Metastasis Of B16 Melanoma Cells Research Articles

Effect of restraint stress on immune system and experimental B16 melanoma metastasis in aged mice

An overnight restraint stress was given to young and old mice and its effect was examined in terms of the number and function of T cells and natural killer (NK) cells in spleen and patterns of lung metastasis of B16 melanoma cells. A great decrease was observed in the number and proliferative activity of splenic T cells in old mice after the stress. The decrease in young mice was rather temporary with a quick recovery. The number of NK cells in spleen was not different between young and old mice before giving the stress, but a significant decrease was observed in the old after the stress. NK activity was always much lower in old than in young throughout the experiment. The pattern of metastasis of B16 melanoma cells was different between young and old mice. Metastatic colonies in lungs were larger in number and bigger in size in young mice than in old mice. After the stress, the number increased and the size unchanged in old mice, while the size increased and the number remained unchanged in young mice. It was shown that the same restraint stress resulted in a more serious influence on the immune cells in old than in young mice and gave rise to a differential effect on the pattern of tumor metastasis between young and old mice.

Enhanced metastasis of B16 melanoma cells by unexpected elevated expression of the metastasis-associated TI-241 (LRF-1-, Jun-Fos-related) gene treated with antisense oligonucleotide.

B16-F10 is a B16 mouse melanoma subline that preferentially metastasizes to the lung following intravenous injection. Previously we isolated TI-241 (LRF-1 homologue related to Jun-Fos) gene that was expressed higher in the high metastatic clone B16-F10 than the low metastatic clone F1. Transfection of TI-241 into F1 converted it into a high-metastatic cell. We studied the effect of antisense oligonucleotide designed to reduce the expression of TI-241 in B16-F10 cells, and observed an unexpected increase in the TI-241 level. The increase in the expression was maximal at 30 h, then it decreased during further culture with or without TI-241 antisense oligonucleotide. This increased TI-241 expression by antisense oligonucleotide was also observed in B16-F1 cells whereas sense oligonucleotide did not affect the expression. B16-F10 cells cultured with TI-241 antisense oligonucleotide showed enhanced experimental metastatic potential to the mouse lungs compared with untreated B16-F10 and B16-F10 cultured with TI-241 sense oligonucleotide.

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine.

We have shown previously that loxoribine exhibits adjuvant activity for B cells, activates natural killer (NK) cells, and enhances the activation of lymphokine-activated killer cells by interleukin-2 (IL-2). In this study, we examined loxoribine for protective effects in a B16 melanoma lung tumor metastasis model. Significant inhibition of B16 metastasis was seen in mice given a single injection of 2 mg loxoribine as late as day 3 of tumor growth but the greatest inhibition (96%) was seen in mice given four injections of loxoribine on alternate days starting the day before tumor injection. In experiments in which both IL-2 and loxoribine were administered, both agents were active when tested alone, but the combination of IL-2 and loxoribine gave significantly greater inhibition of metastasis. Loxoribine partially inhibited the development of tumors in mice that had been depleted of NK cells by the administration of anti-asialo-GM1 or anti-NK1.1 antibodies and in NK-deficient beige mice. In all cases, protection was seen only when smaller tumor inocula were injected. Taken together, these data suggest that both NK and non-NK cell populations or effector mechanisms with antitumor activity were activated by loxoribine. Since substituted guanosine analogs have been shown to have adjuvant activity in B cell systems, we evaluated whether loxoribine was active as an adjuvant in a tumor protection model. Mice immunized with both irradiated tumor cells and loxoribine developed a significantly lower number of lung tumors when challenged by live B16 tumor cells, whereas mice injected with either vaccine or loxoribine alone were not protected. There was a clear dose response seen with both loxoribine and the vaccine preparations. These data suggest that loxoribine may be useful in tumor therapy as an immunomodulator or as an adjuvant for use with tumor vaccines.

