Search

Resistance to immune reactions, innate or acquired, may be one of the mechanisms responsible for the progression of tumors. We have, indeed shown higher numbers of macrophages surrounding low- as compared to high-malignancy cells. In the present study we examined the level of cell surface molecules known to determine sensitivity to macrophages, namely galactose (GAL) and sialic acid (SA) residues. A histochemical assay for identification of SA by electron microscopy showed a higher cell surface content on metastatic (MT) than on primary (PT) tumor cells. The FACS data seen with fluorescent lectins showed a higher binding of Sambucus nigra agglutinin, which identifies SA attached to terminal GAL in -2.6 or -2.3 linkage, in MT than in PT cells. Binding of Maakia amurensis lectin (MAL-1), which identifies SA at position 3 of GAL, showed that the MT cells contain two subpopulations, one binding more MAL-1 and another less. Cell sorting showed a more aggressive behavior of the first population. The comparison of Peanut agglutinin (PNA) binding, which identifies GAL, demonstrated a decreased amount of PNA receptors in MT as compared to PT cells. Western blot analysis of the membranal proteins with different lectins, identified 3 sialylated glycoproteins. The 88 kDa glycoprotein had no significance for metastatic potential. The 130 kDa glycoprotein was higher in MT than on PT cells. The 220 kDa glycoprotein was practically present only on MT cells. The tendency observed was of a higher level of membranal glycoconjugates terminally sialylated with subterminal galactose residues, inMT cells as compared to PT cells. This may explain the recently found decrease in apoptotic cell death with increasing aggressiveness of the AKR lymphoma and suggests a lower sensitivity to macrophages with tumor progression. Treatment based on the reduction in sialic acid content might render the tumor cells more vulnerable to macrophages. We found, indeed, that Wheat germ agglutinin (WGA) injected in vivo, exerted an inhibitory effect on growth of the lymphoma. We found moreover that WGA-treated tumor cells were more sensitive than nontreated cells to macrophages in vitro.

While a direct relation between aging and tumorigenesis is well established, a slower tumor progression rate was reported in old as compared to young cancer patients. The mechanisms responsible for the less aggressive behavior of tumors in the aged, are largely unknown. We have recently shown an increase in apoptotic cell death in tumors derived from aged as compared to young animals in the AKR lymphoma. This was shown by DNA flow cytometry and by the ladder type DNA fragmentation in agarose gel electrophoresis. Analysis of the expression of genes involved in apoptosis in tumors derived from young and old animals showed a lower bcl-2 expression in those from the aged. The Fas antigen, on the contrary, displayed higher expression levels on lymphoma cells derived from old than on those from young mice. Apoptotic cells are recognized and phagocytosed mainly by macrophages. One molecular property of apoptotic cells which is recognized by macrophages is a loss in cell surface sialic acid concomitantly uncovering galactose residues. While comparing the "eat me status" phenotype of the tumor cells derived from young and aged animals, by the use of lectins recognizing sialic acid and galactose residues, FACS analysis showed a decrease in cell surface sialic acid and a gain in galactose residues in aged as compared to young mice. Moreover, Western blot analysis showed that a 130 Kda sialylated membrane glycoprotein was expressed at a lower level in tumors from the old as compared to young mice. Our results, at both the cellular and molecular levels, particularly with regard to molecules recognized by macrophages, indicate that increased apoptotic cell death in tumors from old as compared to those from young animals constitutes, as we have previously suggested, one of the mechanisms of the age-related decrease in tumor progression rate.

The question of whether metastatic potential and drug resistance are related phenotypes was addressed by comparing the biological behavior of the parental B16 melanoma and a multidrug resistant variant derived from it, the B16/Col/R. A more pronounced metastatic spread to lungs was observed in mice inoculated i.v. with the B16/Col/R variant than in those bearing the parental line. In addition, in the mice injected with the drug resistant melanoma, unusual tumor masses were observed. Large abdominal and spinal cord growths were seen with the MDR variant but not encountered in mice inoculated with the original B16 melanoma. We further attempted to test the capacity of the two cell types to perform several cellular functions relevant to the metastatic process. The B16/Col/R cells displayed a higher aggregability and cell motility than did the B16 cells. Adherence to endothelial cells was higher in the parental line than in the B16/Col/R, possibly supporting a more efficient extravasation of the variant cells. The drug resistant variant displayed a higher capacity to grow locally in kidney, spleen, cecum and peritoneum, as compared to the parental melanoma, indicating a higher ability of homing and growth in these potential target organs for metastasis. A correlation between metastatic potential and multidrug resistance appears therefore to exist in the system examined.

