gigrPt(II) was synthesized and purified by a Biophysics Department chemist.** Sterile saline (0.852 NaCI) was used in the preparation of all aqueous solutions of gingt(II), and all solutions were prepared within 1 hour of the time of injection. The compound was administered via the intraperitoneal (IF) route in all experiments. * Zinn's Feed, A. K. Zinn & Company, Battle Creek, Michigan. at Dr. James Hoeschele. 26 Preparation of Hydrocortisone A saline suspension of hydrocortisone acetate (BC) was purchased from a commercial supplier.* The product was used as provided by the supplier and was administered via the subcutaneous (SC) route. All injections‘were given immediately posterior to the right humerus. Preparation of Zymosan Zymosan from saccharamyces cerevisiae yeast was purchased from a commercial supplier.** Sterile saline was used in the preparation of all aqueous suspensions of zymosan, and all suspensions were boiled in a water bath for 1 hour prior to the time of injection. Zymosan was administered via the IP route in all experiments. Transplantation of Sarcoma 180 Sarcoma 180 (S-l80) was maintained in Swiss mice by weekly transfer. The tumors used in BALB/c mice originated from Swiss mice and the number of transfers in the latter strain was recorded in each experiment. The 5-180 tumors were implanted according to Cancer Chemotherapy National Service Center protocols (1962). According to these methods, animals bearing 8- to 16-day-old implants of 8-180 were killed by cervical dislocation. The left axillary region was swabbed with 802 ethyl alcohol and the tumor was carefully and aseptically dissected away from the overlying skin and adjacent normal tissues. The tumor was placed in a sterile 100 x 15 mm Petri dish containing Chloromycetin, and necrotic and hemorrhagic regions were extirpated. Portions of the e HydrocortoneR acetate, Lot No. O324N, Merck, Sharp and Dohme Division of Merck and Company, Incorporated, west Point, Pennsylvania 19486. ** Zymosan, Lot No. 9913-0100 and 7lC-1370, Sigma Chemical Company, 3500 DeKalb Street, St. Louis, Missouri 63118. 27 remaining tumor were cut into 1-3 mm segments, placed in a 13-gauge trocar and implanted subcutaneously in the region of the left axilla of the recipients. Prior to implantation, the left axillary regions were swabbed with 801 ethyl alcohol. The day of tumor implantation was recorded as day 0. The tumors were measured with calipers at weekly intervals. The measurements included 2 diameters at right angles to each other, the results being expressed as the average of the two in millimeters. Regressions were defined as complete disappearance of tumors without reappearance in a period of 90 days. Humoral Antibody Assay A protocol for the performance of the agarose-slide technique, a modification of Jerne's plaque counting technique, was used (Plotz et al., 1968).* Each week 25 m1 of SRBC were collected and placed in 25 ml of Alsever's Solution (Carpenter, 1965). The resultant suspensions were stored at 4°C for 2 weeks prior to use. Animals to be evaluated were given an IP injection of 3 x 108 SRBC which were washed 3 times in .01M phosphate buffered saline (PBS) at pH 7.0. The PBS was freshly prepared prior to each experiment. 0n the fifth day after injection, the animals were killed by decapitation. After exsanguination the spleens were removed and placed in sterile 100 x 15 mm.Petri dishes and stored on ice. The individual spleens were then forced through fine stainless steel mesh (faucet aerators) with a pestle. The homogenate was taken up through a 22-gauge hypodermic needle into a *Provided by Dr. Harold C. Miller, Department of Macrobiology and Public Health, Michigan State University, East Lansing, Michigan. 28 tuberculin syringe containing 1 ml of Eagle's Minimum Essential Medium (MEM)* (pH 6.9 to 7.2). The splenic material was then forced back and forth through the needle until it flowed freely. A 27-gauge hypodermic needle was attached to the syringe, and the forcing procedure was repeated a number of times. Since there was a certain amount of fluid loss during this procedure, at its termination the fluid volume was restored to 1 ml with fresh MEM. The homogenates were then placed in 100 x 16 mm screw cap test tubes and stored on ice. The spleen cells ee were quantitated on an electronic counter. A 12 solution of agarose*** was prepared in distilled water and melted on a hot plate. Upon melting, an equal volume of 2 times con- centrated MEM, prewarmed to S6'C, was added. Thus the final concentra- tion of agarose was 0.52. Using plastic pipettes, a 0.4 ml aliquot of the 0.52 agarosedMEM solution was delivered into Wesserman tubes located in a 48-50'C water bath. A 0.05 ml aliquot of 20X SRBC previously washed 3 times in PBS was suspended in.MEM and delivered into the tdbes as well. Finally, in rapid succession the tubes were removed from the bath, a 0.1 ml aliquot of spleen cells diluted 1:100 with MEM was added, the suspensions were gently agitated on a Vortex mixer and the suspensions were then poured on glass microscope slides which had previously been coated with 0.1% agarose. *Grand Island Biological Company, Grand Island, New York, Catalog No. F-lS. ** Coulter Counter Model B, Coulter Electronics, Hialeah, Florida. eee Agarose-L'Industrie Biologique Francoise S.A., Distributed by Fisher Scientific Company, 15800 West McNichols Road, Detroit, Michigan 48235. 29 Upon solidification, slides were placed face down on trays which were specially made for these experiments by a member of the Biophysics Department.* Briefly, the trays consisted of a flat sheet of plexi- glass 9" x 17" x 1/4". In the middle and along both sides 1/4" from the edge, glass microscope slides were mounted extending the full length of the sheet. The slides were glued to the surface with epoxy resin. Narrow strips of plexiglass 1/2" x 17" x 1/4" were then glued in the middle of the glass slides forming 2 separate compartments on each tray. When the slides containing the agarose-spleen cell mixture were placed on the tray, their ends were in contact with the glass slides of the tray. As a result the agarose-spleen cell mixture was suspended approximately 1 mm.from the floor of the tray. Bach tray held 24 slides. Once placed on the trays, the slides were incubated at 37'C in a humid environment consisting of 95% air and 52 C02. After this initial incubation, the trays were removed, and 2 ml of a 1:10 dilution of guinea pig complement** in MEM were pipetted into each tray compart- ment so that the agarose-spleen cell mixture was immersed. They were then re-incubated at 37‘C for an additional 2 to 3 hours. At the termination of the experiment the complement was poured off the slides, and the plaques were quantitated using bright indirect light and the unaided eye. e Mr. George Moldovan. ee Guinea Pig Complement-Microbiological Associates, Bethesda, Maryland, Catalog No. 30-956. 30 Cell Mediated Immunity Assay Skin allograft rejection was selected as the means to evaluate cell-mediated immune responses. The skin grafting technique used was a modification of that described by wexler (1970). Female DEA/2 mice, 4 months of age, were used as donors. They were anesthetized with 'methoxyflurane* and the hair was clipped with scissors from the lateral abdomen and back. The area was then shaved with a straight razor.** The shaved skin was incised with iris scissors commencing immediately lateral to the lines alba on one side. The remaining dissection was alternately performed with iris scissors and a scalpel. Skin from the lateral abdomen on both sides and back'was removed in one sheet. The donor animal was killed by cervical dislocation at the termination of the dissection. The skin was placed in a sterile 100 x 15 mm Petri dish with the dermis side up, and all remaining subcu- taneous tissues were removed with iris scissors. Rectangular grafts, 5 x 5 mm, were cut from the skin sheet. The grafts were placed in a sterile 100 x 15 mm Petri dish containing 150,000 IU of procaine penicillin G*** and 250 mg dihydrostreptomycin sulphate*** in a total volume of 6 m1. Petri dishes were then placed on ice for the remainder of the experiment. Recipient female BALB/c mice, 10 weeks of age, were anesthetized with methoxyflurane. Hair was then clipped and shaved from a region on the right side extending from the tuber coxae to the thoracic inlet e MetofaneR, Pitman-Moore, Incorporated, Fort washington, Pennsylvania 19034. *e fleck Hair Shaper, Edward Neck and Company, L.I. City, New York 11101. eee ’ W. A. Butler Company, Columbus, Ohio 43201. 