Syngeneic Fibrosarcoma Research Articles

Anti-Tumor Immunity in B Lymphocyte-Deprived Mice

Abstract Immunity to a syngeneic methylcholanthrene-induced tumor was studied in (C57BL/6 × C3H)F1 males, treated continuously from birth with a rabbit anti-mouse IgM serum. Such mice have been shown to be selectively depleted of Ig-bearing lymphocytes and incapable of synthesizing antibodies. In the experiments reported, a heightened resistance of these mice to the syngeneic fibrosarcoma was demonstrated. This resistance was manifest in significantly slower tumor growth at the site of injection, as well as a lower incidence of spontaneous pulmonary metastasis.

Accelerated growth of syngeneic tumors in mice treated with methylcholanthrene.

BALB/c and C3H- mice were given a single injection of methylcholanthrene (MCA) and were tested at various times thereafter for immunologic reactivity and resistance to syngeneic tumor challenge. MCA treatment did not affect the rejection of H-2-incompatible tumor allografts, the rejection of H-2-compatible skin allografts, or the induction of delayed hypersensitivity to DNCB as measured by footpad challenge. In contrast, mice treated with MCA exhibited an increase susceptibility to challenge with several syngeneic ultraviolet-induced and MCA-induced fibrosarcomas. These studies demonstrate that in the autochthonous host the measurement of host immune status by in vivo cell-mediated immune responses to exogenous antigens does not necessarily predict the host response to tumor growth.

Progression of the immune response to solubilized tumor antigens.

Local adoptive transfer assays (LATA) were used to analyze and compare the progression of the immune response in C3H/HeJ mice to irradiated tumor cell vaccine and to crude 3M KCl solubilized antigens extracted from a syngeneic methylcholanthrene-induced fibrosarcoma. Sequential LATA performed 2, 6, 9, 12 and 15 days after soluble antigen pretreatment of spleen cell donors revealed a sinusoidal evolution of lymphoid cell activity. An initial brief period of potent tumor facilitation (days 6-9) was followed by a phase of tumor neutralization (days 9-12) which decayed by day 15. On the other hand, spleen cells from donors sensitized with irradiated tumor cells exhibited consistent tumor neutralization which was sustained throughout 15 days. Thus the tumor growth facilitation observed only in CSA-treated donors may represent a qualitative difference in the immune state induced by soluble, as opposed to cellular, forms of tumor antigen.

Macrophage function in tumor-bearing mice: Dissociation of phagocytic and chemotactic responsiveness

Macrophage function in mice with tumors was examined by two in vitro assays: chemotaxis to lymphocyte- or serum-derived stimuli and phagocytosis of 51Cr-labeled IgG coated sheep erythrocytes ( 51Cr-EAIgG). Resident peritoneal macrophages from mice with intrafootpad syngeneic fibrosarcomas were less responsive to chemotactic stimuli than macrophages from control mice. Chemotactic responses were depressed to about half of normal within 3 days of tumor transplantation and remained depressed until death of the mouse by 6–8 weeks. In contrast to this persistent depression of macrophage chemotaxis, phagocytosis of 51Cr-EAIgG was markedly increased in tumorbearing mice. Macrophage uptake of 51Cr-EAIgG was 4–5 fold greater than normal within 1 week of tumor transplantation, progressively decreased to normal levels by 2 weeks and remained normal through 4 weeks. Increased phagocytic capacity of macrophages from mice with tumors reflected both increased numbers of phagocytic cells and increased numbers of erythrocytes phagocytized per cell. A similar increase in macrophage phagocytosis was observed with cells from mice bearing another tumor but not from mice treated with syngeneic spleen cells, medium or mineral oil. Thus, growing tumors induce significant alterations in mononuclear phagocyte function. Changes observed in vitro with macrophages from tumor-bearing mice may provide cellular mechanisms for the abnormal phagocytic and inflammatory responses of cancer patients.

Antitumor activity of peritoneal exudate cells induced by cell-wall skeleton of Mycobacterium bovis BCG.