Multiple structural and functional cell properties are responsible for the complex phenotype of the metastatic cell. Cells derived from AKR lymphoma malignancy variants were compared with regard to several cellular functions relevant to the metastatic behavior. Metastatic behavior correlated with relevant in vitro cell activities, such as cell motility, homotypic and heterotypic adhesion as well as proteolytic activity. The Variant displaying the highest degree of malignancy, TAU-42, exhibited the most elevated ability to perform almost each of the cell functions examined: motility, aggregability, capacity to adhere to endothelium and extracellular matrix, heparanase and protease activity and ability to grow in the spleen. The TAU-33 variant was second in rank in the performance of the above activities. The TAU-44 variant cells, the third in rank of malignancy, displayed some peculiar functional behavior: while manifesting the lowest homotypic adherence and heparanase activity, they had a high predilection for growth in kidney.

Tumor progression (TP) is often accompanied by evolution of drug resistant clones. Decreased intracellular accumulation of cytotoxic agents is probably the major mechanism of drug resistance. In the present study, we tried to examine the possibility to overcome the resistance to adriamycin (ADR) treatment, by cyclosporin A (CS) in two models of TP in the Lewis lung carcinoma (3LL) system. The first model consisted in the comparison of primary tumor cells (3LL-PT) to metastatic cells (3LL-MT) and the second consisted in comparison of lung metastases of the highly malignant variant D122 to those of the parental 3LL tumor. Cyclosporin had a weak augmenting effect on ADR uptake, in the two more malignant cell variants and no influence on the 3LL-PT cells, according to FACS analysis. Cytofluorometry also showed practically no effect of CS on cell size, unlike the effect of other chemosensitizers, such as membrane active agents. In order to find out whether CS counteracts resistance to ADR despite the fact that it does not increase cytotoxic agent uptake, we examined its effect on in vitro proliferative capacity of the 3LL-PT cells. CS in combination with ADR had a more pronounced effect, as compared to single treatments on cell proliferation. The low effect of CS on ADR uptake according to FACS analysis, and by contrast, its efficiency to overcome resistance to ADR according to the in vitro growth results, suggest that the mechanism of the CS action as a chemosensitizer is not related to the p-glycoprotein (P-G-P), known to be overexpressed in the typical multidrug-resistance (MDR) phenotype. A better understanding of the complexity of MDR mechanisms may contribute to the design of new modalities to overcome this phenomenon, which still limits effectiveness of cancer cure, to the early stages of the disease.

Lectin-histochemical studies were performed on macrophages activated by Nocardia opaca water soluble molecules (NWSM) and elicited by phosphate buffered saline (PBS) as control to demonstrate the pattern of carbohydrate residues. Ten different biotinylated lectins were used as probes and avidin-biotin-peroxidase complex (ABC) was used as visualizer. The lectins wheat germ agglutinin (WGA) and Griffonia simplicifolia agglutinin-I (GS-I) stained macrophages only after activation by NWSM and not macrophages elicited by PBS. Both macrophages did not stain by succinylated wheat germ agglutinin (S-WGA). The difference in lectin-binding patterns between NWSM-activated and PBS-elicited macrophages, suggests that activation of macrophages by the NWSM is associated with modification in the distribution of specific membrane carbohydrates, including enhancement in the expression of sialyl and alpha-galactosyl residues.

The comprehension of tumour progression has advanced due to the use of various models, the most rewarding being probably the study of malignancy variants derived from the same tumour. In the present study the biological behaviours of three AKR lymphoma variants were compared. The three variants, TAU-33, TAU-38 and TAU-39, differed markedly in biological behaviour. The TAU-39 variant formed very large 'primary tumours', TAU-33 produced local growths of intermediate size, and TAU-38 formed small s.c. tumours. However, the metastatic potentials of the variants were inversely related to their ability to produce local tumours. According to various parameters (spread to organs, cachexia and mice mortality rate), the variant of highest metastatic potential was TAU-38, the one of intermediate ability TAU-33 and the TAU-39 had the least aggressive behaviour. A lack of difference in invasive capacity as well as a similar ranking of malignancy by both s.c. and i.v. inoculation indicate a differential behaviour in late metastasis phase. Tumour progression models may contribute to a better understanding of this threatening process and to testing of new treatment modalities suitable for cancer at different stages.

Several immunomodulatory fractions derived from Nocardia have been found to inhibit the growth of several experimental tumors, including Lewis lung carcinoma (3LL). An involvement of both macrophages and lymphocytes in the antitumoral effect of Nocardia fractions has been suggested. The mechanism of the Nocardia delipidated Cell Mitogen (NDCM)-induced tumor-inhibiting effect was investigated further in the present study. Macrophages activated by NDCM exerted a cytotoxic effect on 3LL cells in vitro, indicating a direct influence of macrophages on the tumor cells. These results correlate with previous findings which showed a local accumulation of macrophages (but also lymphocytes) at the tumor site in NDCM-treated mice. In tumor-bearing mice--both treated and non-treated with NDCM--a splenomegaly due to a pronounced extramedullary hematopoiesis was seen. Concomitant with the gradual evolution of the extramedullary hematopoiesis in the red pulp, a depletion in white pulp component was observed, more pronounced in the control 3LL-bearing mice than in the 3LL-inoculated NDCM-treated animals. The disappearance of the lymphatic follicles in 3LL-bearing mice may be responsible for the failure to cope with the tumor. It is therefore possible that by delaying white pulp depletion, NDCM favors a better host defense against the tumor. Examination of lungs in 3LL-bearing mice treated by NDCM showed a rich infiltration of macrophages in the vicinity of isolated tumor cells, probably indicating a defensive role of NDCM-activated macrophages against metastatic spread of the tumor. Although the macrophage appears to be of major importance in the NDCM-induced host response against the tumor, other components of the immune system are probably system are probably also activated by the Nocardia fraction in defense against the neoplasm.

The currently available antitumoral therapeutic modalities are most often inefficient against metastatic disease. The metastatic phenotype has been shown to be largely determined by cell membrane properties. The cell membrane could therefore be considered as a possible target for antimetastatic drugs. In the present study we examined the effect of hyperthermia (the antitumoral effect of which is based, at least partly, on an action on the cell membrane) on the F1 and F10 variants of B16 melanoma. Cells of the more malignant variant, F10, were found to be markedly more sensitive to hyperthermic treatment than those of the less malignant one, F1. One hour in-vitro treatment by supranormal temperatures (ranging from 40 to 46 degrees C) resulted in a differential effect with regard to both proliferating capacity of the cells in vitro and tumorigenic ability following inoculation to mice. Our present results in the B16 melanoma corroborate data obtained by us in another tumour system, the AKR lymphoma. Study of the effect of membrane-acting agents on tumour variants differing in degree of malignancy might result in the finding of antitumoral agents efficient against advanced cancer.

Various Nocardial fractions have been shown to exert inhibitory effects against several experimental tumors, via a host response mechanism. With the aim of obtaining further information on the mechanism of action of these immunomodulators, we examined in the present study the local cellular host response induced by intratumoral administration of one of these fractions, the Nocardia-delipidated cell mitogen (NDCM), at the site of the tumor (Lewis lung carcinoma-3LL). The tumor itself was found to provoke a scanty cellular infiltration surrounding the tumor mass, mainly composed of lymphocytes. The site of the tumor in NDCM-treated mice was surrounded by a very thick rim of inflammatory cells, consisting of macrophages and lymphocytes. Although recognized decades ago, the existence of tumor infiltrating leukocytes still constitutes a puzzle. The composition and significance of these host cells, in both non-treated and treated tumors has not yet been elucidated. Since lymphocytes were also found around non-treated tumors and they apparently did not prevent tumor development, it is possible that macrophages play a more important role in the NDCM-elicited host defense against the Lewis lung carcinoma.

Tumor progression has been reported to be often accompanied by a loss of sensitivity to various elements of the immune system. In the present study, three variants of malignancy of AKR lymphoma were compared as to the host reaction they elicit. Splenectomy caused an acceleration of growth of the low-metastasizing tumor, slightly inhibited the growth of the tumor of intermediate malignancy and significantly inhibited the development of the high-metastasizing lymphoma. These results suggest that splenic elements exert an inhibitory effect on the low-metastasizing tumor and a growth-stimulatory effect on the more malignant ones. While decreased sensitivity to various host defense mechanisms following tumor progression has been amply documented, the present study shows a more severe phenomenon: increased malignancy can be accompanied by a deviation of the host immune reaction from tumor growth inhibition to growth stimulation.

Mice of the randomly bred strain ICR and of the inbred strain BALB/c could be successfully sensitized to chromate by repeated topical applications of a solution of potassium bichromate in dimethyl sulfoxide. The specificity of chromium sensitization was ascertained by ear tests, specific desensitization by intravenous injection of potassium bichromate and by histological ear sections showing marked infiltrates of leukocytes following ear application of chromium-dimethyl sulfoxide in already sensitized animals. It is concluded that mice may provide a useful experimental model for study of contact sensitivity to chromium.

High-mol.-wt levan injected locally inhibits the growth of Lewis lung carcinoma in C57BL mice. The inhibition is dependent on the number of tumour cells injected and on the dose of levan. The inhibition decreases tumour incidence and size as well as prolonging survival. The polysaccharide is most effective when injected daily beginning on the day of tumour-cell inoculation. Treatment begun on later dates is less effective. Treatment begun one day before tumour-cell inoculation enhances tumour growth. Histological studies showed that levan induces an intense polymorphonuclear (PMN) reaction followed by accumulation of vacuolated, levan-laden macrophages. Both PMN and activated macrophages seemed to have an inhibitory effect upon the growth of the tumour. The effector role of PMN was not explained by the histological study. Tumour cells in close contact with levan-laden macrophages appeared mostly necrotic. Administration of levan begun one day before tumour-cell inoculation produced a similar reaction, but the infiltrating cells did not appear to approach and damage the tumour cells. Images Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12

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.

Activated macrophages were found to induce opposite effects on the growth of the F10 variant of B16 melanoma in both in vivo and in vitro systems. Low macrophage numbers had lytic effects and high numbers stimulated the growth of the melanoma. In fact, the number of macrophages per area (namely, their density) was found to determine whether lysis or growth stimulation would occur. Low density macrophages were found to be structurally and functionally different from high density macrophages. Macrophages in sparse cultures were round, separate, and lysed tumor cells, while crowded macrophages appeared spread, with pseudopodia, and caused enhancement of tumor growth.

A combined treatment modality including surgical excision and daily injections of high molecular levan seemed to be effective against Lewis lung carcinoma in C57BL mice. The inhibitory effect of levan on tumor growth in mice treated by resection of the primary tumor was demonstrated as follows: (1) recurrence of primary tumors was less in levan-treated mice compared to untreated animals; (2) metastases were less numerous in the levan-treated animals; (3) levan treatment partly prevented loss in body weight, and (4) it increased the survival rate. Recurrence of primary tumors appeared to be more efficiently inhibited by local levan treatment, while systemic (i.p.) administration was more effective than local treatment in preventing metastases. The mechanism of inhibition of tumor growth was related to accumulation of macrophages around the metastases.

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 influence of duration and of various routes of administration of native levan on blood levels of the drug was studied in mice. The blood levels varied with the doses administered. With a single i.p. injection of 20 mg levan/mouse the blood levels rose more rapidly than with higher and lower doses. In the second and third week of daily injections the rise and fall in blood levels became less pronounced. With treatment longer than 3 weeks spikes of maximal concentrations reappeared. After s.c. injections blood levels rose more slowly than after i.p. treatment. The findings indicate that: 1. high doses slow down absorption of levan; 2. after 7-21 days of treatment the RES (reticulo endothelial system) is more active in removal of levan from the blood stream than at the beginning of the treatment; 3. after treatment of 3 weeks and more the RES appears to be blocked and is again slow in the uptake of levan from the blood stream.

Opposite effects on the growth of the F10 variant of B16 melanoma--inhibitory and stimulatory--were found with varying doses of the polysaccharide levan. Low (0.1 and 1 mg) daily doses inhibited, while high (5 and 10 mg) doses enhanced tumor growth. In combined cyclophosphamide-levan treatment, these opposite effects were more pronounced.

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 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.

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.

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.

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.

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.

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 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.

The mechanism of the antitumoral effect of levan was studied in the C57BL mice-Lewis lung carcinoma system. Modulation of host immune response and a direct inhibitory effect on tumor cells were found. Local treatment was more effective when begun early. It reduced tumor incidence without affecting the size of developing tumors. Systemic treatment was more effective when started late, inhibiting equally the tumor size in all mice. Macrophages are involved in local, lymphocytes possibly in systemic, inhibition. A direct effect on tumor cells was suggested by a decrease in oncogenicity, following preincubation with levan. Levan augments the antitumoral effect of cyclophosphamide in vivo and in vitro.