31 and from the vertebral transverse processes to the linea alba. The area was swabbed with 802 ethyl alcohol. A linear incision was then made parallel to and equidistant from the transverse processes and the lines alba. The incision was 15 mm.in length and extended to the costal arch. Subcutaneous tissue superior to the incision was separated from the overlying skin by blunt dissection. The skin in this region was then elevated with forceps, and the skin graft was placed dermis side down in the undermined region, making sure that the graft remained flat. The overlying skin was then pulled over the graft, and the incision was closed with wound clips.* Seven days after grafting, the skin covering the grafts was incised and dissected away. The animals were observed daily. A superficial layer of desquamation usually formed over the graft area in the days following separation of the protective body skin covering. This layer usually sloughed off prior to graft rejection. When the entire graft was dark and had lost its pliability, graft rejection was con- sidered complete. Treatment of Animals with Combined cis-Pt(II) and Hydrocortisone (HC) Two experiments were performed to ascertain what effect immuno- suppression with HC would have on the anti-tumor properties of gig-Pt(II). Segments of 8-180 tumors, 8 to 10 days old, were implanted in 248 female Swiss mice eight weeks of age. The treatment schedules and doses are listed in Table 1. * AutoclipsR, Clay-Adams, Incorporated, New York 10, New York. 32 Table 1. Treatment schedule for combined hydrocortisone and cis-Pt(II) therapy in Swiss mice bearing 8-180 ——v Treatment Dose Day of Treatment Number of Animals 1. Saline 0.2 ml 1, 8 and 14 0.5 m1. 8 38 2. Egg-Pun) 8 mg/Kg 8 34 3. Hydrocortisone 150 mg/Kg l 40 4. Hydrocortisone 150 mg/Kg 8 24 5. Hydrocortisone 150 mg/Kg 15 24 6. Hydrocortisone 150 mg/Kg l gig-Pun) 8 mg/Kg 8 40 7. ‘gig-Pt(ll). 8 mg/Kg 8 Hydrocortisone 150 mg/Kg 8 24 8.. _c_i§_-Pt(II) s mg/Kg 8 Hydrocortisone 150 mg/Kg 15 24 The animals serving as controls received sterile saline in an equivalent volume as the drug being administered and via the same route, e.g., 0.5 ml gig-Pt(11) IP versus 0.5 m1 saline IP. When‘gig-Pt(II) and HC were administered on the same day, e.g., group 6, the‘gig-Pt(II) was administered 6 hours prior to the HC. All animals which did not have palpable tumors by day 15 of the experiment and those dying prior to that time were eliminated from the experiment. Treatment of Animals with Combined cis-Pt(II) and Zymosan Four experiments were performed in an attempt to ascertain.what effect the administration of zymosan on day 1 of tumor growth would have on the anti-tumor efficacy of cis-Pt(II). Segments of 8-180 tumors, 33 8 to 12 days old, were implanted in 384 female BALB/c mice 8 to 12 weeks of age. Depending on the experiment, the tumors had been trans- ferred at weekly intervals in BALB/c mice 3, 8, 26 or 38 times. The treatment schedules and doses are listed in Table 2. Table 2. Treatment schedule for combined zymosan and gig-Ptul) therapy in BALB/ c mice bearing 8-180 Treatment Dose Day of Treatment Number of Animals 1. Saline 0.2 m1 1 0.5 m1 8 87 2. 9_i_§-Pt(ll) 7 lug/Kg 8 85 3. Zymosan 50 mg/Kg 1 65 4. Zymosan 75 mg/Kg 1 20 5. Zymosan 100 mg/Kg 1 20 6. Zymosan 50 mg/Kg l _<_:_1_a_-rt(II) 7 mg/Kg 8 67 7. Zymosan 75 lug/Kg l gig-Ptfll) 7 Ins/Ks 8 20 8. Zymosan 100 mg/Kg l gig-Ptfll) 7 mg/Kg 8 20 The animals in the control groups received sterile saline via the 1P route and at a volume equivalent to the drug being administered on each treatment day. All animals not bearing palpable tumors by day 15 of the experiment and those dying prior to that time were eliminated from the experiment. The animals were weighed on day l and twice weekly thereafter through day 21 of the experiment. Tumors were measured at weekly 34 intervals. The diameters of the tmors were measured at right angles to each other, and the average of these 2 measuraents was expressed in millimeters. Timing Experiment 1 Three experiments were performed in an attempt to ascertain what influence the day of zymosan administration had on the anti-tumor efficacy of _c_i_s_-Pt(II). In these experiments the zymosan was given prior to the platinum compound. Segments of 8-180 tumors, 8 to 11 days old, were implanted in 300 female BALB/c mice, 8 to 9 weeks of age. These tumors had been trans- ferred at weekly intervals in BALB/c mice 3, 12 or 15 times. The treatment schedules and doses are listed in Table 3. The control animals were treated, all animals were weighed and the tumors measured as previously described. All, animals not bearing palpable tumors by day 15 and those dying prior to that time were eliminated from the experiment. Timing Experiment 2 Two experiments were performed in order to ascertain what influence the administration of zymosan concomitant with and after the gig-Hal) would have on the anti-tumor efficacy of the latter drug. Segments of 8-180 tumors, 11 to 12 days old, were implanted in 120 female BALB/c mice, 8 to 11 weeks of age. These tumors had been transferred at weekly intervals in BALB/c mice 3 or 18 times. The treatment schedule and doses are listed in Table 4. The control animals were treated, all animals were weighed and the tumors were measured as previously described. All animals not 35 Table 3. Treatment schedule for combined zymosan and cis-Pt(II) therapy in BALB/c mice bearing 8-180. Timing study 1 Treatment Dose Day of Treatment Number of Animals 1. Saline 0.2 ml 1,2,4 and 6 0.5 m1 8 30 2. gig-PtUI) 7 mg/Kg a 30 3. Zymosan 50 mg/Kg 1 30 4. Zymosan 50 mg/Kg 2 30 5. Zymosan 50 mg/Kg 4 30 6. Zymosan 50 mg/Kg 6 30 7. Zymosan 50 tug/Kg 1 gig-Ptal) 7 leg 8 3o 8. Zymosan 50 mg/Kg 2 gig-Ptul) 7 mg/Kg 8 3o 9. Zymosan 50 lug/Kg 4 gig-PtUI) 7 mg/Kg 8 30 10. Zymosan 50 mg/Kg 6 gig-Pall) 7 mg/Kg 8 30 36 Table 4. Treatment schedule for combined zymosan and Egg-Pt(II) therapy in BALB/c mice bearing 8-180. Timing study 2 Treatment Dose Day of Treatment Number of Animals 1. Saline 0.5 ml 8 0.2 m1 8.10.12 and 14 10 2. gig-Ptfll) 7 mg/Kg 8 10 3. Zymosan 50 mg/Kg 8 10 4. Zymosan 50 mg/Kg 10 10 5. Zymosan 50 mg/Kg 12 10 6. Zymosan 50 mg/Kg 14 20 7. gig-Pull) 7 mg/Kg 8 Zymosan 50 mg/Kg 8 10 8. .gig-Pt(II) 7 mg/Kg 8 Zymosan 50 mg/Kg 10 10 9. pgig-Pt(II) 50 mg/Kg 12 Zymosan 7 mg/Kg 8 10 10. .C_i_§_-PC(II) 7 mg/Kg 8 Zymosan 50 mg/Kg 14 20 37 bearing palpable tumors by day 15 and those dying prior to that time were eliminated from the experiment. Timing Experiment 3 A single experiment was performed in an attempt to ascertain what effect preimnization and multiple injections of zymosan would have on the anti-tumor efficacy of gig-Pt(II). In addition multiple low dose injections of gig-Pun) were evaluated. Segments of an 8-180 tumor, 8 days old, were implanted in 110 female BALB/c mice, 10 weeks of age. The tumor had been transferred 23 times in BALB/c mice at weekly intervals. Segments from a 10-day- old 8-180 tumor which had been transferred 3 times in BALB/c mice were implanted in 10 female BALB/c mice 10 .weeks old. The treatment schedules and doses are listed in Table 5. All animals were weighed, and the tumors were measured as previously described. All animals not bearing palpable tumors by day 15 and those dying prior to that time were eliminated from the study. Evaluation of the fiIfntegrity of Host Defenses Evaluation of Humoral Antibody Response A total of 115 female BALB/c mice, 8 weeks of age, was used to evaluate the humoral antibody response in animals treated with a coati- naion of zymosan and 5_:l._g-Pt(II), but not bearing tumors. The treatment schedule, day of antigen administration and the day on which the agarose-slide technique was performed are listed in Table 6. A total of 112 female BALB/c mice, 8 weeks of age, was used to evaluate the humoral antibody response in animals bearing 8-180 and 38 Table 5. Treatment schedule for combined zymosan and cis~Pt(Il) therapy in BALD/c mice bearing 8-180. Timing study Treatment Dose Day of Treatment Number of Animals 1. gi_s-Pt(II) 1 mg/Kg 1-7 10 2. £1_.s_-Pt(ll)* 5 mg/Kg 1 and 6 1o 3. gig-Pan) 7 mg/Kg a 10 4. Zymosan 50 ms/Ks -7 10 5. Zymosan 50 mg/Kg 1 10 6. Zymosan 50‘mg/Kg 14 10 7. Zymosan 50 mg/Kg l and 14 10 8° Zymosan 50 ms/Kg -7 _c_1_8_-Pt(II) 7 salt; 8 10 9. Zymosan 50 mg/Kg -7 c_i_s_-Pt(II) 1 Ins/Ks 1-7 ’ 10 10. Zymosan 50 ms/Ks 1 E_i._s_-Pt(II) 1 Ills/K3 1-7 10 11. Zymosan 50 ms/Kg l and 14 EEETPt(II) 7 m8/K8 3 1° * Tumor transferred in BALB/c mice 3 times at weekly intervals. 