IMMUNIZATION AGAINST EHRLICH MOUSE ASCITES CARCINOMA WITH CHEMICALLY DEVITALIZED TUMOR CELLS *

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1 IMMUNIZATION AGAINST EHRLICH MOUSE ASCITES CARCINOMA WITH CHEMICALLY DEVITALIZED TUMOR CELLS * MASAHIRO YOSHIMURA AND TOSHIKO KABURAKI Department of Pharmacology, Research Institute of Tuberculosis, Kanazawa University, Kanazawa Received for publication November 19, 1962 Attempts to induce immunity against Ehrlich ascites carcinoma in inbred mice have been the subject of considerable studies in recent years (1-4). Various procedures were employed with varying degrees of success to prepare altered cancer cells which would be effective in producing resistance to subsequent transplant of the carcinoma cells. McKee et al. (5) failed to induce immunity by pretreatment of mice with Ehrlich ascites cells that had been killed by desiccation, freezing and thawing, mechanical grinding, and supersonic waves. Revesz (6), and Donaldson and Mitchell (7) were successful to protect mice by immunization with X-irradiated carcinoma cells. Recently, Donaldson and North (8) demonstrated that repeated injections of tumor cells inactivated with nitrogen mustard resulted in a high degree of immunity. The present report deals with attempts to induce immunity in dd strain of mice against Ehrlich ascites carcinoma by use of cells treated with various chemical agents. METHODS Animals : Male dd mice, weighing 17 to 21 g, were used in this study. They were kept on a standard pellet diet and water supplied ad libitum. Tumor : Ehrlich ascites carcinoma was maintained in dd mice by weekly intraperi toneal injections of 0.3 ml of ascites exudates. The tumor, when injected intraperitone ally in a dose of 8,000-10,000 cells, grew progressively in the mice, killing the hosts in about 30 days. Tumor cells used for experiments were withdrawn from 2 or more mice that had received intraperitoneal transplants of 2 x 10' cells per mouse 7-10 days pre viously. The pooled ascites fluids were centrifuged for 10 minutes at 1,500 rpm and then washed three times with Krebs solution. The packed cells were suspended in Krebs solution to one-half of the original ascites volume. For challenge transplants the suspension was diluted with Krebs solution, so that 1 ml contained about 3 x 104 cells. Efforts were made to use aseptic technic and to maintain a temperature of not over 5 C throughout the procedure. * This work was supported, in part, by grants from the Japan Anti-Cancer Society, the National Institute of Health, U. S. A., and the Tokyo Biochemistry Research Fund.

2 Tumor cells employed for immunization were prepared by treating the washed tumor cells with the reagents to be studied, under conditions specified below and, after thorough washing, by resuspending them in Krebs solution to give a concentration of 103 cells per ml. Immunization Tests: In each experiment were 2 0roups of 'mice, controls and test animals. Test animals received a single intraperitoneal injection of 0.5 ml of a chemical ly treated tumor cell preparation. Three weeks later the mice were given intraperi toneally a challenging dosage of non-treated viable tumor. An equal number of normal control mice also received the challenge injection. Mice were examined daily for deve lopment of tumor, and death, if occurred, was noted. Experiments were terminated at 90th day after the challenge injection. Animals dying during experimental period and surviving at the termination of experiment were all autopsied,' and gross pathological incidence of organs was observed. Smears made from peritoneal cavity were stained with May-Grunwald-Giemsa solution and examined microscopically for tumor cells. Mice which died from obvious intercurrent disease or by any accident were omitted from consideration. Reagents : The che_ Tlical agents used for preparation of tumor vaccines were (a) acetic anhydride, (b) monoiodoacetic acid, (c) methyl-bis-(q-chloroethyl)-amine-n-oxide (nitrogen mustard N-oxide ; Ni'tromin), and (d) sodium nitrite. RESULTS L Effect of Chemical Agents on Transplantability of Ehrlich Ascites Carcinoma Cells The first series of experiments was designed to establish the dosages of agents neces sary to prepare nonviable tumor vaccine. The conditions employed for chemical treat ment were selected so that upon microscopic examination of tumor cells the alteration of nuclear appearance was minimal. Washed tumor cell suspension was treated with the respective agents as follows : (a) Acetic anhydride. To 20 ml of chilled tumor cell suspension were added 20 ml of a saturated sodium acetate solution and subsequently 2.4 ml of acetic anhydride. The mixture was kept in an ice-water bath for 30 minutes to 2 hours. (b) Monoiodoacetic acid. One part of tumor cell suspension was treated with one part of 0.2 M monoiodoacetate in Krebs solution (adjusted to ph 6 with N-NaOH) at 0 C for 30 minutes to 2 hours. (c) Nitrogen mustard N-oxide (Nitromin). Three different concentrations of Nitromin, i.e., 50, 5, and 0.5 mg/ml, were used. Nitromin was dissolved' and diluted in Krebs solu tion. Nitromin-treatment of tumor cells was carried out at two different temperatures, 0 and 3TC. An aliquot of Nitromin solution was added to an equal amount of tumor cell suspension. The mixture was kept at a temperature for definite periods of time, as indicated in Table 1. (d) Nitrous acid. HNO2-treatment was carried out in ice-water bath. One part of tumor cell suspension was mixed with one part of 4 M NaNO2. The mixture was then

