Role of the thymus and T-cells in slow growth of B16 melanoma in old mice

Clinical observations suggest that tumors grow more slowly in aged subjects. To investigate the influence of age on tumor growth, we injected the same number of cultured B16 melanoma cells into C57BL/6 mice of various ages. B16 melanoma cells, inoculated s.c., grew more slowly in old (18-20-month-old) as compared to young (6-8-week-old) mice. In young tumor-bearing mice there was a significant increase in the number and the proliferative response to phytohemagglutinin and concanavalin A of splenic T-cells as compared to old tumor-bearing animals. There was no difference in the response of B-lymphocytes from old and young tumor-bearing mice to lipopolysaccharide. The positive association between T-cells and the rate of tumor growth was also suggested by the slower growth of melanoma cells in thymectomized or thymectomized and anti-theta antiserum-treated young mice. Finally, the age-associated difference in tumor growth could be transferred by spleen cells from old or young mice to thymectomized and lethally irradiated syngeneic young animals. Young mice with rapidly growing B16 melanoma tumors have increased numbers and proliferative responses of thymic-derived lymphocytes. It is likely that T-cells or their products facilitate the growth of B16 tumor cells.     

Hormonal influences on growth of B16 murine melanoma

The influence of endocrine factors on B16 melanoma growth was investigated in young and old male mice to test the hypothesis that senescent hormonal changes account for the age-associated reduced tumor growth previously observed in our laboratory. Again it was demonstrated that tumors grow more slowly and to a lesser volume in old mice. For a test of whether senescent hormonal changes account for this tumor growth pattern, B16 was implanted into male and female C57BL/6 mice and tumor growth observed. In addition, young adult male mice were castrated and later B16 melanoma growth was determined. Tumor growth was similar in male and female mice. Contrary to our expectations, however, castrated male mice demonstrated larger tumor volumes, despite serum testosterone levels similar to those of old mice. Furthermore, after iv tumor cell injection, the occurrence of pulmonary colonies was greater in castrated mice. These observations indicate the importance of hormonal factors in this commonly explored tumor system. With regard to aging and tumor growth, however, senescent sex hormone changes probably do not account for the slower tumor growth observed in aged animals.     

Effect of melatonin on B16 melanoma growth in athymic mice

The effect of melatonin on the growth of B16 mice melanoma was examined. Male and female BALB/c athymic mice, inoculated with 7 X 10(4) melanoma cells, were given drinking water containing melatonin (5 micrograms/g body weight/day) and 0.5% ethanol. Compared to control animals the melatonin treated male and female athymic mice had significantly smaller tumors on Day 40. The weights of the testes, the ovaries, and the adrenal glands of melatonin treated mice were significantly reduced compared to control animals. These data indicate that melatonin p.o. significantly inhibited the growth of B16 mouse melanoma and that the antitumor effect of melatonin was associated with a significant decrease in gonadal and adrenal weights.     

Autocrine tumor growth regulation and tumor-associated hypoglycemia in murine melanoma B16 in vivo

A substance immunochemically cross-reactive with insulin (SICRI) appears in melanoma B16 growing in diabetic and nondiabetic C57BL/6 mice. Progression of tumor size is paralleled by the increase of SICRI levels in the serum of both diabetic and nondiabetic animals; this increase correlates with a decreased concentration of circulating glucose and an elevated concentration of growth hormone in blood. Melanoma B16 grown under serum-free culture conditions secretes SICRI into the medium. Affinity-purified SICRI stimulates glucose uptake by rat epididymal adipocytes and competes with radiolabeled insulin for binding to these cells. Low concentrations of SICRI enhance growth of cultured melanoma B16 cells, whereas high concentrations of this substance have inhibitory growth effects on these cells. Porcine insulin, human insulin-like growth factors I and II, human growth hormone, platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor have negligible influence on growth of melanoma B16.     

Inhibition of growth of B16 murine malignant melanoma by exogenous interferon

We previously reported that polyinosinic-polycytidylic acid, a potent interferon inducer, inhibits the growth of B16 malignant melanoma in the C57BL/6 mouse. Two experiments were done to evaluate the effectiveness of interferon in tumor inhibition in vivo. In the first, mice were implanted with melanoma and divided into four groups, according to treatment: interferon preparation; interferon control preparation ("breakthrough fraction"); phosphate-buffered saline control; and murine serum albumin control. Daily, each mouse was given i.p. injections of 200,000 NIH reference units (hereafter called units) of interferon or of one of the control substances. The second experiment was similar to the first, except that bovine serum albumin was an additional control. In both experiments, the average tumor volume in interferon-treated mice was statistically significantly smaller than that of each control group. Mouse interferon preparations also inhibited the multiplication of B16 malignant melanoma cells in culture. This inhibition was statistically significant from interferon levels as low as 5 to as high as 5000 units/ml. The degree of inhibition markedly increased from 5 up to 500 units, the inhibition reaching its maximum at this concentration. The inhibitory effect of interferon was abrogated by anti-murine interferon serum produced in a rabbit. These findings suggest that the in vivo inhibition of the growth of B16 melanoma demonstrated with polyinosinicpolycytidylic acid and with exogenous interferon probably results, at least in part, from a direct effect of interferon on the tumor cells themselves.     