39 Table 6. Treatment schedule for evaluation of humoral antibody responses in BALD] c mice treated with zymosan and gig-mu!) but not bearing tumors ' Day of Day Antigen Day Agarose-Slide Treatment Dose Treatment Administered Technique Performed l. Saline (10)* 0.2 m1 1 0.5 ml 8 14 19 2. Zymosan (ll) 50 Ins/Kg 1 14 19 3. _c__1_g_—P:(II) (13) 7 Ins/K8 8 14 19 4. Zymosan 50 lug/K3 1 Lig-PtflI) (12) 7 ml“ 8 14 19 5. Saline (18) 0.2 m1 1 0.5 ml 8 21 26 6. Zymsan (17) 50 mg/Kg 1 21 26 7. c_i_g_-Pt(11) (l7) 7 lag/Kg 8 21 26 8. Zymosan 50 mg/Kg l gig-Pun) (17) 7 tag/Kg 8 21 26 _v_ a Numbers in parentheses are numbers of animals per group. 40 treated with a combination of zymosan and gig-Pt(II). The mice were implanted with segments of an 8-180 tumor 12 days of age. The treat- ment schedule, day of antigen administration and the day on.which the agarose-slide technique was performed are listed in Table 7. Evaluation of Cell Mediated Immune Responses A total of 85 female, BALB/c mice, 8 weeks of age, was used to evaluate cell mediated immune responses in animals treated with a combination of zymosan and gig-Pt(II), but not hearing tumors. The treatment schedule and day on which skin allografts were applied are listed in Table 8. One hundred four female BALD/c mice, 8 weeks of age, were used to evaluate cell mediated immune responses in animals bearing 8-180 tumors and treated with a combination of zymosan and gig-Pt(II). Segments from 8-180 tumors 12 days of age were used to initiate tumor growth. The treatment schedule and day on which skin allografts were applied are listed in Table 9. Animals that died prior to the complete rejection of allografts were eliminated from the experiment. Microscopic framination of Tissues Segments of an 8-180 tumor, 12 days of age, were implanted in 20 female BALB/c mice 8 weeks old. The animals were divided into 4 equal groups and treated with saline, zymosan, gig-Pt(11) or combinations of zymosan and gig-Pt(ll). 0n the 21st day of tumor growth, the mice were killed by cervical dislocation. Selected tissues including spleen, thymus, regional lymph nodes and tumors were removed and fixed in 10% neutral buffered formalin. Tissue sections were cut at 6 microns, stained with hematoxylin and eosin and evaluated histologically. 41 Table 7. Treatment schedule for evaluation of humoral antibody responses in BALB/c mice bearing S-180 tumors and treated with zymosan and cis-Pt(ll) T V v’ Day of Day Antigen Day Agarose-Slide Treatment Dose Treatment Administered Technique Performed 1. Saline (12)* 0.2 m1 1 0.5 ml 8 14 19 2. Zymosan (12) 50 mg/Kg l l4 l9 3. _<_:_i_g-Pt(II) (12) 7 lug/Kg a 14 19 4. Zymosan 50 mg/Kg 1 gig-Pt(II) (12) 7 mg/Kg 8 14 19 5. Saline (12) 0.2 m1 1 0.5 m1 8 21 26 6. Zymosan (13) 50 mg/Kg 1 21 26 7. _c_:_i_s_-Pt(II) (13) 7 mg/Kg 8 21 26 8. Zymosan 50 ms/Kg 1 ‘gig-Pt(ll) (26) 7 mg/Kg 8 21 26 ._Y * Numbers in parentheses are numbers of animals per group. 42 Table 8. Treatment schedule for evaluation of cell-mediated immune responses in BALB/c mice treated with zymosan and gig-Pt(II) but not hearing tumors Day of Day.Allegraft Number of Treatment Dose Treatment Applied Animals 1. Saline 0.2 m1 1 0.5 m1 8 14 10 2. Zymosan 50 mg/Kg l 14 7 3. gig-Pall) 7 mg/Kg 8 14 10 4. Zymosan 50 mg/Kg 1 gig-Pall) 7 lag/Kg 8 14 8 5. Saline 0.2 m1 1 0.5 ml 8 21 ll 6. Zymosan 50 mg/Kg l 21 9 7. gi_s_-Pt(II) 7 lug/Kg s 21 10 8. Zymosan 50 mg/Kg 1 _<_:_i_._§_-Pt(II) 7 mg/Kg 8 21 1o 43 Table 9. Treatment schedule for evaluation of cell-mediated immune responses in BALB/c mice bearing 8-180 tumors and treated with zymosan and cis-Pt(II) Day of Day Allograft Number of Treatment Dose Treatment Applied Animals 10 Saline 0.2 ma 1 0.5 ml 8 14 26 2o Zymosan 50 mg/Kg 1 14 26 3. gag-Pun) 7 mg/Kg 8 14 26 4. Zymosan 50 ESIKS 1 Igig-Pt(II) 7 mg/Kg 8 14 26 Statistical Analysis The 1 way analysis of variance was used to analyze all data and the method of Scheffe was used to establish confidence limits (Lewis, 1966; Scheffe, 1961). RESULTS Combined cis-Pt(II) and Hydrocortisone Therapy The results of combined gig-Pt(II) and hydrocortisone therapy are summarized in Table 10. The gig-Pt(ll) was capable of eliciting regres- sions in 19/33 (572) of the animals treated. The administration of HC 7 days prior to or 6 hours after the platinum compound, however, markedly reduced its anti-tumor activity, e.g., 11/48 (232) and 5/22 (232). Administering the HC 7 days after gig-Pt(ll) reduced the number of tumor regressions to 8/19 (422). Statistically significant differences in mean life span (MLS) were observed between certain treatment groups (Table 10). Combined Zymosan and cis-Pt(II) Therapy The results of the initial experiments utilizing combined zymosan and gig-Pt(II) therapy are summarized in Table 11. There were no spon- taneous regressions in the control animals, nor were any regressions observed in those animals treated with.gig-Pt(Il) alone. Regressions did occur, however, in those animals treated with 50, 75, or 100 mg per Kg of zymosan on day l of tumor growth, e.g., 2/61 (32), 4/17 (242) and 1/17 (62), respectively. The most successful treatment regimen was the administration of 50 mg per Kg of zymosan on day l of tumor growth followed by 7 mg per Kg of gig-Pt(ll) on day 8, e.g., 30/64 (472). 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Numbers of surviving mice are indicated by the number at each po int . 55 The tumors in animals of all treated groups were significantly smaller than those of control animals on days 14 and 21 (P<0.03 to 0.0005). On day 7, however, only those animals treated with zymosan on day l differed significantly from control animals (P<0.04). The tumors from animals treated with SigrPt(II) and those treated with combined zymosan and-gigrPt(II) were significantly smaller on days 14 and 21 than those treated‘with zymosan alone (P<0.0005). These animals treated with combined zymosan and.gi§7Pt(II), and whose tumors ulti- mately regressed, had significantly smaller tumors than all other groups on days 14 and 21 (P<0.008 to 0.0005). The changes in body weight in animals from all experiments in which 50 mg per Kg of zymosan was administered on day 1 and 7 mg per Kg of gig-Pt(II) was administered on day 8 are illustrated in Figure 2. The administration of zymosan on day 1 caused a significant (P<0.02), but transient, loss in body weight by day 4. The weight changes in these animals then paralleled that of the control animals until day 21. At this point they again weighed less than the control animals (P<0.02). The animals treated with gig:Pt(II) on day 8 exhibited a precipitous weight loss after treatment. The body weights remained significantly lower (P<0.0005) than the control animals and those treated with zymosan through day 21. The animals treated with combined zymosan and gigrPt(II) were divided into 2 groups: those with progressively growing tumors and these with regressing tumors. In the group with progressively growing tumors, the weight changes paralleled the gigrPt(II) group, but were not as marked. Their weight loss by days 14 and 21 was significantly (P<0.0005) greater than in the control animals and those treated with zymosan. On day 17 the group treated with both compounds weighed 56 20" 50 715 i wk 50 : 92 K so so 50 so Illa/Kg ZYMOSAN ’..\ ""\ ” 7mg/Kg Q'PTUI) 'I - (PROGRESSIVE rumoa 2A. \ 3 \ GROWTH) :7. Is— 77 \f" \ 77 e ‘ ..... a 77 ‘3‘ :H \ 9° \\67: E \ 9° '- 94 30.5 ml SALINE Q \\ \O” “34 '7' \ ’2 ,,.‘.’3A so mg/Kg zmosm >' \ 5° 73 32 so lug/Kg ZYMOSAN 8 \ 7m9/Kg ClS-"(In 1 a, \\ so (Reoaessmo TUMORS) 9| 2 ”J P: .. 