Peritoneal exudate cells (PEC) induced by oil-attached cell-wall skeleton of Mycobacterium bovis BCG (BCG-CWS) in ACI/N rats were tested for their effect on both in vivo and in vitro growth of syngeneic fibrosarcoma cells (AMC-60). Treatment of rats with intraperitoneal injections of BCG-CWS induced regression of syngeneic ascites tumor and increased the number of survivals. Whole PEC and adherent PEC from rats injected intraperitoneally with BCG-CWS inhibited the uptake of tritiated thymidine into the fibrosarcoma cells in an in vitro cytostasis test. This in vitro cytostatic effect was more marked as the ratio of effector to target cells increased. In addition, when tumor cells were inoculated subcutaneously with BCG-CWS activated PEC, tumor takes decreased markedly. Oil-stimulated PEC and normal peritoneal resident cells were inactive in inhibition of tumor growth in vivo and in vitro.

The effect of azathioprine on host cell infiltration and growth of a murine fibrosarcoma

Azathioprine (AZ) was injected into C57 Black mice before or after IM implantation of the syngeneic fibrosarcoma FS6, and observations were made on tumour growth and on the cellular composition of the fibrosarcomas. When multiple injections were given to the tumour-bearing host the tumours regressed. The percentage of tumour-associated macrophages remained essentially stable as regression occurred whereas the percentage of theta-antigen-positive cells increased. In the case of single IP injections of AZ, changes in the cellular composition of tumours were seen only when AZ was given 1-72 h before implantation but not when it was given after implantation. Compared with controls there was a 2- to 3-week delay in the appearance of large numbers of tumour-associated macrophages. The percentage of theta-antigen-positive cells within the tumours mass was elevated 7 days post AZ treatment but the level declined to control values between days 10 and 13. The significance of the results is discussed in relation to the dependency of the macrophage content of this tumour upon the development of an immune response towards the tumour.

Non-specific cytotoxicity of spleen cells in mice bearing transplanted chemically induced fibrosarcomas

Spleen cells collected from mice bearing transplanted chemically induced syngeneic fibrosarcomas non-specifically inhibited DNA synthesis of sarcoma and lymphoma target cells in vitro. Splenocytes from mice hyper-immunized against a syngeneic sarcoma specifically inhibited DNA synthesis of the tumour used for immunization. The impairment of tumour-cell DNA synthesis was associated in vitro with cytostasis, and lysis of the target cells was not seen. Since treatment with anti-theta serum and complement did not impair cytostatic action of the spleen cells, and since thymus-deprived animals showed similar activity to normal mice, T lymphocytes were not involved in non-specific cytostasis. Removal of phagocytic adherent cells by carbonyl iron markedly inhibited the cytostatic activity of the spleen cells, suggesting a role in this reaction for cells of the monocyte-macrophage series. The presence of an actively growing sarcoma was a prerequisite for the expression of non-specific cytostasis, since surgical excision resulted in complete disappearance of this activity of spleen cells.

Effect of X-irradiation on host-cell infiltration and growth of a murine fibrosarcoma

Whole body X-irradiation (400 rad) of C57BL mice, either before or after i.m. implantation of the syngeneic fibrosarcoma, FS6, influenced both the growth of the tumours and their cellular composition, particularly their macrophage content. Pre-irradiation resulted in slower initial growth of tumours, and a concomitant lack of host-cell infiltration, but when tumours began to grow at a rate parallel to controls infiltration by host cells was demonstrable. Similarly, irradiation of the tumour-bearing host resulted in a temporary cessation of growth, and a decrease in the macrophage content, which did not return to control levels for 2-3 weeks after irradiation. The significance of these results is discussed in relation to the possibility that infiltrating host cells, particularly macrophages, may stimulate the growth of this tumour.