3 TABLE 1., Effect of chemicals on transplantability of Ehrlich ascites carcinoma cells in dd mice. treated with gradual addition of two parts of 0.5 M acetate buffer '(ph 4.0) and allowed to stand in the cold for 15 or 60 minutes. To insure uniformity of chemical exposure, the reaction mixture obtained was at once transferred into another flask, and the reaction proceeded under slow stirring. After completion of chemical treatment, the tumor cells were thoroughly washed and finally resuspended in the original volume of Krebs solution. The chemically treated tumor cells thus prepared were tested for their transplantability by injecting intraperitoneally 0.5 ml (5 x 10' cells) of the suspension into dd mice. In every experiment were included control groups of animals which were given an equivalent quantity of non-treated tumor cells that had been kept under identical conditions without addition of the reagents. The animals were carefully observed to determine the development of tumor according to the scheme described above. Tests were terminated., after 90 days. The results of these viability tests are summarized in Table 1. It is seen from the table that under conditions employed, the tumor cells were readily inactivated by any one of the chemicals tested. Thus, only 2 out of 7 mice injected with cell preparation exposed to 6% acetic anhydride for 30 minutes died of ascites tumor within 90 days after inoculation. Increasing the incubation time with the agent to 1 hour resulted in a complete loss of virulence. The virulence of tumor was not significantly influenced by a 30-minute exposure to 0,1 M iodoacetate, but a l-hour exposure was enough to render

4 the cells totally inactive. The effect of Nitromin varied considerably with temperatures ; the viability of carcinoma was hardly affected by a 1-hour exposure to Nitromin (25 mg/ml) at 0 C, while the cells were completely inactivated following a 15-minute ex posure at 37 C. At lower concentrations (2.5 mg/ml or 0.25 mg/ml) Nitromin exerted little or no effect upon the transplantability of tumor. A 15-minute incubation of the tumor cells with 1 M nitrous acid at 0 C resulted in a total loss of transplantability. II. Immunization Experiments with Chemically Devitalized Tumor Cells Being considered the results of the viability tests aforementioned, the following pre parations of tumor cells were used for immunization : the tumor cells treated with (a) 6% acetic anhydride at 0 C for 1 hour, (b) 0.1 M monoiodoacetate at 0 C for 1 hour, (c) Nitromin (25 mg/ml) at 37TC for 15 minutes, and (d) 1 M nitrite at 0'C for 15 minutes and 1 hour. Groups of dd mice received intraperitoneally 0.5 ml (about 5 x 10' cells) of one of these chemically devitalized tumor vaccines. Control groups of non-vaccinated animals were included in every experiment. Three weeks after vaccination, the mice were chal lenged for immunity by intraperitoneal injection of viable cells. The transplants grew progressively in most of the vaccinated animals except in the groups of animals immunized with the HNO2-treated tumor cells, but there were considerable differences in survival times of mice among the respective vaccinated groups. Results are tabulated in Table 2. TABLE 2. Immunization of dd mice against Ehrlich ascites carcinoma with chemically devitalized tumor cells. As can be seen in the table, mice immunized with iodoacetate-treated tumor pro duced death in all but 2 of 6 mice with a statistically insignificant prolongation of survival time, compared to that of the controls (P>0.02). Mice immunized with acetic