35 \ // ‘13 7mg/Kg ClS-PT(II) ‘ 2 \ 32 / . \ / I5 2 / 99_—-d92 l . I4 3 I 4' 7 1'2 I'4 I'7 2'I ‘ DAYS Figure 2. Changes in body weight in BALB/c mice bearing 8-180 and treated with combinations of zymosan and gig-Pan). 57 essentially the same as the other 2 groups, while on day 21 those treated with both compounds weighed the same as the zymosan group but significantly less (P<0.02) than the saline controls. These animals treated with combination zymosan and gigrPt(II) and whose tumors regressed showed transient weight loss by days 12 and 14. By day 17, however, their average weight was greater than any other group (P<0.0005). The weight loss by days 12 and 14 in this group was significantly less (P<0.004 to 0.0005) than those treated with gisrPt(II) and those treated with both.compounds but whose tumors were progressive in growth. The Evaluation of Host Defenses in Animals Treated with Combinations of Zymosan.and cis-Pt(II) Evaluation of Humoral Antibody Response The ability of animals treated with combinations of zymosan and' Singt(II), but not bearing 8-180, to produce antibody to SRBC's is summarized in Table 15. There were no differences in the groups to which the antigen was administered on day 14. When the antigen was administered on day 21, however, the.animals treated with combinations of zymosan and gingt(II) produced significantly higher (P<0.009) numbers of plaque-forming spleen cells than the control animals. 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H H»: HHHvunialHd H uM\uE en unemeahN .m o.e.m.m H ::02_ eosnownem 0:6Hnneos venouchHso< unoauuona omen n:oauoona menaeHm we nonssz ovHHmIooonoud he: :omHu:< an: we we: A.v.u:ouV 0H OHAmH 61 In the group in which the antigen was given on day 21, the animals treated with combinations of the 2 drugs and whose tumors were regress sing produced significantly greater numbers of plaques than either the control animals or those treated with zymosan. The animals treated with gingt(II) and those treated with combinations of the 2 drugs with nonregressing tumors produced greater numbers of plaques than the controls. There was no significant difference between the numbers of plaques produced by either combination therapy group and those treated with gigrPt(II) alone. Evaluation of Cell Mediated Immune Responses In animals not bearing 8-180, there was no significant difference in the time of skin allograft rejection between treatment groups (Table 17). In those animals bearing 8-180, the animals treated with combina- tions of the 2 compounds rejected allografts earlier than the controls or other treatment groups. This enhanced time of rejection, however, was independent of the fate of the tumor (Table 18). gircroscopic Evgluat ion of Selected Tissues Spleen The spleens of the animals evaluated were characterized by marginal hyperplasia of lymphoid follicles. This change was characterized by proliferation of what appeared to be immature lymphocytes on the margins of the follicles. numerous degenerating cells and occasional macro- phages were also neted in these preliferative zones. The central artery of each follicle was generally surrounded by a thin halo of 62 .:ee: 0:: we nenne unannoum r .u .m .n meson» no: oHsaH:n mo enemas: one mononnnensm :H onenlnz .e n + »H H» » 9:»... H 5 cases”... H wuxwa on neeeahw .m n H »H H» » 9:»... H 8H. 33......de .H » H HH H» H 9:»... on :8 5.2:» .» « H «H H» » H.. m... H H... ».e HHHV 2.3..» .m « H »H «H » 9:»... H HH» 35973.. H aux»: cm :eoeahu .« « H »H «H » 9:»... H 8: anemia-.HH .» « H »H «H H 9:»... a» E 8.8..» .» « H »H «H » H.. n.» H H.. ».o «83 «5H...» .H .m.m.HH:mez wsHumenonH< useauoona soon u:oaueona :eHuoo oz amenweHH< no men me men me he: unease w:HnooA no: use .AHanMImHo one :mmoshu euHa oouoonu ooHa o\mHHmmonoun waHHmon mHmaHa< .o Hamma mfiu mo Houum vnmucmHm I .m.m .n “macaw “mm meaHam mo mumnasa wum mummnuamumm 6H muwnaaz .m mm + «H «H » 9:»... H 5 353.38 H mM\wa om vammoahm .n H» H »H «H » 9:»... H H3 353......» H »i»... o» 058.5» .« « H »H «H » 9:»... H HHHV HHHrmumHo .» m H »H «H H 9:»... on HHHV 58E» .» m .+. HH «H » H... m.» H H... ».o as. »»H?» .H .m.m anwmz wnHumewOHH< uaoaummuy mmoo unmaummua GOHuommum ummquHH< mo ham mo 5mm mo has HHHVumImHo van ammoahu nuHs wmummuu wam owHIm waHumm£ moHa u\mHm mo muHSmwm .mH mHnme 64 mature lymphocytes. This hyperplastic change appeared to be independent of the type of treatment or the disposition of the tumor. Thymus In those animals in which tumors were regressing, the thymus was composed of a wide cortical region of lymphocytes and the normal complement of epithelial cells in the medullary region (Figure 3). The thymuses of animals with progressively growing tumors were con- sistently atrophic. They were characterized by disarranged cortical lymphocytes, many of which.were degenerate. The medullary region was virtually nonexistent (Figure 4). Lymph.Nodes The regional lymph nodes of all groups were generally hyperplastic (Figure 5). The medullary regions were composed of an admixture of lymphocytes, plasma cells and macrophages. Occasionally, macrophages and lymphocytes were noted in the medullary sinusoids (Figure 6). In one of the control animals the tumor had metastasized to the regional lymph node, displacing the normal follicular architecture (Figures 7 and 8). Tumors The microscopic changes in regressing tumors were characterized by degeneration of tumor cells, infiltration of the tumor by lympho- cytes and plasma cells and the formation of a fibrous capsule on the periphery of the tumor (Figures 9 and 10). In progressively growing tumors, there were multifocal regions of necrosis, but the majority of the tumor cells appeared healthy, and lymphocyte infiltration and/or fibroplasia were virtually nonexistent (Figures 11 and 12). 65 Thymus from BALB/c mouse treated with Note the wide cortical region (C) composed of lymphocytes and the more pale staining medulla (M) composed of epithelial cells. Hematoxylin and eosin. x 100. Figure 3. zymosan and bearing a regressing tumor. 66 Figure 4. Thymus from BALB/c mouse treated with gigrPt(II) and bearing a progressively growing tumor. The thymus is atrophic and has lost its normal archi- tecture. Note absence of medulla. Hematoxylin and eosin. x 100. 67 Figure 5. Axillary lymph node from BALB/c mouse treated with zymosan plus gig-Run. There is marked hyperplasia of both the cortex and medulla. Hematoxylin and eosin. x 40. 68 Figure 6. Higher magnification of the medulla of the lymph node in Figure 5. Note macrophages (blunt arrow) and lymphocytes (arrow) in medullary sinuses. Hematoxylin and eosin. x 400. 69 Figure 7. Axillary lymph node from BALD/c mouse treated with saline and containing metastatic tumor cells. Note sheets of tumor cells compressing cortical lymphocytes toward the periphery. Hematoxylin and eosin. x 190. Figure 8. Higher magnification of a portion of the lymph node in Figure 7. Note tumor cells compres- sing and infiltrating cortical lymphocytes. anatoxylin and eosin. x 400. 71 Figure 9. Regressing tumor from BALB/c mouse treated with zymosan. There is proliferation of a fibrous capsule (F) on the periphery and marked lympho- cytic infiltration (L). Hematoxylin and eosin. x 100. 72 Figure 10. Higher magnification of a portion of the tumor in Figure 9. Note fibrous tissue (F) on periphery and lymphocytes (L) infiltrating tumor cells. Hematoxylin and eosin. x 400. 