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Loss of sensitivity to drugs following tumor progression constitutes the main reason for failure of treatment in advanced cancer. In view of the dynamic nature of neoplasms, models of tumor progression should be used for the testing of drugs. In the present study, two variants of malignancy of AKR lymphoma were used as a model of tumor progression to test various treatment modalities. The two variants, TAU-39 (of low-malignancy) and TAU-38 (of high-malignancy) differed in the pattern of local tumor growth as well as in the rate of metastatic spread and mice killing capacity. The efficiency of chemotherapy, immunotherapy and hyperthermia on the two variants was compared. The low-malignancy tumor was more sensitive to adriamycin than the high-malignancy one. Administration of levan-activated macrophages at the tumor site inhibited the growth of TAU-39. However, growth of the high-malignancy variant was stimulated by macrophages. Hyperthermic treatment was, in contrast to the other treatments, more effective against the high - than against the low-malignancy tumor. Models of tumor progression used in tests for antitumoral drugs may help in the discovery of treatment modalities effective also for advanced stages of cancer.

The problem of comparing human cancer, a disease of long evolution, with experimental models in short life span animals is discussed. It is suggested that malignant tumors are more common in long life span animals because of the requirement of long periods of time for tumor progression. Increased malignancy may result from the ability of tumor cells to change, coupled with the long exposure of the neoplastic cells to the selective forces of the organism and the environment. It is proposed that the dynamic nature of tumors and the time required for changes to be expressed, should be taken into account in the study of the changing biological behavior of tumor cells and their changing responses to therapy. The use of transplantable tumors which increase in malignancy during successive transfers, artificially prolongs the existence of the neoplastic cells in isogeneic hosts, permitting thereby tumor progression. Tumor progression, occurs during a period which is accessible to the therapist. This dynamic process should be of primary interest to cancer researchers and therapists.

Previous studies have shown that 1 h preincubation with levan caused a sharp decrease in tumorigenicity of tumor cells, although cell viability was not affected. In the present study, the possibility that levan might change the sensitivity of Lewis lung carcinoma cells to the host immune system and that levan might cause delayed damage were tested. Cell morphology, DNA synthesis, cell multiplication and viability as well as osmotic fragility were followed during several days in culture. Levan was found not to affect cell immunogenicity. The polysaccharide destroyed tumor cells by a rather peculiar mechanism: after a seemingly normal or even enhanced growth in culture during 3-5 days, cells suddenly burst. During the apparently normal growth, several morphological changes were observed: cell volume increased, cytoplasm swelled by apparent water uptake and some cells contained two or more nuclei. Levan-treated cells were found to be more susceptible to osmotic shock than non-treated cells. The reason for the sudden cell death could be a gradual increase in volume, up to a point which is no more compatible with membrane integrity, resulting in cell lysis.

The combined effect of the polysaccharide levan (previously shown to exert a host-dependent as well as direct antitumoural activity) and the cytotoxic agents cyclophosphamide (CY), methotrexate (MTX), vincristine (VINC) and 5-fluoro-uracil (SFU) was studied in Lewis lung carcinoma and AKR lymphoma. Combined chemo- and immunotherapy was applied beginning on the day of tumour cell inoculation. Additive effects were obtained with the combined treatments, compared to single treatments, with all the combinations except MTX-levan in Lewis lung carcinoma, where the combined effect was synergistic. The additive effect was obtained with different doses and routes of chemotherapy, whether local or intraperitoneal. A 2 mg dose of CY combined with levan administered at daily doses of 10 mg, resulted in a 100% prevention of Lewis lung carcinoma growth. It is suggested that the levan may have two beneficial effects: it can exert an inhibitory effect on tumour growth and diminish the deleterious effect of cytotoxic agents on the immune system.

A marked difference in sensitivity to the direct effect of the polysaccharide levan on tumour cells was observed between two variants of malignancy of Lewis lung carcinoma: cells of the more malignant variant (3LL-M) were much more sensitive to the drug than those of the less malignant tumour (3LL). A gradual decrease in tumorigenicity following preincubation with increasing levan concentrations was observed with both variants, but statistically significant inhibition was observed at lower levan concentrations with 3LL-M than with 3LL.