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine

Anti-tumour efficacy of mouse spleen cells separated with Dolichos biflorus lectin (DBA) in experimental pulmonary metastasis of B16 melanoma cells

Anti-tumour effector cells were generated through 4 days culture of normal C57BL/6 splenocytes in a medium containing concanavalin A supernatant and then fractionated with Dolichos biflorus lectin (DBA) into DBA+ (agglutinable with DBA) and DBA- (non-agglutinable with DBA) cells. The DBA- cells, infused intravenously into mice together with B16 melanoma cells, or adoptively transferred into mice 3 days after the injection of B16 cells, caused a marked decrease in the number of lung nodules, while the DBA+ cells exerted no effect. On the other hand, the DBA+ cells exhibited higher cytolytic activity in vitro than the DBA- cells in short-term 51Cr-release assays. Then, we analysed the mechanism of the strong anti-tumour activity of DBA- cells in vivo. We found that DBA- cells showed higher response to recombinant interleukin-2 (rIL-2) than DBA+ cells and proliferated very well with a small amount of IL-2. In addition, DBA- cells adhered more strongly to lung endothelial cells than DBA+ cells in response to rIL-1 or rTNF. Furthermore, DBA- cells produced larger amounts of macrophage activating factor (MAF) including IFN-gamma when cultured with B16 melanoma. Taken together, our results show that DBA- cells are effective in reducing experimental pulmonary metastases not only by the direct lytic activity but also by the indirect killing activity through the activated macrophage.

Combined Treatment of Adriamycin and Dipyridamole Inhibits Lung Metastasis of B16 Melanoma Cells in Mice

The combined effects of adriamycin (ADM) and dipyridamole (DP) on lung metastasis of B16 melanoma cells in mice were investigated. First, the antitumor effects of ADM and DP were examined both in vitro and in vivo. The clonogenicity of B16 melanoma cells was suppressed by the combination of ADM and the nontoxic dose of DP in vitro, and growth of the B16 melanoma solid tumor implanted subcutaneously in mice was inhibited with this drug combination, compared to findings with ADM alone in vivo. To examine events related to experimental lung metastasis, 2 X 10(5) B16 melanoma cells were given intravenously into the tail vein of mice and the efficacy was determined by counting the number of metastatic tumor nodules. DP in doses of 25-100 mg/kg given alone reduced the number of metastasis to about 85% that in the control group. ADM in doses of 1-4 mg/kg for 3 days inhibited the metastasis, in a dose-dependent manner. When combined with DP, the antimetastatic effect of ADM was enhanced and the number of metastasis prominently decreased, compared to single application of the drug (p less than 0.05). We speculate that ADM given concomitantly with DP may have the potential to inhibit metastatic growth of a tumor.

In vivo dynamics of pulmonary lymphoid cell subpopulations generated against pulmonary metastasis: evaluation by broncho alveolar lavage fluid

The dynamics of lymphoid cell subpopulations in bronchoalveolar lavage fluid (BALF) and the systemic lymphoid organs of mice after intravenous injection of B16 melanoma cells were examined with a fluorescence-activated cell sorter. The lymphoid cell subpopulations of BALF and mediastinal lymph nodes showed significant changes in numbers and proportions, while those of other lymphoid organs including inguinal lymph nodes, thymus and spleen, showed little change. In week 1, the cells with a Thy-1.2+, Lyt-1+, L3T4-, Lyt-2- phenotype and asialo-Gm1+ cells in BALF significantly increased and L3T4+ cells slightly increased in number. By week 3, the numbers of Lyt-2+ cells in BALF markedly increased in number (by about 90 times) compared with controls. The number of Thy-1.2+ cells in mediastinal lymph nodes also increased significantly by week 3. Mice that had been pretreated with an immunosuppressive dose of cyclophosphamide were also inoculated intravenously with B16 melanoma cells. In these mice, a significantly increased number of pleural tumors developed and the number of Thy-1.2+ cells in BALF was markedly reduced from week 1 to 3. The results indicate that L3T4 and Lyt-2 double negative T-cells and natural killer (NK) cells may be generated and/or mobilized to the lung in an early phase of experimental metastasis of B16 melanoma cells and that at a later stage, when multiple metastases develop, T-cells with a Lyt-2+ phenotype markedly increase, probably as an expression of a host reaction against proliferating metastatic tumor cells.

In vivo Effects of Recombinant Interferon Alpha A/D Incorporated in Gelatin Microspheres on Murine Tumor Cell Growth

Intraperitoneal (ip) injections of gelatin microspheres containing a very small amount of recombinant human interferon alpha A/D (A/D‐IFN) (IFN‐microspheres) plus free A/D‐IFN improved the survival of mice bearing ascitic Meth A‐R1 cells which we had isolated as IFN‐resistant cells under in vitro conditions. The dose of free A/D‐IFN in one injection was 10,000 IU, which was insufficient by itself for manifesting in vivo antitumor activity. In these mice, in vivo Rl cell growth was suppressed and macrophage recruitment was enhanced in comparison with mice receiving other control agents. Administration of IFN‐microspheres alone was also effective but less than that of IFN‐microspheres plus free A/D‐IFN. Peritoneal macrophages obtained from normal or R1‐bearing mice receiving ip injection of IFN‐microspheres with or without free A/D‐IFN were activated to inhibit the in vitro growth of R1 cells. The intratumoral injection of IFN‐microspheres strongly inhibited the growth of solid R1 tumors. Intravenous injection of IFN‐microspheres was effective in preventing the pulmonary metastasis of B16 melanoma cells. These results indicate that the IFN‐microsphere is much more effective against tumors than free A/D‐IFN.

Synergistic defense system by cooperative natural effectors against metastasis of B16 melanoma cells in H-2-associated control: different behavior of H-2+ and H-2- cells in metastatic processes.

H-2+ and H-2- cells of B16 melanoma were established by repeated fluorescence-activated cell sorting. The H-2- line formed no metastasis in untreated C57BL/6 mice, whereas the H-2+ cells showed evidence of metastatic development. This difference was ascribed mainly to the increased susceptibility of H-2- cells to attack by natural effector mechanisms, particularly asialo GM1+ NK cells. After treatment with both anti-asialo GM1 serum and whole body irradiation (400 rad), numerous colonies of H-2- cells formed in the lung, whereas the metastasis was only marginally enhanced by irradiation and moderately by treatment with anti-asialo GM1 serum. With the H-2+ cells, treatment with each modality significantly increased the number of metastatic colonies. Therefore collaboration of asialo GM1+ NK cells and radiosensitive natural effectors seems to be the main mechanism involved in the synergistic effects on defense against H-2- cell metastasis, and to a lesser extent against H-2+ cell metastasis. Irradiation (1000 rad) to the right lung to abrogate the organ-associated defense increased the colonies, particularly in the H-2+ cells. On the other hand, treatment with anti-asialo GM1 serum increased colonization in the early phase of metastasis with H-2- cells and may have abolished asialo GM1+ NK cells capable of recognizing the reduced expression of H-2 antigens and eliminating H-2- cells in the blood-born phase. Natural defense mechanisms probably exert suppressive effects on the metastasis of H-2+ cells, mainly in the organ-associated phase after extravasation.

Genetically obese mice: resistance to metastasis of B16 melanoma and enhanced T-lymphocyte mitogenic responses.

The metastasis of B16 melanoma cells differed significantly in obese (ob/ob) and lean (+/?) female mice of strain C57BL/6J. When the mice were inoculated subcutaneously with melanoma cells at 10 to 11 months of age, the primary tumor grew more slowly in obese than in lean littermates and the frequency of lung metastasis was greatly reduced. When the mice were injected with the cells at 4 to 7 months, the primary tumor grew at the same rate in obese and lean mice, but the obese mice again showed a significantly reduced frequency of lung metastasis. That this effect was related to an enhanced immunocompetence in obese mice was supported by the finding that splenic lymphocytes of ob/ob mice showed three times the proliferative response to the T-cell mitogen concanavalin A compared with the proliferative response of lean control mice. The ob/ob mouse may provide a model for the study of enhanced immunocompetence in obese individuals.