73 n... w ., m. .., .I w H... :H. x 100. It is characterised by (II). sheets of proliferating neoplastic cells, numerous mitotic figures (arrows) and multifocal areas of necrosis (N) — Progressively growing tumor from male Hemetoxylin and eosin. Figure 11. mouse treated with cis-Pt 74 .3. “gflfifli’dvffiv- 6.. . Figure 12. Higher magnification of a portion of the tumor in Figure 11. Note area of necrosis (N) sur- rounded by pleomorphic, neoplastic cells. Hematoxylin and eosin. x 200. DISCUSSIGN The results of these experiments indicate that the anti-tumor activity of gig-Pull) is, at least in part, dependent upon active host responses. In the first series of experiments hydrocortisone was administered in combination with the platinum compound to ascer- tain if imnosuppression would abrogate the anti-tumor efficacy of the latter drug. A host of investigators have reported that ill-lunc- suppression with corticosteroids decreases the anti-tumr efficacy of various chemotherapeutic and/ or imunotherapeutic compounds when given before (Fugmann et a2. , 1970), at the same time as (Martin at 611., 1962; Fugmann et a1. , 1970) and/or after the administration of these compounds (Stoerck, 1954; Tarnowski and Stock, 1957 ; Mihich and Nichol, 1959; Bradner and Clarke, 1959; Fugmann at al., 1970). Simi- larly, the experiments reported here indicate that the suppression of host responses with hydrocortisone either before, concomitant with or after gig-Hal) markedly diminish the anti-tumor effects of the latter drug. The greatest abrogation occurred when hydrocortisone was administered 7 days prior to or 6 hours after the gig-Pull). In both instances the‘number of tumor regressions was reduced to less than 502 of those ‘in mice treated with gig-Pan) alone. When hydro- cortisone was administered 7 days after the gig-Pull), the anti- tmnor efficacy of the drug was diminished by 261. This indicates that the later immunosuppression occurs after gig-Fall) administration 75 76 the less effective it is in diminishing the anti-tumor activities of the drug. It is difficult to ascribe a specific mechanism by which immuno- suppression with hydrocortisone interferes with the ability of ggngt(II) to promote regressions. As has been previously described, the steroids are known to suppress humoral antibody synthesis and depress cell mediated immunity. It is significant to note that the administration of the steroid on day l or day 8 of tumor growth caused the greatest retardation of gingt(II) activity. It is during this time that host responses are initiated against the tumor. VanCamp (personal connnunication) demonstrated that concomitant immunity does not develop until day 9 of 8-180 growth. Thus, the decrease in tumor regressions observed when hydrocortisone was administered 7 days prior to the gingt(II) may be explained on the basis that the steroids suppressed the host responses against the tumor in their initial stages. When hydrocortisone was administered 6 hours after the gigrPt(II) the antirtumor efficacy of the latter drug was again markedly diminished. This might be explained on the basis that both drugs have immunosup- pressive capabilities and that the combination of the two suppressed host responses to a point where they were ultimately less effective in promoting tumor regressions. Although unmentioned by previous investigators using similar treatment regimens,one cannot rule out the possibility that, since the gig-Pt(II) and hydrocortisone were given on the same day, there might have been a chemical antagonism between the two drugs which in turn nullified the effects of the platinum compound. Without the use of sophisticated chemical analysis, this problem remains unresolved. 77 The fact that the administration of hydrocortisone one week after gigrrt(II) was less effective in reducing the number of tumor regres- sions parallels the observations of Bradner and Clarke (1959). They found that the administration of hydrocortisone on day 13 of tumor growth was less effective in blocking the anti-tumor activity of zymosan than if it were administered earlier. They concluded that by day 13, host responses to the tumor were well under way and less susceptible to immunosuppression. The specific mechanism by which hydrocortisone depresses the antirtumor efficacy of ggngt(II) is at best speculative. It is readily apparent, however, that the oncostatic properties of g§g7Pt(II), like many cancer chemotherapeutic agents, are markedly diminished by the immunosuppressive effects of hydrocortisone. The results of the next series of experiments reinforce the hypothesis that gig:Pt(Il) requires active host responses in order to promote regressions of 8-180. In these experiments it was found that the platinum.compound was ineffectual in promoting regressions of 8-180 implanted in BALB/c mice. In this particular system.the host and tumor have been reported to be histocompatible (Snell st aZ., 1953) and, consequently, host responses against the tumor are not vigorous. Of particular interest in these experiments, however, was the potentiation of anti-tumor activity by combining.gigfrt(ll), an immunosuppressive drug, and zymosan, an immunostimulant. Bradner and Pindell (1965) reported that zymosan was markedly effective in promoting regressions of 8-180 implanted in DBAIZ mice, a histocompatible system, but chemotherapeutic agents were not. In the experiments reported here, zymosan.was found to be minimally successful, but in the proper temporal relationship, the combination 78 of zymosan and gig-Fran appeared to elicit a moderate anti-tumor effect. Although some regressions were noted with various time intervals between zymosan and the platinum compound, the most con- sistently successful treatment regimenwas found when 50 mg per Kg of zymosan was administered on day l of tumor growth followed by 7 mg per Kg of _<_:_:l_._s_-Pt(II) on day 8. The variation in success of this regimen, and possibly the others as well, was ostensibly dependent on numerous factors such as age of animals, number of tumor transfers, size of the tumor on the day of Eli-Full) administration, as well as the time interval between zymosan and gi_s-Pt(ll) In general the combination therapy, although promoting moderate numbers of tumor regressions, did not appear to have any significant effect in prolonging the MLS of non-surviving animals. This is in agreement with the work of Bradner at al. (1958), who found that Swiss mice bearing 8-180 and treated with zymosan either exhibited tumor regressions or died at essentially the same time as control animals. These authors also reported that the tumors of non-surviving zymosan- treated animals grew at essentially the same rate as those in control animals. In the experiments reported here, essentially the same phenomenon occurred. Those animals treated with zymosan and whose tumors did not regress had tumor growth curves which were essentially the same as in the untreated control animals (Figure 1). Similarly, those mice treated with combinations of zymosan and gifi-Ptul), but whose tumors did not regress, had tumor growth curves that paralleled those of animals treated with c_ig-Pt(II) alone (Figure 1). In evaluating the body weight curves (Figure 2) , it was noted that the animals treated with c_:l_§_-Pt(II) had a transient, but marked, 79 loss in body weight immediately following treatment. This appears to be typical of SigrPt(II) therapy since other investigators have made similar observations (Rosenberg and VanCamp, 1970). Although exhibit- ing a similar pattern of weight loss, the animals treated with zymosan and gigrPt(II) lost significantly less weight than those treated with gig:Pt(II) alone. The animals whose tumors eventually regressed were generally in better health, and this could explain their greater tolerance of the gig:Pt(II). 