Serological changes in adjuvant-treated mice, their specificity and relevance to tumor immunity

The effects of a variety of adjuvant protocols on immunoglobulin levels in normal and tumor bearing CBA mice have been investigated together with their ability to elicit immunoglobulin which bind to tumor cells in vitro and inhibit the growth of a transplanted syngeneic MC-induced fibrosarcoma.

Natural anti‐tumor serum reactivity in balb/c mice. I. Characterization and interference with tumor growth

A natural cytotoxic reactivity directed against syngeneic or allogeneic tumor cells was demonstrated in serum of BALB/c mice by an in vitro cytotoxicity test using rabbit serum as the source of complement. The reactivity, studied on syngeneic fibrosarcoma cells, was found to be minimal in mice less than 10 weeks old and to increase progressively with age. T-deprivation determined an increase of reactivity in young mice to levels reached spontaneously only by the serum of 40-week-old mice. The BALB/c serum also revealed natural anti-thymus antibodies. Non-identity between anti-tumor and anti-thymus antibodies was demonstrated by direct cytotoxicity and absorption tests. An inoculum of syngeneic fibrosarcoma cells increased the level of anti-tumor serum reactivity in both normal and T-deprived young mice. The natural anti-tumor cytotoxicity revelaed in vitro seemed to exert a specific in vivo protection as suggested by the indirect correlation found between the level of the natural anti-tumor reactivity and the grwoth of a transplanted fibrosarcoma.

Effects of murine tumors on delayed hypersensitivity to dinitrochlorobenzene. I. Description of anergy caused by transplanted tumors.

In the murine model presented for tumor-associated immune suppression, normal BALB/c mice displayed significant foodpad swelling when sensitized on the flank with 2 mg dinitrochlorobenzene (DNCB) dissolved in dimethyl sulfoxide and challenged in a footpad with 0.05 mg DNCB 10 days later. This reaction in challenged footpads seemed to be a classic delayed hypersensitivity reaction, since it took 24 hours to develop and included an extensive mononuclear infiltrate. The reaction was transmissible from sensitized to normal mice by the transfer of lymphoid cells but not to serum. When sensitized 10 days after tumor inoculation, mice bearing either an allogeneic melanoma or a syngeneic lymphoma or fibrosarcoma did not demonstrate delayed hypersensitivity to DNCB.

Concomitant immunity to pulmonary metastases of a murine fibrosarcoma: Influence of removal of primary tumor by radiation or surgery, of active specific immunization and treatment with Corynebacterium granulosum

Mice having a syngeneic fibrosarcoma growing in the leg develop fewer metastases in their lungs than do normal mice when cells of the same tumor are removed from syngeneic donors and injected intravenously. This concomitant immunity can be greatly augmented by removal of the primary tumor either with local irradiation or surgery, or by treating the tumor host with Corynebacterium granulosum. Treatment of tumor bearers with heavily irradiated tumor cells did not, however, influence the magnitude of the concomitant immunity.

Combination of C. parvum and specific immunization against artificial pulmonary metastases in mice.

We have studied whether specific immunization administered intraperitoneally can augment the activity of C. parvum (0.25 mg intraperitoneally or intravenously) against intravenously injected cells of a syngeneic fibrosarcoma in C3Hf/Bu mice as expressed by the reduction of pulmonary metastases (nodules, colonies) and/or by the prolongation of the survival of recipients. Combination of specific immunization and C. parvum, applied either before or after IV inoculation of viable tumor cells, was more effective than the single treatments. IV injection of a mixture of heavily irradiated and viable tumor cells gave more tumor nodules in the lungs of normal mice than injection of viable cells alone. The metastasis-enhancing effect of admixed irradiated cells was not found in mice previously treated with C. parvum, and was abolished if the immunostimulant was injected after tumor cells. Generation of lung metastases by IV inoculation of fibrosarcoma cells was reduced in mice already having this tumor in the leg. This concomitant immunity to metastases was increased by treating the recipients with C. parvum, but not with irradiated cells; also, the injection of irradiated cells together with C. parvum did not augment the efficiency of the latter. C. parvum was not as effective in T-cell deprived as in control mice, which suggests that in this system, T-cells are required for optimal anti-tumor activity of this immunostimulant. Specific immunization was not effective in T-cell-deprived mice and did not augment the efficiency of C. parvum.

In vitro destruction of tumor cells by macrophages from mice treated with Corynebacterium granulosum.

Peritoneal macrophages from C3Hf/Bu mice treated with killed Corynebacterium granulosum bacteria were tested for their effect on in vitro growth of syngeneic fibrosarcoma cells, tumorigenic mouse L-P59 cells, human malignant melanoma cells, allogeneic fibroblasts, erythrocytes, and epithelial kidney cells. Only the cell cultures having neoplastic properties were destroyed by stimulated macrophages; the rate of tumor cell destruction was greater as the ratio of effector to target cells was increased. Neither irradiation nor trypsinization of macrophage monolayers altered the cytotoxicity of stimulated macrophages. The results indicated that C. granulosum activated macrophages to destroy tumor cells in an immunologically nonspecific manner but had no cytotoxic effect on normal allogeneic cells.

Inhibition of syngeneic fibrosarcoma growth by lymphocytes sensitized on tumor-cell monolayers in the presence of the thymic humoral factor.

The present experiments were performed to investigate the possibility of inhibiting tumor growth in vivo with syngeneic lymphocytes sensitized in vitro on monolayers of the tumor under test, and to study the effect of a thymic humoral factor (THF) in this sensitization process. Monolayers of fibrosarcoma cells were used to sensitize spleen cells from syngeneic donors against the tumor. Such sensitized lymphocytes manifested cytotoxic activity against cells fo the fibrosarcoma in a microassay measuring tumor-cell detachment. However, when the sensitized lymphocytes were mixed with the fibrosarcoma cells and injected into syngeneic mice, enhanced tumor growth was observed in vivo. Addition of thymic humoral factor to the cultures during sensitization resulted in increased cytotoxic activity by the lymphocytes in vitro and a reduction in the tumor enhancement caused by these cells when injected in vivo. Enhanced tumor growth occured when activated lymphocytes of allogeneic as well as syngeneic origin were injected together with the fibrosarcoma cells. Enhancement, which was already apparent when the spleen cells had been sensitized for 24 h, could be circumvented by separate administration of lymphocytes and tumor cells. Syngeneic lymphocytes injected systemically after sensitization for 5 days exerted anti-tumor reactivity against the fibrosarcoma grafted in the foot-pad of syngeneic mice. Tumor growth was further inhibited by systemic injection of lymphocytes which had been sensitized in the presence of the thymic humoral factor.

Tumor Suppression by pyran Copolymer: Correlation With Production of Cytotoxic Macrophages

Intraperitoneal administration of pyran copolymer (pyran-2-succinic anhydride-4,5-dicarboxytetrahydro-6-methylanhydride polymer) protected C3H/HeN male mice against tumor development after intradermal challenge with cells of the transplantable, methylcholanthrene-induced, syngeneic fibrosarcoma 1038. Peritoneal cells from pyran-treated but not from normal animals suppressed tumor development in local passive-transfer experiments. Adherent peritoneal cells from pyran-treated mice were cytotoxic in vitro to several syngeneic murine tumor cell lines including 1038.

Nonspecific Immunotherapy of Murine Solid Tumors With <italic>Corynebacterium granulosum</italic><xref ref-type="fn" rid="fn2">2</xref>

