asialo-gm1-ganglioside and Melanoma

Metastases account for 90% of all cancer-related deaths, becoming a therapeutic problem. Approximately 50% of all uveal melanoma (UM) patients will develop metastases, mainly in the liver. Post-mortem analyses of livers from metastatic UM patients showed two different metastatic growth patterns: infiltrative and nodular. The infiltrative pattern exhibits tumor infiltration directly to the hepatic lobule and minimal angiogenesis. The nodular pattern shows clusters of tumor cells around the portal venules that efface the liver parenchyma. We recently demonstrated Natural Killer (NK) cells play a pivotal role in the control of hepatic metastases and the pigment epithelial-derived factor (PEDF) controls angiogenesis in the liver using our established ocular melanoma animal model. In this study we investigated the role of NK cells and PEDF in the development of metastatic growth patterns, as this can contribute to the development of novel therapeutics specific towards each growth pattern.. Our in vivo work showed two distinct metastatic growth patterns, the infiltrative and nodular, recapitulating the post-mortem analyses on human liver tissue. We discovered NK cells control the infiltrative growth. In contrast, PEDF controlled anti-angiogenic responses, showing higher MVD values compared to NK-depleted and WT animals. The myeloid lineage, comprised of monocytes, macrophages, and myeloid-derived suppressor cells, was reduced in the absence of NK cells or PEDF.. Our animal model recapitulates the metastatic growth patterns observed in the human disease. We demonstrated a role for NK cells in the development of the infiltrative growth pattern, and a role for PEDF in the development of the nodular pattern. The understanding of the complexity associated with the metastatic progression has profound clinical implications in the diagnostic and disease-management as we can develop and direct more effective therapies.

Topics: Animals; Antibodies; Cell Line, Tumor; Eye Proteins; Female; G(M1) Ganglioside; Gene Knockout Techniques; Killer Cells, Natural; Liver Neoplasms; Macrophages; Melanoma; Mice; Mice, Inbred C57BL; Monocytes; Myeloid-Derived Suppressor Cells; Neoplasm Transplantation; Nerve Growth Factors; Serpins; Uveal Neoplasms

The possibility that endocrine factors may influence the clinical course of malignant melanoma is suggested by the superior survival data of women. In preclinical models we observed a higher rate of colony formation by human melanoma cells in male compared to female SCID mice, but only in the case of the liver and not in other organs. The gender difference could be seen at an early phase of colony formation. On the other hand, in our human melanoma cell lines we failed to detect steroid receptor protein expression, and treatment with sex hormones did not considerably influence their in vitro behavior. Investigating the possible contribution of host cells to the observed gender difference, we performed in vivo blocking experiments applying pretreatment of the animals with Kupffer cell inhibitor gadolinium chloride and the NK cell inhibitor anti-asialo GM1 antibody. While Kupffer cell blockade enhanced melanoma liver colonization equally in the two sexes, a more prominent increase was observed in female than in male mice in the case of NK cell inhibition. Further supporting the importance of NK cells in the lower liver colonization efficiency of melanoma cells in females, gender difference in colony formation was lost in NSG mice lacking NK activity. Although in humans no organ selectivity of gender difference in melanoma progression has been observed according to data in the literature, our results possibly indicate a contribution of natural host defense mechanisms to gender difference in survival of patients with melanoma or other tumor types as well.

Topics: Animals; Apoptosis; Cell Adhesion; Cell Proliferation; Cytotoxicity, Immunologic; Female; Flow Cytometry; G(M1) Ganglioside; Gonadal Steroid Hormones; Humans; Immunoenzyme Techniques; Killer Cells, Natural; Kupffer Cells; Liver Neoplasms, Experimental; Male; Melanoma; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, SCID; Receptors, Steroid; Sex Factors; Tumor Cells, Cultured