0n the other hand, those animals with progressively growing tumors also demonstrated less weight loss than those animals treated with the platinum compound alone. This would suggest that pre—treatment with zymosan protected the host from the subsequent toxic effects of gigrPt(II) to some degree. Fitzpatrick and DeCarlo (1964) reported that zymosan could decrease the lethal effects of total-body irradiation. They observed that, if zymosan was given prior to the irradiation of cancer patients, there was faster recovery from the radiation therapy and better hematopoietic recovery. This was believed to be due to an increased function of the RES. They made no mention of protection against weight loss, however. The relationship between zymosan and gig:Pt(II) in promoting tumor regressions is unknown. Zymosan is known to stimulate the production of macrophages, increase phagocytosis, elevate properdin levels and have an adjuvant effect on the production of antibodies to various antigens. gigrPt(II) exhibits cytotoxic effects against neoplastic cells but has immunosuppressive capabilities. Martin at al. (1964) sug- gested that the synergism between zymosan and cyclophosphamide in reducing the recurrence rate of spontaneous mammary tumors in mice was based on the cross-reactivity between antibodies produced by zymosan and antigenic determinants on tumor cells. This cross-reactivity was 80 believed to alter the integrity of the tumor cells, rendering them more susceptible to the cytotoxic action of the cyclophosphamide. Since gingt(II) is thought to resemble the alkylating agents in mechanism.of action, this hypothesis deserves mention. In the experiments reported in this manuscript, it was observed that occasional regressions were obtained with zymosan alone whereas SggyPt(II) was completely ineffective. Consequently it would appear that a more likely possibility would be that the platinum compound altered the integrity of the tumor cells making them more susceptible to zymosan stimulated host responses. The means by which zymosan stimulates host defenses to ultimately mediate tumor regressions is presently unknown. Bradner at al. (1958) reported that zymosan promoted tumor regressions in Swiss mice through the medium of host defense mechanism rather than by direct inhibitory action of the tumor. This conclusion was 'based on the following evidence: 1) the effect (tumor regression) was considerably delayed beyond the time the treatment was administered; 2) the response was quantal in nature, as opposed to the overall tumor suppressive action seen with many chemical agents; 3) zymosan was more effective at low doses than at high doses; 4) zymosan was not effective against 8-180 in tissue culture. In comparing Bradner's observations with those reported here with combination zymosan and §_i_s_-Ft(II) therapy, one notes definite similarities with regard to the delay in tumor regres- sions and the quantal nature of these regressions. Bradner and his co-workers ruled out the possibility of immediate direct stimulation of antietumor antibody by zymosan because treatment prior to tumor implantation, i.e., before the host had experienced tumor antigen, was still successful in promoting what appeared to be 81 tumor specific immunity. They also observed that the tumor loss effect could be abrogated within 48 hours by giving an initial low dose of zymosan followed by a high dose. The reversal phenomenon was cone sidered to be too rapid for a typical acquired antibody reaction. Finally, they reportedrthat animals receiving large doses of zymosan did not have a significantly higher rate of tumor regressions than untreated control animals. Thus it was hypothesized that the anti- tumor activities of low doses of zymosan were not due to the production of anti-tumor antibodies but rather to the production of some inter- mediary which altered the balance between tumor and host, shifting it in favor of the host. They speculated that this intermediary might be properdin. Numerous investigators have attempted to correlate properdin levels with anti-tumor activity, some even before Bradner's publica- tion. Southam.and Pillemer (1957) discovered that patients with advanced cancer had low properdin levels and readily accepted cancer cell homografts. Normal individuals who were capable of rejecting cancer cell homografts, however, had normal properdin levels. These investigators were quick to point out, however, that the ability to reject cancer hemografts in no way reflected the defense mechanism which controls the growth of spontaneous tumors. While Southam.and Pillemer worked with human patients, Herbut and Kraemer (1956) found that colonic carcinomas taken from humans could be transplanted into rate if multiple injections of zymosan were given prior to transplan- tation. They speculated that the zymosan reduced the levels of properdin leading to a loss of natural resistance to the heterografts. When properdin levels were quantitated, however, they were found to be so variable that the investigators concluded that natural resistance to 82 the transplanted tumor was not mediated through the properdin system (Herbut st 42., 1958). In addition to the transplantation experiments, some investigators attempted to elucidate the effect of properdin on tumor cells both in viva and in vitro (Sekiguchi at al. , 1962; Diller et a1. , 1963; Tokunaga at al. , 1962). From these studies it was generally concluded that properdin‘was not a significant factor in determining the outcome of neoplastic disease. Another possible mechanism by which zymosan promotes tumor regressions may be in its ability to stimulate the proliferation of macrophages. Alexander (1970) hypothesized that tumors are not rejected because there are too few suitable macrophages to produce appropriate levels of antitumor cytophilic antibody. He based his hypothesis on the fact that many skin tumors have disappeared if inflammation was induced in the vicinity of the tumor. Zymosan is known to be capable of causing macrophage proliferation and enhancing the phagocytic index in animals and humans bearing advanced tumors (Diller et a1. , 1963; Kampschmidt and Upchurch, 1968). Kampschmidt and Upchurch (1968) reported that zymosan was capable of markedly stimulating the reticuloendothelial system of tumor-bearing rats. Consequently, the authors suggested that, with appropriate stimula- tion, the normally depressed RES of tumor-bearing animals could be stimulated. Shinichiro and Shinoki (1968) suggested that the enhanced production of phagocytic cells in the peritoneal cavity after zymosan. administration was responsible for the ultimate regression of a rat ascites tumor. They found that transfer of these cells to unsensi- tized animals inhibited subsequent tumor implants. It was also noted that, at least in their experiments, the number of macrophages was 83 less important than the time at which they were given, e.g., 3 days prior to tumor implantation was most successful. A third possible mechanism by which zymosan might promote_tumor regressions is through the production of antibodies which, in turn, cross-react with tumor antigens (martin et aZ.,‘l964). Allegedly, heat stable agglutinins were produced against zymosan when it was injected in combination with an adjuvant into rabbits (Blattberg, 1957). It was also observed that zymosan enhanced the bactericidal activity of rabbit serum against E. coli B, and it was suggested that antibodies produced against zymosan might cross-react with antigenic determinants on the bacteria. Finally, zymosan may act as an adjuvant, enhancing the titers of anti-tumor antibodies. Cutler (1959) reported that zymosan enhanced hemolysin in rats particularly when administered 48 hours before, at the same time as, or 48 hours after antigen administration. As has been previously mentioned,.gigrPt(II), while demonstrating marked cytotoxicity for tumor cells, has also been shown to be an immunosuppressor. This is not surprising since virtually all cancer chemotherapeutic agents usually suppress immune responses. Recently, however, it has been reported that under certain conditions, many of these agents can, in fact, enhance immune responses. Buskirk at at. i (1965) reported that cytarabine and S-Fluoro-Z-deoxyuridine (FUDR) cause immunologic enhancement at high doses given as a single dose but suppression at low doses given daily. He also observed that uracil mustard and KTS were not immunosuppressors when given at thera- peutic doses. Chanmougan and Schwartz (1966) demonstrated that there was enhancement of immune response after the termination of a one-week treatment course of 6MP. Uracil mustard at therapeutic levels and 84 x—irradiation have also shown immunologic enhancement (Haines at al., 1967; Taliaferro, 1964). Schwartz (1967) reported that 6-MF, prednisone and amethopterin could, under given circumstances, aggravate the graft versus host reaction. This led him to suggest that virtually all cytotoxic materials can enhance immunologic responses. The mechanism by which these cytotoxic drugs elicit immunostimu- lation is obscure. It has been suggested, however, that the nucleic acids released from injured cells may stimulate lymphoid tissues which in turn enhance antibody formation (Chanmougin and Schwartz, 1966). Stolfi at al. (1971) proposed that the success of