5 anhydride-treated cells produced death in 10 of 12 animals but with a markedly pro longed survival time (P<0.01). Similarly, survival time of mice vaccinated with Nitro min-treated cells was significantly longer than that of the controls (P<0.01), although death occurred in 6 of 8 mice. In contrast to these results, mice immunized with nitrite treated tumor showed sufficient resistance to protect nearly 50 per cent of animals from lethal inoculum of viable tumor. The data, further, indicated no significant difference in the immunizing potency between the 15-minute and 1-hour HNO,-treated tumor vac cines. DISCUSSION The results presented here showed that the Ehrlich ascites carcinoma cells lethally damaged with various chemical agents differed widely in the potency to induce resistance to subsequent transplant of viable untreated tumor cells. This diversity of immunizing effect may be presumably attributed to possible antigenic differences of the chemically altered tumor cells. In this respect, however, it should be considered that, as shown by other workers, the degree of immunity induced by vaccination of devitalized tumor cells deeply depends upon the dose of agents used. Thus, in the Donaldson and North's ex periments (8) a certain amount (0.01 mg) of nitrogen mustard was shown to be highly effective in rendering Ehrlich carcinoma cells antigenic, although larger quantities reduced the effect. Larson (4) demonstrated that Ehrlich ascites cells treated with doses of 2,000 to 4,000 r of X-irradiation gave an optimum production of resistance in mice, while 8,000 r was ineffective. In our experiments different amounts of each of the chemicals were not studied in detail so that optimum conditions for individual agents may not have been achieved. The fact that nitrous acid was more effective than any other chemicals tested, seems to be of particular interest, when considered together with our previous observations (9, 10) that the nitrous acid-killed vaccine of a virulent strain of human tubercle bacillus produced a high degree of immunity against subsequent challenge infection in guinea pigs. Because of the complexity of interactions of nitrous acid with cell constituents, including not only proteins but also nucleic acids, mechanisms responsible for its effec tiveness upon cellular and bacterial antigenic potentialities remained yet obscure. Attention may be directed to a significant degree of protection induced by Nitromin devitalized cells, since this indicates the possibility that the lethally damaged tumor cells occurring in vivo during the course of chemotherapeutic treatment of experimental cancer in animals may stimulate immunobiologic responsiveness in the hosts and thereby exert, at least partially, its beneficial effect on regression of tumor. In numerous attempts, going back to the early period of cancer research, to treat cancer patients by active immunization, some investigators have unsuccessfully utilized formalinized or phenolized autologous cancer tissue to vaccinate patients (11). Ass stated by Witebsky (12), a successful attempt to immunize against cancer, perhaps, depends upon the occurrence of antigen which the host would refuse to recognize as self. At

6 any rate, ever increasing number of successful immunization attempts by vaccination of altered tumor cells against experimental tumors, as exemplified by the experiments presented here, together with the occasionally reported clinical observations of spontane ous regression of malignant tumor in patients, encourage us with the impression that the prospect of active immunization seems still promising in the study for controlling cancer. SUMMARY Ehrlich ascites tumor cells were exposed to the action of acetic anhydride, mono iodoacetic acid, Nitromin and nitrous acid. Tests for viability by transplantation of the treated tumor cells to dd mice showed that tumor cells lost their transplantability fol lowing exposure to (a) 6% acetic anhydride at 0 C for 1 hour, (b) 0.1 M monoiodoacetate at 0 C for 1 hour, (c) Nitromin (25 mg/ml) at 37TC for 15 minutes, and (d) 1 M nitrite at 0 C for 15 minutes. Immunization experiments carried out with the lethally damaged tumor cells revealed (a) that a single intraperitoneal injection of the nitrite-inactivated cells caused an en hanced resistance against subsequent viable transplants, resulting in reduction of the mortality to about 50 per cent of the recipients; (b) that similar pretreatment either with the Nitromin or acetic-anhydride inactivated cells caused some prolongation of survival times of mice; (c) and that, in contrast to these positive findings, no significant degree of protection was demonstrated following prior treatment of mice with the monoiodo acetate-inactivated cells. Acknowledgement : The authors wish to express their sincere gratitude to Prof. H. Okamoto, Department of Pharmacology, School of Medicine, and Prof. R. Ito, Depart ment of Pharmacology, Research Institute of Tuberculosis, Kanazawa University, for their encouragement, kind advice, and revision of the manuscript. REFERENCES 1) WOLGLEM, W.H.: Cancer Rev. 4, 129 (1929) 2) HAUSCHKA, T.S.: Cancer Res. 12, 615 (1952) 3) MILGROM, F. : Ibid. 21, 862 (1961) 4) LARSON, W.M.JR., SLATER, C.R. AND McKEE, R.W. : Proc. Soc. exp. Biol., N.Y. 108, 140 (1961) 5) McKEE, R.W. et al.: Ref. DONALDSON, D.M. AND MITCHELL, J.R. (7) 6) REvEsz, L.: J. Nat. Cancer Inst. 20, 1157 (1958) 7) DONALDSON, D.M. AND MITCHELL, J.R.: Proc. Soc. exp. Biol., N.Y. 101, 204 (1959) 8) DONALDSON, D.M. AND NORTH, J.A. : Fed. Proc. 19, 392 (1960) 9) YOSHIMURA, M.: Jap. J. Tuberc. 4, 145 (1956) 10) MUKOZAKA, K.: Ibid. 5, 101 (1957) 11) SOUTHAM, C.M.: Cancer Res. 21, 1302 (1961) 12) WITEBSKY, E. : Ibid. 21, 1216 (1961)