A number of methods have been developed to assess the impact of a xenobiotic on the various components of the immune system. For risk analysis, it is necessary to determine what degree of chemically induced immune perturbation translates into altered host resistance. Natural killer (NK) cells play a pivotal role in the innate immune system with the ability to lyse cells infected with intracellular pathogens and certain tumors without previous exposure to the antigen. Spontaneous NK activity in B6C3F1 mice could be incrementally and consistently decreased by 20 to > or =80% by the intravenous administration of a range of dilutions of anti-asialo GM1 (AAGM1) antibody. The decrease in spontaneous NK activity following a single iv administration of AAGM1 antibody persisted for up to approximately 3 weeks when the initial suppression (e.g., 24 h after AAGM1 antibody injection) was almost 100%. Treatment with AAGM1, however, did not appear to perturb the function of other immune cells, based on results of the plaque assay, the mixed lymphocyte response, the cytotoxic T lymphocyte assay, the reticuloendothelial system clearance of sRBC assay, and the Streptococcus pneumoniae host resistance assay. Following a > or =80% decrease in spontaneous NK activity in mice, challenge with > or =1 x 10(3) B16F10 melanoma cells resulted in an increase in tumor burden based on the number of lung nodules. However, following challenge with 1 x 10(5) melanoma cells, a significant increase in tumor burden in mice was not observed until spontaneous NK activity had been decreased by > or =50-60%. Altered host resistance is a function not only of the magnitude of the decrease in NK activity but also of the magnitude of the challenge to the host.

Topics: Animals; Antibodies; Body Weight; Cell Count; Cells, Cultured; Crosses, Genetic; Cytotoxicity Tests, Immunologic; Dose-Response Relationship, Immunologic; Erythrocytes; G(M1) Ganglioside; Immunocompromised Host; Immunosuppression Therapy; Immunosuppressive Agents; Killer Cells, Natural; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Neoplasm Transplantation; Organ Size; Spleen; Streptococcus pneumoniae; T-Lymphocytes; Xenograft Model Antitumor Assays

In an attempt to enhance the anti-tumour immune response, the co-stimulatory molecules B7-1 or B7-2 were expressed on the surface of B16 melanoma cells. B7-expressing tumours grew more slowly in both syngeneic immunocompetent mice and athymic T cell-immunodeficient nude mice. The delay in growth of B7-expressing tumours was dependent on natural killer (NK) cells, as reductions in tumour growth rates were minimized in mice depleted of NK cells. Systemic immunity to B16 melanoma was examined by vaccination with irradiated tumour cells. Inoculation with irradiated B16 B7-1 cells failed to protect against a subsequent challenge with live parental B16 cells, but conferred partial protection against challenge with live B16 B7-1 cells. In contrast to the local anti-tumour reaction, this protective response was dependent on T cells. The results presented here reveal some of the mechanisms involved in the in vivo response to a poorly immunogenic tumour modified to express co-stimulatory molecules.

Topics: Animals; Antibodies; Antigens, CD; B7-1 Antigen; B7-2 Antigen; G(M1) Ganglioside; Killer Cells, Natural; Lymphocyte Depletion; Melanoma; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Nude; T-Lymphocytes; Transfection

The optimal conditions were examined for selective re-N-acetylation with 14C or 3H.acetic anhydride of de-N-acetylated aminosugar-containing glycosphingolipids. Re-N-acetylation, which is nearly quantitative within 10 minutes in methanol, occurs selectively up to a maximal 100% yield when using a molar ratio of 5 mol of acetic anhydride per mole of aminosugar present in the glycosphingolipid. Above this molar ratio, it was observed some O-acetylation of carbohydrates which could be removed by mild alkali treatment. The method allows the choice of 14C- or 3H-labeling of glycosphingolipids with a final specific radioactivity which depends solely on the one of acetic anhydride. The binding of specific antibodies to glycosphingolipids, which was abolished upon de-N-acetylation, was again detectable after re-N-acetylation with radioactive acetic anhydride, suggesting that the native structures were recovered. This procedure of radiolabeling offers safety, rapidity and broad applicability to alkali-stable aminosugar-containing glycosphingolipids.

Topics: Acetylation; Animals; Brain Chemistry; Carbon Radioisotopes; Cattle; Chromatography, High Pressure Liquid; Erythrocytes; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Isotope Labeling; Melanoma; Tritium

Antitumor effect of N-1554 (alpha-dihydrodecaprenyl phosphate containing eight trans internal isoprene residues) against B16-F10 melanoma in syngeneic C57BL/6 mice was examined. B16-F10 cells were inoculated into the footpad of mice and N-1554 was intraperitoneally administered after the inoculation. The drug significantly inhibited the tumor growth in the footpad and dramatically reduced the pulmonary metastasis from the tumor. The antitumor effect of N-1554 was almost abolished when the immunosuppressant carrageenan or anti-asialo GM1 antibody was administered to mice. In addition, pretreatment of host mice with N-1554 reduced the growth of subcutaneously inoculated B16-F10 melanoma. These results suggest that enhancement of host immune system may be involved in the antitumor effect of N-1554.