cancer chemothera- peutic agents in promoting tumor regressions may, in fact, have an immunologic basis. They suggested that, concomitant with tumor cell destruction, there is a drug-induced lymphoreticular depression followed by a period of lymphocytic propagation. Due to the tumorie cidal activity of the drugs, large amounts of tumor cell antigens should be available during this latter period. Thus, conceivably, the proliferating and differentiating lymphocytes could become immunologically committed to these antigens at this time. Currently, the subject of chemoimmunotherapy, i.e. , cowination chemotherapy and specific or nonspecific immunotherapy, is of great interest to the cancer therapist. Mathe (1971) recently reviewed the rationale behind its use. He cited numerous experiments in which various chemotherapeutic agents were used in combination with'both specific and nonspecific immunostimulants. In virtually all of the experiments, the combined therapy elicited greater numbers of cures or extended the life span to a far greater degree than either chemotherapy or immunotherapy alone. Mhthe suggested that the a priori fear that chemotherapeutic agents would negate the effects of immunostimulation 85 was not justified if the temporal relationship between the drugs and immunostimulants were appropriate. As an example, it was found that chemotherapeutic agents are much more immunosuppressive if given in daily doses than if administered as one injection. The effects of appropriate timdng were demonstrated by Chanmougin and schwartz (1966), who found that if rabbits were treated with 6-MP and then rested for 5 days they would produce hyperimmune responses to antigen. Currie and Bayshawe (1970) demonstrated the importance of timing between the adjuvant (Cbrynebaoterium pavum) and the chemotherapeutic agent (cyclophosphamide). They found that giving the adjuvant 12 days after a single dose of cyclophosphamide resulted in complete and lasting regressions in 702 of the animals but that the results were negative if the interval between adjuvant and drug was shortened or lengthened. Alexander (1970) concluded that, if immunotherapy was to be effective, the tumor size must be reduced since immunotherapy is most effective when relatively few tumor cells are present. Thus it was suggested that chemotherapy should precede immunotherapy. Mathé (1970) appeared to be in complete agreement with this philosophy and, in fact, stated that immunotherapy followed by chemotherapy is not theo- retically recommended. He based this on the premise that immunotherapy 'makes lymphocytes commence cyclic division, making them.more susceptible to destruction by chemotherapy. He noted that administration of Cbrynebaoterium pavum and ECG prior to chemotherapy was generally ineffective. The results of the experiments reported here appear to be in direct conflict with this notion. The most successful mode of therapy was when zymosan preceded gig:Pt(II). In concert with these observa- tions were those of Sokoloff at al. (1961). They found that zymosan 86 administered prior to Mitomycin C considerably increased the inhibitory effects of the drug against Sarcoma 180. This was not the case, however, when zymosan was administered at the same time as or after the drug. Since 8-180, a nonspecific transplantable tumor, was used in both the experimentsreported here and in Sokoloff's experiments, it could be concluded that there are behavioral differences between transplantable tumors and.syngeniec or autochthonous tumors. The credi- bility of this argument breaks down, however, with the report of Martin et a1. (1964). These investigators found that the most effective treatment regimen'invdecreasing‘the.recurrence rates of spontaneous mammary tumors in mice consisted of a combination of surgery, cyclo- phosphamide therapy and zymosan. Of particular interest is the fact that the initial injection of zymosan.was always given prior to surgery and the cyclophosphamide, i.e., days -9 to ~11, and that two other injections were administered during the period of cyc10phosphamide therapy. 'More recently-Martinqet al. (1970) found that zymosan enhanced the cure rates of spontaneous murine mammary tumors when used in combination with surgery and 4 chemotherapeutic agents, i.e., Streptonigrin, Thioguanine, Endoxan and Mitomycin C. Zymosan was administered in 5 injections: the first 3 days prior to surgery and the others at 14-day intervals. The last 4 injections were during the period in which the chemotherapeutic agents were being used. Conse- quently, it would appear that at least with zymosan, treatment prior to and/or during chemotherapy is of value. Taking into consideration the properties of zymosan and gig:Pt(II), the following hypothesis as to their combined mechanism of action can be made. It is proposed that, although zymosan is capable of increas- ing properdin levels, stimulating the RES and/or producing 87 cross-reacting antibodies, these augmentors of host responses are insufficient to promote tumor regressions in the majority of animals treated with zymosan alone. The addition of gig-Ptal) to animals previously treated with zmosan, however, may alter the integrity of the tumor rendering it more susceptible to subsequent attack by zymosan stimulated host defenses. Although varying numbers of tumor regressions were observed with virtually all combinations of zymosan and gig-Pull) therapy, the most consistently successful treatment regimen was found when zymosan was administered 7 days prior to the platinum compound. This suggests that, during this 7-day period, a population of lymphoid cells is produced which are less susceptible to the imnosuppressive effects of gig-Pun). The platinum compound is known to exert its maximum imunosuppressive effects within 2 days after antigen administration (Berenbaum, 1971) . Consequently, the shorter the interval between the administration of the compounds, the greater the chance of suppres- sing the immune responses stimulated by zynosan. In fact, experiments utilizing shorter intervals were less successful. Since S-lOO'is a nonspecific tumor, attempts to evaluate tumor specific immunologic responses were considered inapplicable. Conse- quently, the integrity of host responses in animals treated with combinations of zymosan and gg-Ptul) were evaluated indirectly by measuring responses to SRBC's and skin allograft rejection times. The results of the experiments measuring host responses to SRBC's were of some interest. In the studies in which the treated animals did not bear tumors, the only significant difference noted was that between animals receiving combination therapy and those receiving saline. This difference was only present in those animals who 88 received antigen on day 21 (Table 15). This suggests that there was a slight enhancement of immune responses with combined therapy as contrasted to zymosan or gigrPt(II) alone. The results of those experiments involving animals bearing 8-180 were even more rewarding. When the antigen was administered on day 14, those animals treated with saline or zymosan produced plaque- forming spleen cells far in excess of those treated with gigrPt(II) or combinations of the two compounds. This indicated that: 1) the immunosuppressive capabilities of gingt(II) were still prevalent at 6 days aftesggreatment and 2) pre—treatment with zymosan did not protect against the immunosuppressive effects of gingt(II). When the antigen was administered on day 21, however, the results were completely reversed. Those animals treated with saline or zymosan had marked reduction in.plaque numbers. This was particularly true in the former group. Those animals treated with gig:Pt(II) or combina- tions of the two drugs, on the other hand, had an enhanced plaque— forming ability as compared to the other groups. This was in spite of progressively growing tumors which, in their own right, are believed to be immunosuppressive (Esber et aZ., 1972). It is of interest to note that the enhancement in response to SRBC's was observed in both mice treated with gingt(II) and those treated with zymosan and gingt(II) and that there were no significant differences between the two treatment groups. The enhancement with the drug alone is suggestive of that noted with other chemotherapeutic agents pre- viously described. The fact that combined therapy did not elicit greater responses in those animals with progressively growing tumors is prdbably based on the status of the tumor. The animals that did not respond favorably to the treatment in regard to either the tumor 89 or the SRBC antigen might be considered "poor reactors." On the other hand, those animals whose tumors were regressing appeared to respond more favorably to the antigen. However, one cannot rule out the possibility that the enhanced response to SRBC's in this latter group may have been due to a nonspecific response. Since these animals were responding favorably to the tumor, it would be suspected that their RES activity might be accelerated; thus, their response to SRBC's might be enhanced as well. When cell mediated immunity was evaluated, a different set of responses was observed. In these experiments the only treatment to accelerate skin allograft rejection was the combination of zymosan and gi§7Pt(II). This acceleration was noted only in those animals bearing 8-180. In these experiments it appeared that combination therapy was capable of enhancing allograft rejection, and the status of the tumor did not appear to play a significant role. Because of the number of animals dying prior to graft rejection, however, the significance of these data is questionable. The histologic changes in the lymphoid organs and tumors taken from animals treated with saline, zymosan, giggPt(II) or combinations of zymosan and gigrPt(II) were nonspecific. They were dependent on the status of the tumor and independent of the type of treatment. The hyperplastic response of spleens and regional lymph nodes was marked in all groups and indicated that, even in the controls, the 8-180 tumor evoked a host response. In those animals with progressively growing tumors, the thymuses were atrophic. On the other hand, those animals with" regressing tumors had normal appearing thymuses. Of particular interest were the cellular reactions in regressing tumors. These were compatible with those found in a homograft rejection 90 irrespective of treatment. Consequently, it was concluded that the success of the therapeutic regimen was not dependent upon the speci- ficity of the treatment, but rather the ability of the drug or combina- tion of drugs to alter the balance between the tumor and host so that it was favorable to the host. SUMMARY The role of host defenses in mediating the regression of Sarcoma 180 (8-180) tumors in mice treated with Sigrnichlorodiammineplatinue(II) [gigrPt(II)] was investigated. It was found that the marked anti-tumor efficacy of gigyPt(II) against 8-180 implanted in Swiss mice was reduced when hydrocortisone (HO), an immunosuppressive drug, was admonistered 7 days before, 6 hours after or 7 days after the platinum compound. This reduction was most dramatic when BC was administered 7 days before or 6 hours after the p1atinum.compeund. In another series of experiments it was found that gigrPt(II) was ineffective in promoting regressions of 8-180 implanted in.BALB/c mice. The administration of zymosan, a nonspecific immune stimulant, in combination with gigrrt(ll), however, promoted significant numbers of tumor regressions. This was particularly true if the zymosan was administered on day l of tumor growth followed in 7 days by a single injection of SigrPt(II). Prom.the results of the previously described experiments it was concluded that the anti-tumor efficacy of gi§7Pt(II) is, at least in part, dependent on an active host response directed against the tumor. The immunologic integrity of BALB/c mice treated with a combina- tion of zymosan and Sigrrt(ll) was studied. Humoral antibody produc- tion was evaluated by the agarose-slide technique using sheep red blood cells as antigen. There was virtually no difference in spleen 91 92 cell plaque forming ability between control and treated animals when the antigen was administered on day 14 of the experiment. When the antigen was adndnistered on day 21 of the experiment, however, those animals treated with a combination of zymosan and gigrPt(II) had sig- nificantly higher numbers of plaque forming spleen cells than the saline treated animals. Similar studies were performed on animals bearing 8-180. When antigen was injected 14 days after tumor implantation, plaque forming cells were found in significantly higher numbers in the animals treated with zymosan or saline than in those treated with gigrPt(II) or SggyPt(II) plus zymosan. When antigen was administered on day 21 of the experiment, however, those animals treated‘with.gigrPt(II) or ‘gigrPt(II) plus zymosan had significantly higher numbers of plaque- forming cells than the other two groups. Thus it was concluded that, depending on the time of antigen administration, treatment with gigrPt(11) or zymosan plus gigrPt(II) may have some stimulatory effect on humoral antibody responses. Cell mediated immune responses were evaluated by skin allograft rejection. Allografts were rejected earlier in animals bearing 8-180 and treated with a combination of zymosan and gigrPt(II). Consequently, it was concluded that combination therapy may stimulate cell mediated immune responses. Selected tissues from animals bearing 8-180 and treated with saline, zymosan, gingt(II) or a combination of zymosan and gigrPt(II) were evaluated histologically. Spleens and regional lymph nodes from all groups were markedly hyperplastic, particularly in the marginal zones of the lymphoid follicles. The thymuses of animals with regres- sing tumors had a normal appearing architecture with a somewhat hyper- plastic cortex. In contrast, the thymuses of those animals with 93 nonregressing tumors were atrophic and the demarcation between cortex and medulla was obscured. Regressing tumors, regardless of treatment, were characterized by marked lymphocytic infiltration and were surrounded by a proliferating fibrous capsule similar to that seen in homograft rejection. This suggested that ultimate tumor regression may be mediated via immuno- logic mechanisms rather than a specific attack on tumor cells by the therapeutic agents. 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Cong. on Chemo., Avicenum, Prague, 1971: in press. Wardlaw, A. C., and Pillemer, L.: The properdin system and imnity. J. Exptl. Med., 103, (1956): 553-575. ‘Welsch, C. W.: Growth inhibition of rat mammary carcinoma induced by gig-Platinum diamminodichloride. J. Natl. Cancer Inst., 47, (1971): 1071-1078. ‘Wexler, M. R.: Simple method for skin grafting in mice. Israel J. 104 Woodman, R. J., venditti, J. M., Schepartz, S. A., and Kline, I.: ICRF-159 (1,2-Bis (3,5 Diosopiperazin-l-YL)-Propane) Activity against intracerebrally inoculated (NSC-129943). L1210. Therapeutic superiority against IPL1210 in combination with gig-Platinum(II) diamminodichloride (gig-Pt(II): NSC- 119875). Proc. Am. Assoc. Cancer Res., 12, (1971): 24. VITA VITA The author was born July 29, 1939, in Healdsburg, California. His primary and secondary education was completed in that locale in 1957. The author entered the University of California at Davis in September, 1957. While enrolled at this institution he married Phyllis Ann Loonan at Healdsburg, California, in January, 1964. In June, 1964, he received the D.V.M. degree. The author was commissioned as a let Lieutenant in the United States Army in September of 1964 and was stationed at the Armed Forces Institute of Pathology until August of 1968. During this time he was Assistant Chief of the Vivarium and a resident in the Veterinary Pathology Division. In August of 1968, the author accepted a position as research pathologist for the Dow Chemical Company. The author enrolled in the Department of Pathology at Muchigan State University in June of 1969 and was granted a departmental post- doctoral fellowship. From 1970 through 1972 he received a postdoctoral fellowship from Engelhard Industries anthatthey Bishop, Inc., and performed his research in the Department of Biophysics. The author is a.member of the A.V}M.A., A.C.V.P., and Sigma Xi. 105 "Imlfl'lfllfiflliilfiiilifllfifi'ias