Int. J. Cancer: 11, 186-190 (1973) THE EFFECT OF SPLENECTOMY ON FIBROSARCOMA “METASTASES ” IN LUNGS OF MICE! Luka Mitas * and Hamza Musacic Laboratory for Transplantation and Tumour Immunology, Institute “ Rudjer Boskovie ”, and Central Institute for Tumours and Allied Diseases, Zagreb, Croatia, Yugoslavia Cells of @ methylcholanthrene-induced fibrosarcoma, injected intravenously into syngeneic C37BI mice, generated tumour nodules (“ metastases”) in the lungs. The number of tumour nodules was reduced in recipients whose spleens had been previously removed. This effect of splenectomy was specific (thymectomy did not affect the number of pulmonary metastases), and was greater if fewer tumour cells were injected. It was also observed that active immunization against the fibrosarcoma caused stronger inhibi- tion of pulmonary metastases in splenectomized than in sham-operated mice. One of the reasons for progressive growth of an antigenic tumour is the ability of serum anti- bodies to protect tumour cells from a destructive effect of immune lymphocytes (Hellstrém and Hellstrém, 1969, 19705; Hellstrém er al., 1969; Bubenik et al., 1970; Jagarlamoody e7 al., 1971). As the spleen is the major organ producing circulating antibodies (Rowley, 1950; Adler, 1965) it might play an important role in this tumour enhancement. Removal of the spleen was found to decrease humoral anti-tumour antibody responses and inhibit the growth of several trans- plantable tumours (Méller, 1965; Ferrer, 1968a,b; Ferrer and Mihich, 1968; Pollack, 1971). Also, sera from splenectomized tumour-bearing mice were less effective in blocking the destruction of ‘tumour cells in vitro by immune lymphocytes than. sera from sham-operated tumour hosts (Hell- strém er al., 1970). In this report we describe a marked inhibitory effect of splenectomy on the ‘umber of experimental metastases generated in lungs of mice by syngeneic methylcholanthrene- induced fibrosarcoma cells inoculated intra- venously. The assay of lung metastases was first described as a quantitative test for transplantabi- Received: July 20, 1972. lity of tumours in rats by Williams and Till (1966), and in mice by Hill and Bush (1969) in studies of the radiation response of solid tumours. MATERIAL AND METHODS Mice Male mice of C57BI/H inbred strain were used when 3-4 months old. They were produced in our Institute's breeding unit by strict brother x sister mating. The animals were maintained on a standard pellet diet and tap water ad libitum, Tumour The studies were carried out with third- generation isotransplants of a fibrosarcoma originally induced by methylcholanthrene in a CS7BI mouse (Milas e¢ al., 1972). Tumour-cell suspension Suspensions of tumour cells used for im- munization were prepared by mincing non- necrotic tumour pieces and passing the mince through nylon gauze, Resulting cells were centrifuged three times for 5 min at 185x¢ in fresh saline (Milas e¢ al., 1970). + Presented at the Seventh Congress of the Yugoslav Physiological Society (Symposium on Immunology), Belgrade, 26-29 September, 1971, * Present address: Section of Experimental Radiotherapy, The University of Texas, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025, USA. 186SPLENECTOMY AND PULMONARY METASTASES ‘The suspension for intravenous injection was prepared by trypsin digestion of minced tumour tissue. Approximately 1 g of tissue was mixed with 20 ml of 0.25% trypsin solution in saline and stirred in a magnetic stirrer for 30 min, Non- digested tumour tissue was allowed to settle in the beaker, and the upper two thirds of the suspension were removed and centrifuged as above. Viability of the cells was over 90% as determined by trypan blue exclusion, ‘Spleen-cell suspension Spleen suspensions were prepared by teasing and filtering the tissue through nylon gauze (Milas ef al., 1970). Viability of the cells was over 80%. Immunization The mice were immunized by three consecutive injections of 2 x 107 heavily irradiated (10,000 R) tumour cells at 7-day intervals. The first injection was given SC into each inguinal and axillary region, The second and third injections were administered IP. Splenectomy Mice were anaesthetized with Evipan Sodium (* Bayer ”) given intraperitoneally at a dose of 1 mg/l0 g body weight. Through a left lateral incision the spleen was gently exteriorized, and the pedicle tied with silk. The wound was closed with silk sutures for the peritoneal and skin incisions. Control animals were