A single intraperitoneal (ip) or intravenous (iv) injection of Corynebacterium granulosum into C3Hf/Bu mice shortly after subcutaneous (sc) injection of cells from a strongly antigenic syngeneic fibrosarcoma induced by 3-methylcholanthrene caused complete and lasting regressions of 100 and 70% of resulting tumors, respectively. Treatment with this bacterium sc only slightly inhibited the growth of some tumors. C. granulosum given iv to mice 3 days after the sc injection of fibrosarcoma cells caused complete regressions of 39 of 45 tumors; two iv injections with this immunostimulant given 1 month apart were no more effective than a single injection. Intralesional treatment of fibrosarcomas 8 mm in diameter induced complete regressions of tumors in 30% of the animals, whereas sc treatment contralateral to the growing tumor only slightly reduced tumor growth. Intraperitoneal growth of a fibrosarcoma was efficiently controlled (58-80% survival of mice) if C. granulosum was given ip, but not iv, 3 days after inoculation with tumor cells. Again, two injections of C. granulosum (given ip 4 days apart) were only as effective as a single injection. Treatment with C. granulosum iv at 3, 7, 14, or 21 days after sc inoculation of a weakly antigenic, spontaneously arising mammary carcinoma (MC-1) strongly inhibited tumor growth. Three complete but temporary tumor regressions were observed. The subcutaneous growth of another spontaneous mammary carcinoma (MC-2), which contained fairly strong tumor-specific antigen(s), was also significantly inhibited if C. granulosum was given 3,7, or 14 days after, but not 7 days before, tumor cell inoculation. However, pretreatment of mice with the immunostimulant significantly protected the mice against artifically induced pulmonary metastases of this tumor.

Brief Communication: Embryonic Antigens Shared Between Chemically Induced Lymphosarcomas and Fibrosarcomas of the Mouse2

An antiserum obtained by the immunization of C57BL/HeDp mice with a pool of C3HF/Dp 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced fibrosarcomas exerted a specific cytotoxic activity in vitro on C57BL/HeDp chemically induced lymphosarcomas. Conversely, C57BL/HeDp spleen cells sensitized against C3Hf/Dp chemically induced lymphosarcomas or embryo cells were cytotoxic for plated cells of syngeneic DMBA-induced fibrosarcomas. Absorption studies with antiembryo and antilymphoma antisera showed that embryonic antigens were shared between lymphosarcomas and fibrosarcomas and that all serologically defined antigens present on lymphoma cells, including virus-related antigens, were also on fibrosarcoma cells.

Protection by corynebacterium granulosum against radiation-induced enhancement of artificial pulmonary metastases of a murine fibrosarcoma.

Intraperitoneal treatment of C3Hf/Bu mice with killed Corynebacterium granulosum bacteria before intravenous inoculation of cells from a syngeneic fibrosarcoma greatly reduced the number of metastatic nodules in the lung. In contrast, the number of pulmonary metastases was significantly increased in mice previously given whole-body irradiation of 200–600 rads of γ-rays. Pretreatment of animals with C. granulosum before irradiation not only abolished this effect but also produced a response equal to that in mice treated with the immunostimulant alone. C. granulosum protected the mice against radiationinduced enhancement of pulmonary metastases if given within 2, but not at 4, weeks before irradiation. However, if mice were first irradiated and then, 1 or 2 days later, treated with C. granulosum, the radiation effect on the formation of pulmonary metastases was not influenced.

Tumor resistance in rats immunized to fetal tissues

Inbred Fischer 344/N rats were immunized with irradiated cells of either a MCA-induced syngeneic fibrosarcoma (MCA-R), syngeneic second trimester fetal tissue, or allogeneic, Marshall 520 splenic lymphocytes. Animals injected intradermally with soluble antigen extracts (SAE) of MCA-R and normal Fischer 344/N tissues were evaluated for development of delayed cutaneous hypersensitivity reactions (DCHR). Fischer rats immune to allogeneic tissue gave no positive DCHR to either SAE. Ten of 11 rats immune to MCA-R and 6 of 9 immune to second trimester fetal tissue gave positive reactions to MCA-R SAE but none to normal tissue SAE. In a second experiment immunized rats were challenged subcutaneously with viable MCA-R tumor cells. Significant resistance to tumor growth was demonstrated by animals immune to MCA-R and by animals immune to second trimester fetal tissue. This resistance was unrelated to sex. Immunological similarity between fetal antigens and tumor associated transplantation antigens is suggested.