Potentiation by Carbon Tetrachloride of the Immunosuppressive ERects of Fibrosarcoma and Toxic Material Produced by Fibrosarcoma Cells A. O. B. Wong, R. Mankovitz, and J. C. Kennedy 3,

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4,5

SUMMARY-SaD2-AG fibrosarcomas growing in DBA/2 mice caused nonspecific immunodepression. Injection of soluble material produced by cultured SaD2-AG cells into normal DBA/2 recipients caused nonspecific immunodepression and signs of systemic toxicity. These in vivo effects were enhanced by prior treatment of the recipients with carbon tetrachloride (CCI4). In contrast, material released by syngeneic fibroblasts was neither immunosuppressive nor toxic to normal mice or mice previously treated with CCI4. A soluble toxic material released by the fibrosarcoma cells may be responsible for at least some of the systemic effects of localized SaD2-AG tumors growing at a site not involving vital structures, and a protective host mechanism whose maximum capacity can be decreased by the administration of CCI4 may minimize the toxic effects of the cancer-derived material.-J Natl Cancer Inst 55: 1113-1118, 1975.

It is a common observation that severe systemic abnormalities may be associated with small and apparently localized cancers. Although certain types of cancer may produce material with hormone-like activity (1), the spectrum of systemic abnormalities usually observed in cancer patients cannot readily be attributed to the activity of any known hormone. These abnormalities may include depressed immunologic reactivity (2-5), depressed hematopoiesis (6-8), decreased liver catalase activity (9-11), abnormal iron metabolism (7, 11, 12), depressed formation of certain muscle proteins (13, 14), and lower production of various hormones (11, 15, 16). Many of these apparently unrelated systemic effects of cancer may be caused by a nonspecifically toxic material released into the circulation by the malignant cells (17). Material extracted from tumors by chemical treatment or released by tumor slices in short-term cultures can reproduce in vivo some systemic abnormalities associated with the malignancy itself (11, 18-21). However, since tumors often contain many reactive host cells in addition to the cancer cells (22, 23), it is not certain that the material in question was produced or released by the malignant cell component of the tumors. The question of whether the host or the cancer cells produce certain biologically active materials found in body fluids of patients or animals with cancer (24-37) is likewise unsettled. Although the effect of material found in the supernatants of pure cultures of cancer cells was tested in various in vitro systems with varying results (38-40), there have been no reports that material produced by pure cultures of cancer cells can reproduce in vivo any of the systemic effects of the corresponding in vivo cancer. We reported (41) that pure cultures of several different types of murine cancer cells produce material that is strongly and nonspecifically immunosuppressive when tested in vitro. We now present evidence that this material is nonspecifically immunosuppressive and toxic when tested in vivo. The toxicity and the immunosuppression can both be potentiated by prior treatment of the test animals with carbon tetrachloride (CCI 4 ) , an indication that normal animals may have some mechanism

capable of making harmless such biologically active cancer-derived materials. MATERIALS AND METHODS

Mice.-DBA/2 mice (The Jackson Laboratory, Bar Harbor, Me.; 9-15 wk old were matched for age in each experiment. Cancer cells.-SaD2-AG, an azaguanine-resistant mutant of the DBA/2 fibrosarcoma SaD2, was selected because it is strongly immunosuppressive in vitro (41). The SaD2 parent line and the azaguanine-resistant mutant had been doned and then carried as a pure line in tissue culture for many months. The SaD2-AG cells were grown in 800-ml glass tissue culture flasks (Rochester Scientific Co., Rochester, N.Y.) in Eagle's minimum essential medium (MEM) containing 10% fetal calf serum and harvested by treatment for 3 minutes at room temperature with 0.2% trypsin (Di£Co Laboratories Inc., Detroit, Mich.) in a potassium chloride and sodium citrate buffer. The washed cells were counted in a Neubauer chamber. Cultures were tested and found free of mycoplasma at the beginning and end of the series of experiments. Fibroblasts.-Fetal fibroblasts were obtained from DBA/2 donors and maintained in culture for about 8 weeks before use. Culture and harvesting conditions were identical to those used for the SaD2-AG cells. Preparation of supernatant material from cell cultures.-This was described in (41). The SaD2-AG cells or fetal fibroblasts were maintained in serum-free MEM for 24 hours. The medium was then withdrawn, centrifuged at 1,200xg to remove cells and large fragments, passed through a 0,45-p. micropore filter Millipore Corporation, Watertown, Mass.), concentrated approximately 100-fold by means of a UM-lO Diaflo membrane (Amicon Corp., Lexington, Mass.), washed by passage of fresh medium through the same membrane, and finally centrifuged at 100,OOOxg for 2 hours. All procedures were done at 4° C. The amount of supernatant material injected into a mouse was expressed in terms of "cell equivalents." The cell equivalent of the total volume of concentrated supernatant obtained from a monolayer of cells was the total number of cells in that monolayer. Dilutions of the concentrated supernatant were then expressed as appropriate fractions of the total number of cells from which the original supernatant was obtained. Evaluation of in vivo immunodepression.-Mice were given 4 X 108 sheep erythrocytes (SRBC) via the tail vein 4 days before assay, at which time the spleens were reReceived February 5, 1975; accepted July 2, 1975. Supported by the National Cancer Institute of Canada. Division of Cancer Research, Department of Pathology, Queen's University and the Kingston General Hospital, Kingston, Ontario, Canada. 4 Address reprint requests to Dr. Kennedy. 5 The excellent technical assistance of Mrs. Frances Paul and Mrs. Arja Karvinen is gratefully acknowledged. 1

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JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.5, NOVEMBER 1975

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WONG, MAN KOVITZ, AND KENNEDY

moved and the direct anti-sheep erythrocyte hemolytic plaque-forming cells (PFC) (anti-SRBC PFC) were assayed by the method of Cunningham and Szenberg (42). The ratio of the PFC responses of treated mice to those of normal controls provided a measure of the degree of immunodepression caused by the treatment. CC14 injections.-CCI 4 and paraffin oil T. Baker Chemical Co., Phillipsburg, N.].) were mixed to produce a solution containing 0.4 ml CCl,/ml solution. The standard procedure was to inject 0.25 ml of this solution sc into the left leg of each mouse 24 hours before immunization with SRBC.

a.