similarly operated, but the spleens were not removed (sham- splenectomy). Thymectomy A longitudinal skin incision was made on the midline of the neck of mice anaesthetized as above. The muscles were slightly pushed aside with ophthalmic forceps, a glass tube was inserted behind the sternum and both thymic lobes were removed by gentle suction. The wound was closed by suturing the skin, Mor tality with this operative procedure did not exceed 10%, Transfer of immunity with cells of sera One week after immunization with tumour cells the mice were bled (see below) and cell suspensions prepared from their spleens. Spleen cells were mixed with tumour cells at a ratio of 2,000:1 and incubated at 37°C for 1 h. The admixture was then diluted with Hank’s solution so that each mouse received intravenously 5 x 10 viable tumour cells plus 10° viable spleen cells in 1 ml of medium. The same was done with the spleen cells from normal controls. Sera from tumour-immunized and normal mice were prepared as previously described (Milas er al., 1972), The sera were added to viable tumour cells in such a way that 0.3 ml of serum corresponded to 0.1 ml of media containing 5x10! viable tumour cells. After 1 h of incubation at 37°C, the mixture was diluted with media to contain $10! tumour cells in 1 ml and in- jected intravenously into recipients. Tumour nodules (“ metastases” ) in lung Tumour cells suspended in 1 ml of Hank’s solution were injected into the lateral tail vein of mice. Twenty-five days later the recipients were killed and their lungs removed and fixed in Bouin’s solution. Tumour nodules were counted as white round-shaped nodules on the surface of the yellowish lung under a dissecting microscope (magnification 6 times). Statistics The results were statistically evaluated accord- ing to Student's Hest. RESULTS Mice were splenectomized and three weeks thereafter inoculated intravenously with viable fibrosarcoma cells, In the lungs of these mice tumour cells generated more “ metastases ” than in lungs of sham-operated mice: twice and three times more after injection of 5 x 10* and 2.5 x10" tumour cells respectively (Table 1). In the following experiment we investigated the effect of specific immunization against the fibrosarcoma on pulmonary metastases in splen- ectomized mice. Immunization of the mice was begun a week after splenectomy, and a week after it was completed, the mice were injected with viable tumour cells. Table II shows that immuni- zation induced significantly higher resistance in splenectomized than in control mice. Thus, 5 x 10* tumour cells generated 7.4.1.1 tumour nodules in the lungs of splenectomized and immunized mice as compared with 16.4:£1.5 nodules immunized sham-operated mice. 187MILAS AND MUJAGIC TABLE I NUMBER OF FIBROSARCOMA NODULES IN LUNGS OF NORMAL OR SPLENECTOMIZED MICE Number of tumour [Number of tumour nodules in lung aber of um Stes (end number) of recipients = P (tes) . Sham-spleneetomized (9) 26-29 46.653 7 eee Splenectomized (10) 9-46 23.9437 Su s 2 ‘Sham-splenectomized (11) 13-46 26.1-43.2 esl, Splenectomized (11) 1-18 9041.7 nod TABLE IT SPECIFICITY OF SPLENECTOMY, AND THE EFFECT OF ACTIVE IMMUNIZATION OF NORMAL AND SPLENECTOMIZED MICE ON THE NUMBER OF FIBROSARCOMA NODULES IN LUNG Number of tumour nodules in ung Status (and number) of recipients (eres) Range Mean: SE Shamsplenectomized (9) 50-92 9.3248 Splenectomized (10) 54.244.0 <0.05 ‘Thymectomized (9) Poe 44-98 76.1463 NS1 ‘Thymectomized +-Splenectomized (7) 37-59 46.3:.3.5 <0.005 Immunized to tumour 16.4415 Splenectomized+Immunized to tumour (11) <0.001 = <0.001 =0.001 TAdLd "Not significant. Thymectomy performed 4 weeks before tumour transplantation had no effect on the number of pulmonary metastases. Thymectomy plus splenec- tomy produced the same effect as splenectomy alone (Table 11). Table III shows the effect of lymphoid cells and sera from tumour-immunized and normal mice on the number of experimental metastases in Tung. Tumour cells mixed with immunized lymphoid cells produced significantly fewer colonies (p<0.001) than did tumour cells alone or a mixture of tumour cells with non-immunized lymphoid cells. On the other hand, tumour cells incubated in serum from tumour-immunized animals generated more tumour nodules in comparison with tumour cells suspended in media (p = 0.05). A similar increase in pul- monary metastases, although not significant, was obtained with tumour cells incubated in serum from normal mice. DISCUSSION