Topics: Animals; Antibodies; Antineoplastic Agents; Carrageenan; Cell Line; G(M1) Ganglioside; Glycosphingolipids; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred C57BL; Polyisoprenyl Phosphates

Surface galactosyltransferase (GT) has been described on a variety of cells where it is believed to be involved in cell-cell and cell-substratum adhesion. Here we show that B16 metastatic murine melanoma cells exhibit a 5-fold higher cell surface GT activity than their nonmetastatic counterparts, although total GT activity in NP-40 solubilized cells is similar for both melanoma variants. Interestingly, on living cells, this cell surface GT almost exclusively galactosylates an endogenous glycoprotein (Mr = 110,000). This metastasis-associated GT is specific for terminal D-N-acetylglucosamine, catalyzes the formation of a beta 1-4 linkage, does not recognize polylactosaminoglycans, and has its specificity altered from D-N-acetylglucosamine to D-glucose by alpha-lactalbumin; yet the Mr = 110,000 protein is not a major substrate when exogenous bovine GT is used on the outside of living cells. In addition to this protein-specific endogenous GT activity, another cell surface GT activity that selectively galactosylates glucosylceramide is also prominent. Endogenous galactosylation of both protein and glycolipid substrates is reduced when the membrane is solubilized by the detergent NP-40 but remains unaltered in the presence of digitonin, which permeabilizes but does not dissolve the membrane. These data suggest that the GTs and their substrates are associated on the cell surface. Chloroquine treatment of intact cells leads to a 4-fold and a 3-fold increase in galactosylation of the Mr = 110,000 protein and glucosylceramide, respectively, suggesting that these two substrates normally reside mostly in the lysosomal or Golgi compartments. The increased expression of lysosomal membrane proteins on the surfaces of highly metastatic cells may, in part, also explain the galactosylation differences observed. These studies further suggest that increased surface localization of certain glycosyltransferases with highly restricted in situ substrate specificities may be a common feature of highly metastatic tumor cells.

Topics: Animals; Antigens, CD; Cell Membrane; Chloroquine; Chromatography, Thin Layer; G(M1) Ganglioside; Galactose; Galactosyltransferases; Glycolipids; Glycoproteins; Glycosphingolipids; Glycosylation; Isomerism; Lactalbumin; Lactosylceramides; Melanoma; Mice; Molecular Weight; Neoplasm Metastasis; Protein Processing, Post-Translational; Tumor Cells, Cultured

The effects of pure recombinant human interferon alpha A/D (IFN alpha A/D) on natural killer (NK) activity and the experimental lung metastasis of B16-F10 melanoma were studied. Treatment of C57BL/6 mice with IFN alpha A/D augmented splenic NK activity and also inhibited the experimental lung metastasis of B16-F10 melanoma in a dose-dependent manner. The augmentation of NK activity and the inhibition of experimental lung metastasis by IFN alpha A/D were completely abolished in anti-asialo GM1-pretreated mice. These results suggested that the effector cells which inhibited melanoma metastasis in the present system were mainly NK cells, and that it was by activating NK cells that IFN alpha A/D had its effect. We next studied the timing of IFN alpha A/D administration for the most effective prevention of melanoma metastasis. The inhibitory effect of IFN alpha A/D was most pronounced when it was given 12 hr before or at the same time as melanoma inoculation. This suggested that melanoma cells were susceptible to NK cells only for a short period of time after intravascular invasion.

Topics: Animals; G(M1) Ganglioside; Glycosphingolipids; Interferon Type I; Killer Cells, Natural; Lung Neoplasms; Macrophages; Male; Melanoma; Mice; Mice, Inbred C57BL; Neoplastic Cells, Circulating; Recombinant Proteins; Spleen; Time Factors

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.

Topics: Animals; Cell Line; Cell Survival; Female; G(M1) Ganglioside; Glycosphingolipids; H-2 Antigens; Immune Sera; Immunization, Passive; Immunologic Deficiency Syndromes; Killer Cells, Natural; Lung Neoplasms; Lymphatic Metastasis; Lymphocyte Cooperation; Melanoma; Mice; Mice, Inbred C57BL; Phenotype; Spleen