RESULTS Effect of eCI, Injection Into Normal Mice

We selected 0.25 ml of 40% CCl, dissolved in paraffin oil as our standard dose. When given sc into the hind leg, this dose resulted in definite macroscopic liver damage but did not have a detectable effect on either the general appearance and behavior of the recipients or their ability to produce PFC in response to an iv injection of SRBC given 24 hours after the CCl,. Table I summarizes data from 14 consecutive experiments in which 4-12 mice were given the standard dose of CCl, and then immunized with SRBC 24 hours later; PFC responses 4 days later were compared with those of a similar number of age-matched controls given SRBC at the same time. There was no consistent difference between the two groups; the maximum difference in either direction was about 25%, except for one puzzling experiment in which the mice given CCl, produced much stronger PFC responses than did the controls. However, it was safe to conclude that injection of the standard dose of CCl, did not reduce the ability of a recipient to produce PFC in response to an injection of SRBC given 24 hours later. Effect of eCI4 Injection in Mice With SaD2-AG Fibrosarcomas

Typically, mice inoculated sc in a right hind leg below the knee with 2x 106 SaD2-AG cells remained in TABLE

good general condition for at least 30 days, despite the enlarging tumor. Between 30 and 40 days post injection, the mice became less active, crouched in a hunched position, developed ruffled fur, ate poorly, and exhibited decreased responsiveness to immunologic stimulation. Shortly after this, some mice developed a few macroscopic lung secondaries. All mice eventually died of their disease, but the cause of death appeared to be general debilitation rather than either direct extension of the original tumor into a vital area or widespread dissemination and growth of secondary neoplasms. Text-figure 1 illustrates the effect of SaD2-AG tumors of various ages on the ability of the hosts to produce PFC in response to an injection of SRBC. Mice were given SaD2-AG cells as described above; age-matched controls were set aside for later use. After 5, II, 21, 38, or 48 days, the treated and normal control mice were given SRBC. The splenic PFC were assayed 4 days later. It was apparent that 5, II, or 21 days after injection of the SaD2-AG cells, the PFC responses of the recipients were not impaired. However, there was definite immunodepression after 38 or 48 days. In other similar experiments, the SaD2-AG tumors grew for 30 days without causing detectable immunodepression. Text-figure 2 summarizes data from an experiment designed to examine the effect on the anti-SRBC PFC response of injections of CCl 4 into mice with SaD2-AG tumors of various ages. Each time point involved 4 groups of 12 mice each. Two of these groups had been inoculated sc in a right hind leg with 2 X 106 cultured SaD2-AG cells. Four, 10, or 20 days later, mice from one SaD2-AG group and an equal number of mice from one control group were given the standard dose of CCI 4 • One day later, the mice of all 4 groups were immunized with SRBC; their spleens were assayed for anti-SRBC PFC 4 days later. As expected, these young SaD2-AG fibrosarcomas were not immunosuppressive by themselves, and injection of CCl 4 alone had little effect on the PFC responses. However, the combination of the standard dose of CCl 4 plus the presence of an SaD2-AG fibrosarcoma caused definite immunodepression, even when the tumor was only 10 days old and so small that it produced no visible swelling.

I.-Effect of CCI, alone on PFfJ responses in vivo

Age of mice when CCl, was injected (wk)

CCl,-treated mice b

9 9 9 9 9

31,171 (7)d 174,850 (4) 43,200 (5) 264,371 (7) 204,260 (9)

Average number PFC/spleen a Controls 28,171 193,000 49,400 284,100 163,490

CCl,-treated c control

(7) d (5) (5) (7) (9)

10 10 10 10

66,000 109,445 76,860 53,677

(12) (11) (5) (9)

79,616 (12) 125,400 (12) 61,520(5) 51,555 (9)

11 12 12 14 15

49,633 116,240 70,800 84,900 67,160

(12) (5) (5) (6) (5)

65,583 121,040 34,000 81,971 73,666

(12) (6) (6) (7) (6)

1.11 0.90 0.87 0.93 1.25 0.83 0.87 1.25 1.04 0.76 0.96 2.08 1.04 0.91

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Age of tumor (days) at • Assayed 4 days after SRBC injection. • Standard dose of CCI. given 1 day before SRBC injection. A PFC/spleen from CCl s-treated mice e verage value of ~ 1.06. PFC/spleen from control mice d Numbers in parenthese« ~ No. of spleens/group.

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l.-Anti-SRBC PFC responses of DBAj2 mice with SaD2-AG tumor on leg, as a function of age of tumor at SRBC injection. Bars represent I SD; figures in parentheses indicate number of spleens assayed per point.

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POTENTIATION OF CANCER-INDUCED IMMUNOSUPPRESSION BY CCL

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Potentiation by carbon tetrachloride of the immunosuppressive effects of fibrosarcoma and toxic material produced by fibrosarcoma cells.

SaD2-AG fibrosarcomas growing in DBA/2 mice caused nonspecific immunodepression. Injection of soluble material produced by cultured SaD2-AG cells into...
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