Pulmonary metastases are a suitable index for quantitating and anti-tumour immunological reaction. The number of metastases is reduced in 188 specifically immunized mice when compared to that in normal mice (Milas and Withers, 1970; Milas and Mujagic, 1972), and the reduction shows a linear dose response (Silobréic and Milas, 1972). The test was found to be more sensitive than the subcutaneous transplantation of tumour cells (Silobréic and Milas, unpublished data) and enables us to study the immunity of tumour metastases. Using this test in the present experiments we demonstrated that the spleen plays an important role in tumour growth, Removal of the spleen markedly inhibited development of fibrosarcoma “metastases” in the recipient’s lung. This is in agreement with the results obtained with tumours growing in the subcutaneous tissues (Moller, 1965; Ferrer 1968a,b; Ferrer and Mihich, 1968; Pollack, 1971). The tumour-protective effect of the spleen depended on the number of injected cells: i.e., the fewer the cells injected, the better the protection, Furthermore, the effect was specific for the spleen, since thymectomy did not affect the number of pulmonary metastases. Also, tumour metastases were much more inhibited in actively immunized mice which were previouslySPLENECTOMY AND PULMONARY METASTASES TABLE UL THE EFFECT OF SYNGENEIC SENSITIZED CELLS OR IMMUNE SERA ON THE NUMBER OF FIBROSARCOMA NODULES IN LUNG Number of tumour nodules in tung (Cet injections Number of (test) i" eelpients Range Mean :SE x Tumour cells ue, 9 22-62 44.3439 Tumour cells+-spleen cells from mice im- munized to tumour : 10 6-22 1541.7 <0.001 ‘Tumour cells+spleen cells from normal mice 10 27-46 35.7419 NS* Tumour cells +serum from mice immunized to tumour ae 10 42-62 S3SL19 Tumour cells +serum from normal mice. . 10 28-60 49.643.1 1 Not significant. splenectomized than in sham-operated mice immunized to tumour. These results, however, do not define the mechanism(s) whereby the spleen promoted tumour metastases. Having in mind the fact that the spleen represents the largest organ involved in immune responses in mice (Bard and Pilch, 1969), particularly in humoral responses (Rowley, 1950; Adler, 1965), it could be antici- pated that inhibition of tumour metastases in splenectomized mice was also immunological in nature. Spleen cells from mice immunized against the fibrosarcoma were cytotoxic to the tumour cells, but the serum from these mice showed neither cytotoxic nor enhancing activity when mixed with tumour cells. This agrees with the observations of Hellstrém and Hellstrém (19704) that the sera from tumour-immunized mice do not possess blocking activity against tumour cells grown in vitro, On the other hand, these investigators observed that sera from individuals with tumours did prevent destruction of tumour cells by immune lymphocytes (Hell- strém and Hellstrém, 1969, 1970; Hellstrém et al., 1969), but the protection was less pro- nounced than with the sera from splenectomized tumour-bearers (Hellstrém er al., 1970). The serum blocking activity seems to be due to antigen-antibody complexes formed in the presence of the growing tumour (Sjégren er al., 1971). Therefore, we suppose that the tumour Protective effect of the spleen in the present experiments might be explained by the production of humoral antitumour antibodies which formed antigen-antibody complexes in mice, even im- munized ones, in which the tumour was growing. Another possibility might be that removal of the spleen altered the distribution of tumour cells within the mice affecting the growth of tumour nodules in lung by a non-immunological means. Further experiments are, however, needed to elucidate the exact role of the spleen in the pro- motion of pulmonary metastases, ACKNOWLEDGEMENTS The authors express their great appreciation to Mrs. Adela Lechpammer and to Mrs. Nevenka Ujgic for their excellent technical assistance. L'EFFET DE LA SPLENECTOMIE SUR LES “ METASTASES” DU FIBROSARCOME DANS LES POUMONS DES SOURIS Les ceilules d'un fibrosarcome induit par le méthylcholanthréne, injectées par voie intraveineuse a des souris syngénéiques C57BL, ont formé des nodules tumoraux (“ Métastases”) dans les poumons des hotes. Le nombre de nodules tumoraux était plus faible chez les souris splénectomisées. Cet effet de la splénectomie est spécifique; la thymectomie n'a pas d'influence sur le nombre des métastases pulmonaires. 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