This study was undertaken in an attempt to understand the mechanism of antitumor action of pyrimidinones alone and in combination with cyclophosphamide (CY). Pyrimidinones such as 2-amino-5-bromo-6-(3-fluorophenyl)-4(3H)pyrimidinone (ABMFPP) were relatively nontoxic toward murine L1210 leukemia cell growth in vitro with the concentration of drug required for a 50% inhibition of cell growth being greater than 50 micrograms/ml. In contrast, ABMFPP showed anti-B16 melanoma activity in vivo which was sensitive to X-irradiation of the hosts. These results collectively suggest that pyrimidinones may act differently from conventional cytotoxic antitumor agents. Multiple i.p. injections of ABMFPP (125 mg/kg/injection) significantly augmented the cytotoxicity of both natural killer cells and macrophages in peritoneal exudates. The augmentation of both effector cell populations was delayed, but was more pronounced when animals received a dose of CY (100 mg/kg) prior to ABMFPP injections. The combination of CY and ABMFPP also showed a synergistic anti-P388 leukemia effect which appeared to be related to the initial reduction of the tumor burden by CY and the marked augmentation of the cytotoxicity of both natural killer cells and macrophages by ABMFPP. The antitumor activity of ABMFPP against B16 melanoma was almost completely eliminated when animals received a dose of 400 rads X-irradiation 5 days prior to tumor inoculation or a dose of 200 rads X-irradiation followed by several injections of anti-asialo monosialoganglioside antibody. The administration of anti-asialo monosialoganglioside alone also markedly reduced the anti-B16 melanoma activity of ABMFPP. The magnitude of reduction of the antitumor effect of ABMFPP by radiation and/or anti-asialo monosialoganglioside antibody directly correlated with the inhibition of the ABMFPP-mediated augmentation of immune responses. These results strongly suggest that the antitumor effect of ABMFPP alone or in combination with CY is at least in part mediated through its augmentation of natural killer cell and/or macrophage activities.

Topics: Animals; Antibodies; Cell Division; Cyclophosphamide; Cytosine; G(M1) Ganglioside; Glycosphingolipids; Killer Cells, Natural; Leukemia L1210; Leukemia P388; Leukemia, Experimental; Macrophages; Melanoma; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred DBA

Mice treated with anti-asialo GM1 (asGM1) serum exhibited increased formation of experimental metastases in lung and liver after i.v. challenge with B16 melanoma or Lewis lung carcinoma. This increased metastasis formation coincided with decreased splenic NK activity and increased survival of i.v. injected radiolabeled tumor cells. In contrast, the injection of mice with the pyran copolymer maleic anhydride divinyl ether (MVE-2) augmented NK activity in the spleen and significantly depressed the formation of experimental metastases in the lungs and liver. However, a single or double administration of anti-asGM1 antiserum to MVE-2-pretreated mice failed to inhibit the immunoprophylaxis associated with MVE-2 administration, although it did decrease splenic NK activity and also increased the survival of i.v.-injected radiolabeled tumor cells. To address the mechanism for this dichotomy, we examined NK activity not only in the spleen but also in the blood, lungs, and livers of MVE-2-treated mice. Levels of NK activity in the lungs and liver were several-fold higher than those observed in spleen and blood. However, MVE-2-augmented NK activity in lung and liver was more resistant to depletion by the standard regimen of anti-asGM1 treatment than was NK activity in blood and spleen, and required two high-dose administrations of a higher titered antiserum for depletion of the augmented response. This high-dose regimen removed all detectable NK activity from the lung and liver, and concomitantly eliminated the metastasis-inhibiting effect of MVE-2. These data are consistent with a role for organ-associated NK cells in inhibiting metastasis formation during the extravasation and/or early postextravasation phases of the metastatic process. The results also suggest that biologic effects of NK activity in spleen and blood can be dissociated from those mediated by NK activity in other organs by use of different treatment regimens with anti-asGM1 serum. Finally, because NK activity in target organs can be augmented to an even greater extent than in the blood and spleen by at least some biologic response modifiers (BRMs), organ-associated NK activity should be considered as a possible mechanism for the therapeutic effects of BRM treatment.

Topics: Animals; Antineoplastic Agents; Cytotoxicity, Immunologic; Female; G(M1) Ganglioside; Glycosphingolipids; Growth Substances; Immune Sera; Killer Cells, Natural; Liver Neoplasms; Lung Neoplasms; Lymphoma; Male; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Organ Specificity; Pyran Copolymer