Inhibition of B16-BL6 melanoma growth in mice by methionine-enkephalin

The antitumor effect of methionine-enkephalin [( Met]enkephalin) was demonstrated in C57BL/6J mice inoculated with B16-BL6 melanoma cells. Local subcutaneous tumor growth was inhibited with a 50-micrograms dose daily for 7 or 14 days. The antitumor effect of [Met]enkephalin was inhibited by the administration of the opioid receptor antagonist naloxone. Naloxone alone had no significant effect on tumor growth.     

Dietary glycine inhibits the growth of B16 melanoma tumors in mice.

Dietary glycine inhibited hepatocyte proliferation in response to the carcinogen WY-14,643. Since increased cell replication is associated with hepatic cancer caused by WY-14,643, glycine may have anti-cancer properties. Therefore, these experiments were designed to test the hypothesis that dietary glycine would inhibit the growth of tumors arising from B16 melanoma cells implanted subcutaneously in mice. C57BL/6 mice were fed diet supplemented with 5% glycine and 15% casein or control diet (20% casein) for 3 days prior to subcutaneous implantation of B16 tumor cells. Tumor volume was estimated from tumor diameter for 14 days. Tumors were excised, weighed and sectioned for histology post-mortem. B16 cells and endothelial cells were cultured in vitro to assess effects of glycine on cell growth. Statistical tests were two-sided and a P-value of 0.05 was defined as a significant difference between groups. Weight gain did not differ between mice fed control and glycine-containing diets. B16 tumors grew rapidly in mice fed control diet; however, in mice fed glycine diet, tumor size was 50-75% less. At the time of death, tumors from glycine-fed mice weighed nearly 65% less than tumors from mice fed control diet (P < 0.05). Glycine (0.01-10 mM) did not effect growth rates of B16 cells in vitro. Moreover, tumor volume and mitotic index of B16 tumors in vivo did not differ 2 days after implantation when tumors were small enough to be independent of vascularization. After 14 days, tumors from mice fed dietary glycine had 70% fewer arteries (P < 0.05). Furthermore, glycine (0.01-10 mM) inhibited the growth of endothelial cells in vitro in a dose-dependent manner (P < 0.05; IC50 = 0.05 mM). These data support the hypothesis that dietary glycine prevents tumor growth in vivo by inhibiting angiogenesis through mechanisms involving inhibition of endothelial cell proliferation.     

Effect of anti-B16 melanoma monoclonal antibody on established murine B16 melanoma liver metastases

The administration of anti-B16 monoclonal antibody of the IgG2b isotype to mice bearing established B16 melanoma liver metastases caused a significant and consistent reduction of up to 90% in the number of these metastases. No reduction in the number of metastases was noted when antigenically unrelated tumor or nonspecific immunoglobulin were employed. The antibody-mediated antitumor effect was completely abrogated by total body irradiation of the host. Treatment of the tumor-bearing host with antiserum directed against asialo GM1 prior to anti B16 antibody administration, abrogated the therapeutic effect indicating the involvement of a radiosensitive, ASGM1-positive cell in the tumor regression. The antitumor effect of the antibody treatment could be augmented by the concomitant administration of recombinant interleukin-2. The effect seen may have possible application in the treatment of liver metastases in humans by combined immunotherapy using recombinant interleukin-2 and specific antitumor monoclonal antibodies.     

Effect of medroxyprogesterone and an aorta-derived cell growth inhibitor on B16 melanoma in mice

An aorta-derived inhibitor of endothelial cell and tumor cell growth and medroxyprogesterone, which depresses collagenase expression in vivo, were tested alone and in combination against B16-F10 melanoma in C57BL/6 mice in such doses that either agent alone had little effect. Together, these agents retarded growth of subcutaneously transplanted tumor cells and reduced the number and size of pulmonary tumors after iv tumor cell injection. Of the treatments used, only the aortic factor administered alone prolonged life in mice with pulmonary tumors.     

Clonal origin of metastasis in B16 murine melanoma: a cytogenetic study

A cell line isolated from the B16 melanoma and carried in continuous culture for 8 years (the parent line) exhibited great heterogeneity in terms of marker chromosome content. A lung metastasis from a C57BL/6 mouse inoculated im with cells of this line showed karyotypic homogeneity. Inoculation iv of cells from the parent line produced numerous tumor foci in various organs. Cytogenetic analyses of 18 such lesions led to the following conclusions: Cells from each metastatic colony exhibited relatively homogeneous karyotypic characteristics, indicating that metastases are of clonal origin; many parental cells with different marker chromosomes had metastatic potential; and some genomes maintained homogeneity longer than others.     

B16 melanoma development, NK activity cytostasis and natural antibodies in 3 and 12 month old mice.

Three types of natural immune responses against malignant cells were studied in vitro: Cytotoxicity mediated by splenic NK cells; cytostasis mediated by splenocytes and binding of naturally occurring antibodies to various tumour targets. These responses were studied in untreated 3 and 12 month old mice and in mice of both age groups inoculated with B16 melanoma cells. The results showed that in normal mice NK activity decreases with age, cytostatic activity remains unchanged and the titre of natural antibodies increases. Twelve-month old mice were shown to be appreciably more resistant than 3 month old mice to the development of tumours from subthreshold numbers of B16 tumour cells. In mice injected with threshold amounts of the B16 tumour, there was no change in any of the responses in the tumour-free period, but there was a decrease in NK activity and an increase in cytostatic activity when a large tumour mass developed. An increase in the titre of natural antibodies in young mice injected with the tumour was also seen. The correlation between these changes and tumour appearance and development is discussed.     

B16 melanoma in hairless mice: a model for thermographic research

Little is understood concerning the mechanism of tumor-induced thermographic abnormalities observed in man. An ideal animal model is lacking. In an effort to create such a model we have worked with hairless mice, subcutaneously inoculated with B16 melanoma cells. This report documents the progress of that work and the subsequent development of a totally satisfactory system for the study of such tumors in a hairless animal.     

Phenotypic diversity of murine B16 melanoma detected by anti-B16 monoclonal antibodies

A panel of monoclonal antibodies (MoAbs), produced against the murine B16 melanoma, has been used to characterize its phenotypic diversity. Six MoAbs that did not bind to primary cultures of kidney, brain or liver, spleen cells, thymocytes, 3T3 fibroblasts, melanin, or transferrin receptors were selected for further evaluation. Five MoAbs, which recognized surface antigens expressed on parental B16 cells and the B16-F1, B16-F10, B16-F10 FLR, and B16-BL6 sublines, did not appear to cross-react with each other, suggesting that they identified antigenically distinct epitopes. Four MoAbs, designated as IB16-2, IB16-4, IB16-8, and IB16-10, recognized B16 surface antigens that were variably expressed over short periods of time. This variable expression was independent of the cell cycle and was characteristic of four B16 sublines. Two of these MoAbs, both of the IgG2b isotype, fixed rabbit and guinea pig complement and were cytolytic in the presence of rabbit complement. One MoAb, designated IB16-6, recognized a surface antigen consistently expressed on greater than 90% of cells of both the parental tumor and the sublines. This MoAb bound to several murine and one human melanoma cell line, but not to other histopathological types of tumors or normal tissues. The cellular antigen that this antibody recognized was not detected in the cytoplasm, did not modulate in the presence of IB16-6, and was sensitive to trypsin, pronase, alcohols, acetone, and detergents, thereby suggesting that it was a protein. Our data are among the first that directly show the extent of phenotypic diversity of the B16 melanoma and sublines that have been derived from it.     

Effects of sodium cyanate in mice bearing B16 melanoma

Summary Sodium cyanate injected IP at a dose level of 200 or 250 mg/kg caused a 90% or greater inhibition of the incorporation of [³H]thymidine into DNA of B16 melanoma transplanted SC in mice. Despite the inhibitory effect of sodium cyanate on precursor incorporation into DNA, no significant effect on host survival was observed when sodium cyanate was administered as a single agent in the diet, in drinking water, or by IP injection to mice that had received IP transplants of B16 melanoma. The action of melphalan and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in prolonging the survival time of melanoma-bearing mice was not enhanced by combined treatment with sodium cyanate. However, combined injections of sodium cyanate and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) increased the survival of tumor-bearing mice significantly more than injections of BCNU alone at a lower dose than the maximum tolerated one. These data and other studies suggest that B16 melanoma may be less responsive to the action of sodium cyanate than are murine leukemic cells or rat hepatomas.The work described in this paper was supported by NIH grant CA-35315 and a research award from the Veterans Administration     

Polyethylenimine-mediated in vivo gene transfer of a transmembrane superantigen fusion construct inhibits B16 murine melanoma growth

Immunotherapy has been proposed as a therapeutic strategy in advanced-stage melanomas in which other therapeutic options have little effect. The Staphylococcus enterotoxin A (SEA) has been used to stimulate an antitumoral immune response but its use is hampered by severe systemic side effects. Here, we show that SEA can be targeted to melanoma cells to limit these side effects. More specifically, we used a nonviral vector, the cationic polymer, polyethylenimine (PEI), to express a transmembrane SEA fusion construct (pSEA-TM) in B16F10-induced subcutaneous melanoma in mice. The efficacy of this in vivo transfection was enhanced by concomitant infusion of epinephrine to induce local vasoconstriction. In these conditions, repeated injections of pSEA-TM/PEI complexes elicited a significant response, as evidenced by tumor growth inhibition, without systemic adverse effects. T cell infiltration of the tumors, together with positive lymphocyte proliferation tests, suggested local and systemic immune responses. Altogether, PEI-mediated targeting of SEA to melanoma tumor cells in vivo efficiently stimulates the antitumor immune response without inducing the side effects observed with systemic administration of SEA.     

Innate immunity based cancer immunotherapy: B16-F10 murine melanoma model

Background

Using killed microorganisms or their parts to stimulate immunity for cancer treatment dates back to the end of 19^th century. Since then, it undergone considerable development. Our novel approach binds ligands to the tumor cell surface, which stimulates tumor phagocytosis. The therapeutic effect is further amplified by simultaneous application of agonists of Toll-like receptors. We searched for ligands that induce both a strong therapeutic effect and are safe for humans.

Methods

B16-F10 murine melanoma model was used. For the stimulation of phagocytosis, mannan or N-formyl-methionyl-leucyl-phenylalanine, was covalently bound to tumor cells or attached using hydrophobic anchor. The following agonists of Toll-like receptors were studied: monophosphoryl lipid A (MPLA), imiquimod (R-837), resiquimod (R-848), poly(I:C), and heat killed Listeria monocytogenes.

Results

R-848 proved to be the most suitable Toll-like receptor agonist for our novel immunotherapeutic approach. In combination with covalently bound mannan, R-848 significantly reduced tumor growth. Adding poly(I:C) and L. monocytogenes resulted in complete recovery in 83% of mice and in their protection from the re-transplantation of melanoma cells.

Conclusion

An efficient cancer treatment results from the combination of Toll-like receptor agonists and phagocytosis stimulating ligands bound to the tumor cells.

     

Non-specific and specific active immunotherapy in a B16 murine melanoma system.

Conflicts amongst reports concerning the efficacy of both nonspecific and specific attempts at immunotherapy may be ascribed to different animal models utilizing tumors of different immunogenicity. We have selected the B16 mouse melanoma model as the example of a spontaneously occurring neoplasm that is histocompatible with the host and does have tumor-associated antigens. Attempts to alter tumor growth or survival with nonspecific active immunotherapy as well as with specific active immunotherapy were not successful. Nonspecific active pre-immunization failed to alter tumor take or growth. Specific active immunotherapy both with and without adjuvant did decrease tumor take and prolong host survival. The effects were increasingly documented at lower tumor cell inoculums and became less apparent with increase in the tumor cell challenge.     

Suppression of growth and hepatic metastasis of murine B16FO melanoma cells by a novel nutrient mixture

Highly metastatic melanoma is resistant to existing therapies. Our main objective was to investigate the effect of a nutrient mixture (NM) on B16FO tumor growth and hepatic metastasis. Tumor growth was studied in athymic nude male mice, 5-6 weeks old, inoculated with 10(6) B16FO melanoma cells subcutaneously and fed either a regular diet or one supplemented with 0.5% NM. Four weeks later, the mice were sacrificed and their tumors excised, weighed and processed for histology. Metastasis was studied in C57BL/6 mice, which received 10(6) B16FO melanoma cells by intrasplenic injection, as well as a regular or 0.5% NM-supplemented diet for 2 weeks. Survival was studied in C57BL/6 mice receiving 10(6) B16FO melanoma cells intraperitoneally (i.p.) followed by the regular, NM-supplemented, or regular diet in addition to being administered with 2 mg NM injection 3 times per week. NM inhibited the growth of B16FO melanoma cells by 50%. Lesions in the two groups were consistent with malignant melanoma. Mice were injected with B16FO cells in the spleen. Those fed the regular diet developed large black spleens and livers indicating growth in the spleen and metastasis to the liver. In contrast, mice supplemented with NM showed less growth in spleen, but also reduced metastasis to the liver. The survival time of mice receiving NM supplementation and B16FO cells i.p. was greater than in mice which were fed the regular diet. To confirm effects in vivo, we investigated the effect of NM on murine B16FO melanoma cells in vitro, including cell proliferation by MTT assay, morphology by hematoxylin and eosin (H&E) staining and apoptosis using live green caspase detection kit. In vitro, NM was not toxic at 100 microg/ml concentration, but exhibited 44% toxicity over the control at 500 and 1000 microg/ml. H&E did not indicate any changes up to 100 microg/ml. NM induced slight apoptosis at 100 microg/ml, moderate at 500 and extensive at 1000 microg/ml concentration.