The polysaccharide levan (polyfructose) has previously been shown to exert an inhibitory effect on the growth of several murine tumors. This activity is mediated by a host reaction, involving mainly macrophages but also other elements of the immune system. It was not clear, however, whether levan-activated macrophages act by a direct cytocidal effect on the tumor cells or via the activation of a specific immune response to the tumor. In the present study, the possibility of a direct cytotoxicity of levan-activated macrophages against Lewis lung carcinoma cells was tested by coculture in vitro. It was found that levan-induced (as well as paraffin oil induced) macrophages actually exert a direct cytotoxic effect on Lewis lung carcinoma cells. The tumor cell killing is mediated by cell to cell contact. A cytoplasmic bridge was often seen between the macrophage and the tumor cell. The remaining tumor cells in the lysed area appear slender, shrunken and non-dividing.

Experimental allergic encephalomyelitis was induced in male guinea pigs of the N13 strain by inoculation of an emulsion of basic protein of myelin with complete Freund's adjuvant. The disease was also passively transferred to other animals of the same strain by i.p. injection of sensitized lymphocytes obtained from donors inoculated with the antigenic emulsion eight days previously. Daily administration of levan markedly reduced the incidence and severity of the disease in actively sensitized animals. Little, if any, effect was obtained by levan treatment of animals with the passively transferred disease. The findings indicate that the previously reported inhibitory effect of levan on the development of experimental allergic encephalomyelitis is probably due mainly to a derangement of the afferent path (stage of sensitization). Administration of levan also causes a depletion of lymphocytes in the lymph nodes and this may presumably also affect the efferent path.

The polysaccharide levan exerted a direct effect on Lewis lung carcinoma cells in vitro. Tumor cells incubated with levan showed a pronounced decrease in oncogenic properties when inoculated sc in the syngeneic hosts (C57BL mice), although the viability of cells as determined by the trypan blue exclusion test was not diminished. The effect of pretreatment of inoculated mice with levan. Radiolabel incorporation into DNA, RNA, and protein was not affected in cells incubated with levan, but transport properties were selectively altered: Thymidine, uridine, and leucine transport was enhanced, whereas deoxyglucose transport was unchanged. The results indicated that levan exerted a direct antitumor effect on Lewis lung carcinoma cells in vitro.

Immunotherapeutic agents have often been found to provoke opposite effects on tumor growth--inhibitory or stimulatory--depending on dose, timing or route of administration. The reason for these opposite effects is not yet known. Levan (polyfructose), an immunomodulatory polysaccharide, has been found to exert opposite effects on the growth of the F10 variant of B16 melanoma. Low doses inhibit and high doses enhance the growth of this tumor. Cyclophosphamide (CY) augments the inhibitory effect of the polysaccharide. In order to elucidate the mechanism of these opposite effects, we tried to determine the changes induced by levan at inhibitory and stimulatory doses, alone or in conjunction with CY, on the lymphatic and hematopoietic systems of B16-F10 melanoma-bearing mice. In a previous study we reported the effect of these treatments on the morphology of spleen and lymph nodes (Leibovici, Kopel, Siegal & Gal-Mor (1986). Int. J. Immunopharmac., 8, 391). In the present study, we examined the effect of the treatments on bone marrow and peripheral blood composition. The growth of the tumor itself, as well as the various treatments, induced very marked changes in both bone marrow and blood. Tumor inoculation produced a sharp leukopenia and anemia followed by a restoration of both white and red blood cells. In the bone marrow, the tumor caused a gradual decrease in lymphocyte number. CY accentuated the severe leukopenia caused by the tumor. Lymphocyte depletion was prolonged, while restoration of granulocytes was achieved by day 7. A similar pattern of changes was observed in the bone marrow. With levan, opposite effects were observed in blood and bone marrow with the two doses in relation to the number of the cells of the lymphoid and myeloid lines: while 0.1 mg (tumor inhibitory) doses caused a more active restoration of lymphocytes as compared to 10 mg (tumor stimulatory) doses, an opposite effect was seen on the myeloid series--the high dose induced a more pronounced granulocytosis than the low dose. In the combined treatment, the low levan dose accelerated lymphocyte restoration in bone marrow compared to CY, while the high dose delayed the recovery of these cells. The results of the present study in conjunction with our previous study may explain the basis of the intriguing tumor inhibitory-stimulatory effects of some immunomodulators. Moderate increases in myeloid cell series appear to favor tumor inhibition and high increases favor tumor stimulation. In addition, the results of this study suggest that a regulatory relation might exist between the proliferation of the lymphoid and myeloid cell series.

Levan was shown to inhibit lymphoma development in AKR mice. Growth of tumor was inhibited at the site of injection and in metastases. Levan caused a decrease in the incidence of tumors, and it reduced pleomorphism, mitoses, and invasiveness of tumors in comparison to nonlevanized mice. Evidence of tumor cell destruction was observed in levanized mice. The effect of levan on tumor development was dose dependent.

The delaying effect of continued administration of levan on mouse skin homograft rejection was found to be due mainly to inhibition of thrombotic occlusion of the nutrient blood vessels. Levan administration did not appear to affect epidermal regeneration, but it seemed to inhibit degranulation of mast cells, resorption of plasma extravasated during the operation and growth of granulation tissue. The delayed rejection caused by levan treatment was apparently caused by infiltration of the graft by activated mononuclear cells from the blood stream. The phenomenon is believed to represent a late stage of the graft rejection process, its apperance depending on the survival of the graft beyond the stage of vascular occlusion.

This study represents an attempt to characterize differences in the cell surfaces of cells of high - and low - malignancy by use of lectins. The effect of concanavalin A (con A), Phaseolus vulgaris lectin (PHA) and the galactophilic lectin of Pseudomonas aeruginosa (PA-I) on cell agglutination, viability and tumorigenicity of two variants of malignancy of AKR lymphoma was tested. Cell viability was not affected by the lectins. Agglutination of the two variants was similar with all lectins and was specifically inhibited by the respective sugars. However, the effect on tumorigenicity of the two variants differed markedly: it was strongly reduced by PHA in the less malignant variant, while the more malignant one was hardly affected. The effect of con A was, on the contrary, more accentuated on the high-malignancy variant. The effect of PA-I was in the same direction as con A, but reduction in oncogenicity of both variants was feeble.

Pretreatment with the polysaccharide levan of Lewis lung carcinoma and AKR lymphoma cells affected unequally different stages of tumor development. While levan pretreatment sharply reduced the evolution of tumors from subcutaneously inoculated cells, no such inhibition was observed with pretreated cells inoculated intravenously. Since levan is known to affect the cell membrane, it is concluded that different cell membrane properties may be involved in the various stages of tumor progression.

In vitro treatment of Lewis lung carcinoma cells with the polysaccharide levan was shown previously to reduce the oncogenicity of these cells by acting on the cell membrane. In the present study the direct effect of levan on the tumorigenicity of Lewis lung carcinoma in C57BL male mice was tested at different doses and intervals and temperatures of incubation. A dose-dependent decrease in tumorigenicity was observed: Doses of 0.5, 1, and 2 mg levan/2 X 10(5) cells/0.2 ml reduced tumor incidence by 30, 50, and 70%, respectively. Some degree of inhibition was observed even with a dose of 0.004 mg. The inhibitory effect was rapid; 5 minutes of incubation resulted in the same reduction in oncogenicity as 60 minutes. Inhibition at 0 degree C was as effective as inhibition at 37 degrees C. The lack of temperature effect indicates that enzymatic activity probably is not involved. The data presented here, together with previous data, suggest that levan induces a physical, rather than a chemical, change in the affected tumor cells. The polysaccharide appears to be loosely, noncovalently bound to the cell membrane.

The effect of high-molecular-weight levan on skin graft rejection was studied. Daily ip administration of 15-30 mg levan was shown to delay rejection in Balb/c and C57BL recipient mice. An increase in MST value by 3.6 days was obtained in Balb/c mice and of 5.6 days for C57BL.

The effects of cyclophosphamide (CY) and levan at low (0.1 mg) and high (10 mg) doses (previously found to inhibit and stimulate, respectively, B16 melanoma growth) were studied. in single and combined administration, on the blood and bone marrow of C57BL mice. Very marked changes in the cellular composition of blood and bone marrow were induced by the various treatments. CY caused a sharp depletion of leukocytes in blood and bone marrow followed by a restoration of granulocytes while lymphocytes remained low. Levan caused a sharp dose-dependent depletion in bone marrow lymphocytes. Blood lymphocytes were not affected, however, by the polysaccharide. With the high levan dose, after a temporary granulocytopenia and monocytopenia, prominent granulocytosis was observed. While CY affected equally the different types of cells, levan decreased the number of lymphocytes and erythroblasts to a greater degree than the number of granulocytes. In the combined CY-levan treatments, the low levan dose generally attenuated the suppressive effect of CY, while the high concentration aggravated it.

High molecular levan, a polyfructoside, has a dose-dependent inhibitory effect on the primary immune response to sheep red cells (SE) in Balb/c mice, when given as from 1--2 days prior to the antigen injection. A slight stimulation of the immune response was observed when levan was given shortly before or 1 day after the antigen.

Combined treatment with levan and cyclophosphamide (CY) was applied intratumorally to Lewis lung carcinoma tumors in C57B1 mice on day 5 after tumor cell inoculation. Although on that day levan alone had no effect and CY alone caused only temporary regression of tumors, the combined CY-levan treatment caused 60% cure. Levan given before CY was almost as effective as simultaneous administration of both drugs on day 5. Delaying the administration of levan after CY treatment reduced the therapeutic effect. It is concluded that timing in relation to tumor cell inoculation as well as the order of administration of drugs, determine the degree of therapeutic efficacy. In tumor chemo-immunotherapy the final effect is probably due not only to the anti-tumoral effect per se of each drug, but also to the fact that the two agents have an effect on the immune system. Since the immune response may change during tumor development, the effect of timing of administration of an immunomodulator and certainly to two immunomodulators, can be crucial.

The route and schedule of treatment with high-molecular levan markedly influences its inhibitory effect on the growth of transplanted AKR lymphoma. Injections of levan into the site of the primary tumor were more effective in inhibiting tumor growth and preventing tumor-associated weight loss and mortality than were i.p. injections. Local levan injections inhibited metastatic spread only in mice treated from Days 0 and 2. Levan i.p. was more effective in inhibiting metastases in animals started on treatment 7 to 13 days after tumor inoculation than in animals in which levanization was started earlier. Local injection of levan before inoculation of tumor enhanced tumor growth and shortened life-span in comparison to nonlevanized animals. In mice treated with levan for a short period only, the inhibitory effect on tumor growth slowly vanished within 2 weeks. Some animals treated for 5 to 8 months remained completely free of tumor. The results indicate that the effect of levan on tumor development is mainly topical and depends on the concentration of the polysaccharide in the site. The tumor growth period from 0 to 5 days appears to differ from the following period in the reaction to levan treatment. The nature of this difference is not clear, but possible explanations are discussed.

Morphological changes in spleen and lymph nodes of C57B1 mice induced by the cytotoxic agent cyclophosphamide and the polysaccharide levan, separately and in combination were studied. In the spleen, an early decrease (phase 1) and a late increase (phase 2) in weight were found to result from all drug administrations. The hypocellularity of phase 1 was due to a depletion in the white pulp affecting mainly the B-region. Splenic weight decrease was parallel to B-cell depletion and most marked in animals exposed to cyclophosphamide with levan. The splenomegaly observed during phase 2 with all treatments was due to extramedullary hematopoiesis in the red pulp. In the lymph nodes, administration of cyclophosphamide and levan produced opposite effects on the B-cell region: cyclophosphamide eliminated the germinal centers for 8 days, but levan seemed to enhance B-cell activity. In animals given both cyclophosphamide and levan, inhibition of B-cell activity began earlier than with cyclophosphamide alone. Levan does not antagonize the suppressive effect of cyclophosphamide on the B-cell component at the early phase when the drugs are given together.

High molecular levan (polyfructoside) inhibits the skin tuberculin reaction in guinea pigs as judged by the degree of induration and erythema. The effect is dose-dependent. No effect on cellular infiltration was observed in histological studies. The lymph nodes of levan-treated animals were smaller and exhibited a much milder granulomatous reaction than those of non-treated animals.

AKR lymphoma cells derived from spontaneous tumors were serially transplanted at identical inocula. The degree of malignancy and sensitivity to the polysaccharide levan and methotrexate were tested at each transfer by assessing the lag of development of primary and distant tumors and survival of mice. A progressive increase in malignancy, accompanied by a loss of sensitivity to levan, were observed following serial passage of the lymphoma. Sensitivity to methotrexate was not affected. It is reasoned that, since serial passages permit a longer exposure of the tumor cells to selective forces of the internal milieu of the organism, better reflecting the situation in a long-life span animal, spontaneous and serial-passage tumors could serve as models for cancer therapy in humans.