The simultaneous injection of monoclonal antibody 9.2.27, directed against a chondroitin sulfate proteoglycan preferentially expressed on human melanoma cells, and 2 X 10(7) mononuclear splenocytes, eradicated established, progressively growing human melanoma tumors in nude mice. Neither splenocytes nor antibody alone achieved significant tumor regression. The cells responsible for tumor elimination are most likely natural killer (NK) cells: they are present in splenocytes of T cell-deficient nude mice, and cloned cells with NK activity are able to suppress tumor growth. Moreover, splenocytes treated with anti-asialo GM1 and complement or harvested from NK-deficient C57BL/6 beige mice did not cause tumor rejection. Furthermore, treatment of BALB/c nude mice just before injection with anti-asialo GM1 antiserum, which is known to eliminate NK activity in vivo, resulted in better tumor growth. In addition, evidence is presented that cells with NK activity are probably the effectors responsible for melanoma target cell lysis in vitro: Antibody-dependent and -independent cell-mediated lysis of M21 melanoma cells was suppressed when splenocytes were preincubated with complement and antibodies specific for cell surface antigens of NK cells, i.e., anti-asialo GM1, anti-Qa5, and anti-NK1.1. Moreover, splenocytes of C57BL/6 beige mice were not able to lyse M21 cells in vitro. These results strongly support the conclusion that cells with NK activity are indeed responsible for the antibody-dependent destruction of M21 melanoma cells in vivo and in vitro.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Cell Line; G(M1) Ganglioside; Glycosphingolipids; Humans; Immunization, Passive; Killer Cells, Natural; Melanoma; Mice; Mice, Nude; Spleen

We have studied the experimental metastasis of H-2+ and H-2- melanoma sublines in H-2b/b and H-2a/b hosts by enumerating pulmonary colonies 20-50 days after i.v. inoculation of tumor cells. In H-2b/b hosts, the H-2+ "B16-S" cells gave rise to a moderate number of metastatic colonies (mean: 6.3 +/- 6). The "BL16-L" sublines that had lost the expression of MHC class-I antigens, according to FACS-analysis and quantitative absorption tests, gave no metastases under the same conditions. Pretreatment of the H-2+ met+ B16-S with interferons (beta or alpha + beta) increased their H-2 antigen expression and the number of metastatic colonies (mean: 25 +/- 16). Interferon pretreatment of B16-L cells partially restored their H-2b expression and induced them to form a small number of metastatic colonies. The reduction in pulmonary colonization by the H-2 negative B16-L cells could be attributed to their rapid elimination by natural killer cells, already observed within 24 hr of inoculation of radiolabelled cells. H-2- B16-L cells were more susceptible than H-2+ B16-S cells to in vitro lysis by poly I:C-treated splenocytes, and they acquired full metastatic abilities if the hosts were treated with anti-asialo GM-I serum. In H-2a/b heterozygous hosts, the H-2+ B16-S cells also failed to metastasize. Reduced pulmonary colonization was evident by 24 hr after injection in comparison with H-2b/b hosts, and could be reversed by anti-asialo GM-I treatment of the hosts. In vitro, H-2a/b splenocytes were more cytotoxic to the B16 cells than syngeneic effectors. The results are discussed in relation to a recent hypothesis on a surveillance mechanism for elimination of cells on the basis of their lack (or insufficient expression) of host MHC genes.

Topics: Animals; Antibodies; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; H-2 Antigens; Heterozygote; Homozygote; Immunity, Innate; Interferon Type I; Killer Cells, Natural; Lung Neoplasms; Melanoma; Mice; Mice, Inbred Strains; Neoplasm Metastasis

The role of asialo GM1 positive cells was studied in artificial and spontaneous pulmonary metastases as well as in tumor growth by using B-16 melanoma cells in C57BL/6 mice. Single administration of 50 microliters of anti-asialo GM1 antibody resulted in the significant decrease of NK activity in the spleen cells of C57BL/6 mice lasting 13 days from the following day of administration. The anti-asialo GM1 antibody was evaluated in terms of for its effect on pulmonary metastases with regard to the timing of administration. Treatment with anti-asialo GM1 antibody 1 day before or on the day of tumor inoculation resulted in a substantial increase in the number of artificial pulmonary metastases. In the experimental system of spontaneous metastases, the anti-asialo GM1 antibody most effectively increased the number of pulmonary metastases when administered 1 to 2 weeks before the amputation of the tumor primary site. In addition, in mice treated with anti-asialo GM1 antibody, the acceleration of the growth of the transplanted tumor was observed. These results strongly suggest that asialo GM1 positive cells not only inhibit pulmonary metastases acting mainly on circulating tumor cells but also suppress the growth of transplanted tumor.

Topics: Animals; Antibodies; G(M1) Ganglioside; Glycosphingolipids; Killer Cells, Natural; Lung Neoplasms; Male; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental