asialo-gm1-ganglioside and Lung-Neoplasms

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.

Topics: Animals; Antigens, Ly; Autoantibodies; Cell Transformation, Neoplastic; Chemokines, CXC; Chronic Disease; Colitis; Disease Models, Animal; Female; G(M1) Ganglioside; Galactosylceramides; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Depletion; Melanoma, Experimental; Mice; Mice, Transgenic; Neoplasms; NK Cell Lectin-Like Receptor Subfamily B; Tumor Burden

Combination treatment consisting of IL-2 together with anti-IL-2 mAbs results in markedly larger increases in the numbers of CD8(+) T cells, dendritic cells (DCs) and NK cells in vivo compared with the results observed with injections of IL-2 or the antibodies alone. We previously showed that this combination treatment overcomes the problems associated with the short half-life of IL-2 in vivo. Importantly, the combination treatment but not IL-2 or the anti-IL-2 mAbs alone protected the mice against tumor metastases in the lungs. Here we have investigated which cell types are responsible for this protective immunity against tumors. We analyzed tumor metastases in mice that were depleted of DCs, CD8(+) T cells or NK cells. DC-deficient, diphtheria toxin receptor-expressing mice injected with diphtheria toxin as well as B cell- and T cell-deficient RAG-2-knockout mice were protected against tumors after they were administered the combination treatment. On the other hand, mice that were depleted of NK cells using anti-asialo-GM1 antibodies did not exhibit the anti-tumor activity after treatment with IL-2 combined with anti-IL-2 mAbs. Thus, these data demonstrate that NK cells, but not DCs, or CD8(+) T cells mediate the anti-tumor effect induced by this combination treatment. Therefore, combining neutralizing anti-IL-2 mAbs with IL-2 may be clinically useful to effectively enhance IL-2-mediated NK cell activities.

Topics: Animals; Animals, Genetically Modified; Antineoplastic Combined Chemotherapy Protocols; CD8-Positive T-Lymphocytes; Dendritic Cells; G(M1) Ganglioside; Heparin-binding EGF-like Growth Factor; Immunity, Cellular; Immunotherapy; Intercellular Signaling Peptides and Proteins; Interleukin-2; Killer Cells, Natural; Lung Neoplasms; Melanoma, Experimental; Mice; Neoplasm Metastasis; Neoplasm Transplantation

Bovine lactoferrin (bLF), a milk protein known to have bacteriostatic properties was examined for its preventive effects on colon and other organ carcinogenesis and experimental metastasis. (Experiment 1) The influence on colon carcinogenesis was investigated in male rats treated with azoxymethane (AOM), then received 2 or 0.2% bLF for 36 weeks. Significant reduction in the incidence (27% and 46% of the control, respectively) and number of adenocarcinomas of the large intestine was observed. (Experiment 2) In BALB/c mice bearing subcutaneous (s.c.) implants of colon carcinoma 26 (Co 26Lu). bLF demonstrated significant inhibition of spontaneous lung metastasis (approximately 43% of the control). Number of cytotoxic asialoGM1+ and CD8+ cells in white blood cells increased (171% and 122% of control, respectively) after treatment. Results of those experiments indicate that bLF remarkably prevents colon carcinogenesis and lung metastasis of colon carcinoma cells, possibly due to increasing cytotoxic cells in the peripheral blood.

Topics: Animals; Azoxymethane; Cattle; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Coculture Techniques; Colonic Neoplasms; G(M1) Ganglioside; Lactoferrin; Leukocytes; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Rats, Inbred F344; Tumor Cells, Cultured

The effects of oral administration of bovine lactoferrin (bLF) and its hydrolysate on the lung colonization by colon 26 carcinoma were investigated. At doses of 100 or 300 mg/kg/day for seven successive days, bLFs demonstrated a significant inhibitory effect on experimental metastasis, which indicated effectiveness before and after tumor implantation. Oral administration of bLFs augmented CD4+, CD8+, and asialoGM1+ cells in the spleen and peripheral blood. Their cytotoxic activities against Yac-1 and colon 26 carcinoma were enhanced by bLF. In the small intestinal epithelium, CD4+ and CD8+ cells were markedly increased, and, simultaneously, enhanced production of interleukin-18 (IL-18) was confirmed in the intestinal epithelial cells. In this model, intravenous injection of murine IL-18 showed significant inhibition of the lung colonization by colon 26 carcinoma. These results suggested that inhibition of experimental metastasis by oral administration of bLF and pepsin hydrolysate of bLF might be due to enhanced cellular immunity, presumably mediated by enhanced IL-18 production in the intestinal epithelium.

Topics: Administration, Oral; Animals; Carcinoma; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Fluorescent Antibody Technique; G(M1) Ganglioside; Humans; Immunity, Cellular; Interleukin-18; Intestinal Mucosa; Killer Cells, Natural; Lactoferrin; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Specific Pathogen-Free Organisms; Spleen; Tumor Cells, Cultured

SCID mice were inoculated with five human-mouse heterohybridomas derived by fusion of human lymph node lymphocytes from lung cancer patients with murine myeloma cells or human-mouse heteromyeloma cells, and the production of their human monoclonal antibodies (MAb) in the mouse ascites was investigated. In a comparison of the effects of pretreatment by i.p. (intraperitoneal) injection of pristane and anti-asialo GM1 serum on the antibody production of three of the hybridomas, pristane pretreatment resulted in substantial antibody production by all three, and pretreatment with anti-asialo GM1 serum resulted in similar or slightly lower levels of antibody production by two of the hybridomas but none by the third. In a second series of experiments using four of the hybridomas with pristane pretreatment, the co-injection of either penicillin G and streptomycin or kanamycin together with the hybridoma at the time of i.p. inoculation resulted in enhanced MAb production by the two heterohybridomas that had been propagated in medium containing hypoxanthine-aminopterin-thymidine (HAT) but not by the two that had been propagated in HAT-free medium.

Topics: Animals; Anti-Bacterial Agents; Antibodies, Monoclonal; Ascites; G(M1) Ganglioside; Humans; Hybridomas; Immunosuppressive Agents; Lung Neoplasms; Mice; Mice, SCID; Terpenes

Either folic or folinic acid enhanced the antimetastatic activity of recombinant murine interferon beta (rMulFN beta) toward highly metastatic colon carcinoma 26 (Co 26Lu). Folinic acid administered with rMuIFN beta markedly increased asialoGM1+CD4+ and asialoGM1+CD8+ T cell production in the peritoneal cavity but not in the thymus and spleen. Peritoneal cells expressed killing activity toward Co 26Lu cells in vitro. In athymic nude mice, the above combination produced many asialoGM1+CD4+ and few asialoGM1+CD8+ T cells in the peritoneal cavity, but did not decrease lung metastatic colonies. AsialoGM1+CD4+ T cells would thus appear to have no or only very weak killing activity toward these tumor cells. The antimetastatic activity of folinic acid with rMuIFN beta was significantly decreased with anti-asialoGM1 and anti-CD8 antibodies. Inactivated CD8+ and asialoGM1+ cells cease to have killing activity toward Co 26Lu cells as shown by Winn's assay. AsialoGM1+CD8+ cell production was markedly induced in the peritoneal cavity by treatment with rMuIFN beta and folinic acid. AsialoGM1+CD8+ T cells may be inhibiting lung metastasis of Co 26Lu. Folinic acid and interferon are used in combination therapy with 5-fluorouracil for biochemical modulation. Folinic acid with interferon, as adjuvant therapy, may promote the induction of CD8+ T cell production with consequent prevention of metastasis.

Topics: Animals; Antineoplastic Agents; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Colonic Neoplasms; Drug Combinations; Folic Acid; G(M1) Ganglioside; Interferon-beta; Leucovorin; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Peritoneal Cavity

Depletion of both natural killer 1.1+ (NK1+) intermediate alpha beta T-cell receptor (int T) cells and NK cells by in vivo treatment with anti-NK1 antibody greatly increased hepatic metastases of intravenously injected EL4 cells as well as pulmonary metastases of 3LL cells in C57BL/6 mice. However, depletion of NK cells alone by anti-asialo GM1 (AGM1) antibody treatment did not increase the metastases in either organ. Interleukin-12 (IL-12) administration into mice induced strong cytotoxicities of NK cell-depleted liver and lung mononuclear cells (MNC) comparable to those without NK-cell depletion and inhibited metastases in either organ. In contrast, in both NK cell- and NK1+ int T-cell-depleted mice, IL-12 could not induce cytotoxic activity of liver and lung MNC and metastases in both organs increased with or without IL-12 treatment. These results confirmed the fact that NK+ int T cells are more potent antitumour effectors than NK cells against experimental haematogenous tumour metastases.

Topics: Animals; Cytotoxicity, Immunologic; Flow Cytometry; G(M1) Ganglioside; Interleukin-12; Killer Cells, Natural; Leukocytes, Mononuclear; Liver Neoplasms; Lung Neoplasms; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; T-Lymphocyte Subsets

We studied the effect of protein-bound polysaccharide PSK on metastatic colonization of BALB/c mice after intravenous injections of different syngeneic murine H-2 positive and H-2 negative tumor clones. The tumor lines used were different clones from chemically induced fibrosarcomas (GR9.B9, an H-2 negative clone from GR9 tumor, and B7.1.B4, an H-2 positive clone from B7.1 tumor). These clones were selected because of their different sensitivity to NK cytotoxicity, which was related to MHC class I expression. Pretreatment of mice with PSK inhibited metastatic colonization derived from B9 H-2 negative tumor cells. In contrast, lung colonization of PSK treated mice injected with B7.1.B4 H-2 positive tumor cells was higher, and differences in the number of colonies between untreated and PSK treated mice were small. In several experiments the effect of PSK was attenuated to a greater degree when high numbers of cells were injected. Abrogation of NK cells with anti-asialo GM1 serum significantly increased (in all tumors and at different cell doses) the number of metastatic colonies in comparison with untreated mice injected with tumors, regardless of the cell dose used. These results clearly suggest that NK cell activation in vivo by the protein bound polysaccharide PSK abrogates metastasis formation in mice. Abrogation was dependent on the H-2 phenotype even when pretreatment consisted of a single dose of PSK. This effect, related to the NK sensitivity of the tumor target, can be used to predict the effect of PSK in vivo.

Topics: Animals; Antibiotics, Antineoplastic; Clone Cells; Cytotoxicity, Immunologic; Dose-Response Relationship, Immunologic; Fibrosarcoma; G(M1) Ganglioside; H-2 Antigens; Immune Sera; Injections, Intravenous; Killer Cells, Natural; Kinetics; Lung Neoplasms; Lymphocyte Subsets; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Proteoglycans; Sarcoma, Experimental; Spleen; Tumor Cells, Cultured

Treatment of Swiss albino mice with histamine enhanced the clearance of natural killer (NK)-cell sensitive YAC-1 lymphoma and B16/F10 melanoma cells from lung tissue in vivo, but did not affect the elimination of NK-cell-insensitive P815 mastocytoma cells. The effect of histamine was apparently mediated by H2-type histamine receptors (H2R) since it was blocked by ranitidine, and H2R antagonist. Histamine did not affect clearance of tumour cells in animals depleted of NK cells in vivo by treatment with antibodies to asialo-GM1 or NK1.1. The effect of histamine was time-dependent: pretreatment with histamine for 3 h significantly augmented the clearance of YAC-1 cells, whereas, pretreatment with histamine for 5 min was ineffective. Histamine potentiated the anti-tumour properties of NK-cell activators such as interleukin-2 (IL-2) or interferon-alpha (IFN-alpha) in vivo. None of these lymphokines significantly affected the clearance of YAC-1 cells unless animals were concomitantly treated with histamine. Treatment with ranitidine alone reduced the in vivo clearance of YAC-1 cells from lungs but did not affect the clearance of NK-cell-insensitive P815 cells. Effects of ranitidine on NK-cell function in vivo were not shared by a chemical control to ranitidine, AH20239AA, thus indicating that the inhibition of NK-cells results from H2R antagonism rather than non-specific toxicity. It is concluded that histaminergic mechanisms may be involved in the regulation of NK cell function in vivo.

Topics: Animals; Antibodies; Cytotoxicity, Immunologic; Female; G(M1) Ganglioside; Histamine; Histamine H2 Antagonists; Immunity, Cellular; Interferon-alpha; Interleukin-2; Killer Cells, Natural; Lung Neoplasms; Lymphoma; Melanoma, Experimental; Mice; Ranitidine; Receptors, Histamine H2

This study was conducted to examine the effect of gamma-interferon (IFN-gamma) on experimental metastasis formation by murine colon 26 adenocarcinoma in BALB/c mice. We found that the number of experimental lung metastases was increased after colon 26 cells were pretreated for 1 h with as little as 1 OIU/ml of IFN-gamma. 5-[125I] iodo-2'-deoxyuridine-radiolabeled colon 26 cells pretreated with IFN-gamma remained at higher level in the lung at 24h after intravenous injection than when the cells were not pretreated. In vivo elimination of asialo GM1-positive cells increased the number of lung metastases and, in such mice, there was no longer a difference in metastatic ability between control and IFN-gamma-treated cells. Colon 26 cells were completely resistant to lysis by isolated splenocytes. Splenocytes incubated in vitro with interleukin 2 exhibited moderate cytotoxicity against colon 26 cells, but there were no significant differences between control and IFN-gamma-treated cells. Colon 26 cells pretreated with IFN-gamma demonstrated resistance to tumor necrosis factor alpha-mediated growth inhibition. The enhancement of metastases by IFN-gamma was dependent on de novo protein synthesis since the enhancement was abolished by cycloheximide. Taken together, the data suggest that the metastatic ability of colon 26 cells pretreated with IFN-gamma is significantly higher due to the resistance to asialo GM1-positive cells accompanied with de novo protein synthesis.

Topics: Adenocarcinoma; Animals; Antibodies; Cell Survival; Colonic Neoplasms; Cycloheximide; G(M1) Ganglioside; Interferon-gamma; Killer Cells, Lymphokine-Activated; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Osmotic Fragility; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Metastasis is a critical problem in the treatment of human lung cancer. Thus, a suitable animal model of metastasis of human lung cancer is required for in vivo biological and preclinical studies. In this study, we tried to establish a suitable model for this, using SCID mice. Neither human SCLC H69/VP cells (5 x 10(6)) nor squamous-cell carcinoma RERF-LC-AI cells (1 x 10(6)), injected through a tail vein, formed metastases in untreated SCID mice. Pre-treatment of SCID mice with anti-asialo GM1 serum resulted in only a few metastases of H69/VP cells, but pre-treatment with anti-mouse IL-2 receptor beta chain Ab (TM-beta 1) resulted in numerous lymph-node metastases 56 days after tumor inoculation. H69/VP-M cells, an in vivo-selected variant line, formed significant numbers of lymph-node metastases even in SCID mice pre-treated with anti-asialo GM1 serum. SCID mice depleted of NK cells by treatment with TM-beta 1 showed different patterns of metastasis when inoculated intravenously with the 2 different human lung cancer cell lines (H69/VP and RERF-LC-AI cells): H69/VP cells formed metastases mainly in systemic lymph nodes and the liver, whereas RERF-LC-AI cells formed metastases mainly in the liver and kidneys, with only a few in lymph nodes. A histopathological study showed that the metastatic colonies consisted of cancer cells. The numbers of metastatic colonies formed by the 2 cell lines increased with the number of cells inoculated. TM-beta 1 treatment of SCID mice efficiently removed NK cells from peripheral blood for at least 6 weeks, whereas, after treatment of the mice with anti-asialo GM1 serum, NK cells were recovered within 9 days. These findings suggest that NK-cell-depleted SCID mice may be useful as a model in biological and pre-clinical studies on metastasis of human lung cancer.

Topics: Animals; Antibodies; Disease Models, Animal; G(M1) Ganglioside; Humans; Immune Sera; Killer Cells, Natural; Liver Neoplasms; Lung Neoplasms; Lymphatic Metastasis; Male; Mice; Mice, SCID; Neoplasm Metastasis; Receptors, Interleukin-2; Tumor Cells, Cultured

Advanced metastatic renal cell carcinoma has been shown to be responsive to immunotherapy but the response rate is still limited. We have investigated the therapeutic potential of systemic interleukin-4 (IL-4) administration for the treatment of pulmonary metastases in the murine Renca renal adenocarcinoma model. Renca cells were injected iv in Balb/c mice to induce multiple pulmonary tumor nodules. From Day 5, Renca-bearing mice were treated with two daily injections of recombinant murine IL-4 for 5 consecutive days. IL-4 treatment induced a significant reduction in the number of lung metastases in a dose-dependent manner and significantly augmented the survival of treated animals. Immunohistochemistry studies, performed on lung sections, showed macrophage and CD8+ T cell infiltration in the tumor nodules 1 day after the end of IL-4 treatment. The CD8 infiltration increased by Day 7 after IL-4 treatment. Granulocyte infiltration was not detectable. To clarify further the role of the immune system in IL-4 anti-tumor effect, mice were depleted of lymphocyte subpopulations by in vivo injections of specific antibodies prior to treatment with IL-4. Depletion of CD8+ T cells or AsGM1+ cells abrogated the effect of IL-4 on lung metastases, whereas depletion of CD4+ T cells had no impact. These data indicate that CD8+ T cells and AsGM1+ cells are involved in IL-4-induced regression of established renal cell carcinoma.

Topics: Animals; Antigens, Differentiation, T-Lymphocyte; Antigens, Ly; Carcinoma, Renal Cell; CD8-Positive T-Lymphocytes; Female; G(M1) Ganglioside; Immunologic Factors; Interleukin-4; Kidney Neoplasms; Lung Neoplasms; Lymphocyte Depletion; Lymphocyte Subsets; Lymphocytes, Tumor-Infiltrating; Macrophages; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Recombinant Proteins; Thy-1 Antigens; Time Factors

The role of different tilorone analogs in the abrogation of the metastatic spread of H-2 positive and H-2 negative tumor clones was studied. Pre-treatment of BALB/c mice with RMI 10,874DA compound completely abolished lung colonization of an H-2 negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. This effect was also evident when clones were treated with other tilorone analogs (R11,567DA or R11,513DA). Other H-2 positive and H-2 negative chemically induced fibrosarcoma clones were also tested. The effect was not due to direct toxicity of the tilorone analog on tumor cells, but instead was dependent on NK cells; this was suggested by the finding that treatment of mice with anti-asialo GM1 abrogated the effect of the tilorone analog (RMI 10,874DA compound). Interestingly, the inhibition of lung colonization after intravenous injection was again observed regardless of the H-2 phenotype of the tumor clones, and H-2+ and H-2- clones were similarly inhibited. In vitro assays of NK sensitivity of tumor clones showed that lysis varied depending on the H-2 phenotype of tumor clones, indicating an absence of correlation between in vivo and in vitro results.

Topics: Animals; Cytotoxicity, Immunologic; Fibrosarcoma; G(M1) Ganglioside; H-2 Antigens; Immune Sera; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Tilorone

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.

Topics: Adjuvants, Immunologic; Animals; G(M1) Ganglioside; Guanosine; Interleukin-2; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Vaccines

An intravenous injection of bacterial lipopolysaccharide (LPS) into mice exerted prominent antimetastatic activities against a NK-resistant weakly immunogenic NFSa fibrosarcoma. The number of visible metastases in the lung was increased by a pretreatment of anti-asialoGM1 (asGM1) antibody or silica, but pretreatment of asGM1 antibody or silica scarcely affected the antimetastatic activities of LPS. The pulmonary retention of radiolabeled tumor cells showed that LPS accelerated the detachment of the tumor cells from the lung, and that this acceleration was not suppressed by anti-asGM1 antibody. Sialic acids of lung endothelial cell surface, essential components of various receptors, was diminished by the i.v. injection of LPS. These results suggested that the antimetastatic effect of LPS against NK-resistant NFSa cells was partly the result of modulations of lung endothelial cell surface.

Topics: Animals; Antibodies; Fibrosarcoma; G(M1) Ganglioside; Killer Cells, Natural; Lipopolysaccharides; Lung Neoplasms; Male; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Sialic Acids; Silicon Dioxide; Specific Pathogen-Free Organisms

The role of the chemical compound RMI 10,874DA (3,6-bis[2-(dimethylamino)-ethoxyl]-9H-xanthene-9-one dihydrochloride) in the abrogation of the metastatic spread of tumor cells was studied. Pre-treatment of BALB/c mice with the RMI 10,874DA compound (referred to below as tilorone analogue) completely eliminated lung colonization of an H-2-negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. Other murine tumors, including H-2-positive and H-2-negative chemically induced fibrosarcoma clones and B16 melanoma, were also sensitive to the treatment; orally administered tilorone analogue given one day before the i.v. injection of tumor cells markedly inhibited lung colonization. The effect was not due to direct toxicity of tilorone analogue on tumor cells, but instead it was dependent on NK cells; this was suggested by the finding that anti-asialo GM, treatment of mice abrogated the effect of tilorone analogue. Kinetic studies of splenic NK activity in tilorone-treated mice showed a rapid boosting of NK-cell activity, the greatest stimulation occurring the day before removal of splenocytes for 51Cr-release assay against YAC-I target cells. These kinetics correlated with the inhibition of in vivo lung colonization after tilorone analogue treatment. Inhibition of experimental tumor metastasis was dose-dependent and was observed when animals were treated the day before or the day after tumor-cell injection. Furthermore, repeated treatment of mice with this tilorone analogue significantly reduced lung colonization.

Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Dose-Response Relationship, Drug; G(M1) Ganglioside; Immune Sera; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms, Experimental; Spleen; Tilorone; Time Factors; Xanthenes; Xanthones

Preincubation of murine colon 26 colon adenocarcinoma cells with gamma-interferon (IFN-gamma), but not alpha-interferon, produced a significant increase in experimental pulmonary metastases in syngeneic BALB/c and T-cell-deficient BALB/c nude mice. The enhancement was seen after as little as 1 h of exposure to 1 unit/ml of IFN-gamma and persisted for at least 72 h following removal of the cytokine. IFN-gamma exerted its effects by increasing the pulmonary retention of cells during the first 6 h following tumor cell injection. During this period all cells visualized in the lung were trapped in pulmonary capillaries. The enhancement was not due to modulations in class I major histocompatibility complex surface antigen expression; nor was it due to alterations in cell size, adhesion to components of the extracellular matrix in vitro, heterotypic or homotypic adhesion, sensitivity to lysis by activated peritoneal macrophages, osmotic fragility, enhancement of surface class II major histocompatibility complex antigen expression, or enhancement of intercellular adhesion molecule-1 (ICAM-1). Colon 26 was completely resistant to natural killer cell-mediated lysis in vitro, and IFN-gamma did not modulate the ability of colon 26 to form conjugates with isolated splenocytes. In vivo elimination of anti-asialo GM1 + cells increased pulmonary metastasis, and in such mice, there was no longer a difference in metastatic potential between control and IFN-gamma-treated cells. We conclude that low doses of IFN-gamma generated at the site of the tumor by host-infiltrating cells or during cytokine therapy could enhance the survival of tumor cells in the circulation and enhance their metastatic potential.

Topics: Adenocarcinoma; Animals; Antibodies; Cell Adhesion; Cell Division; Colonic Neoplasms; Extracellular Matrix; Female; G(M1) Ganglioside; Gene Expression; Glycosphingolipids; Histocompatibility Antigens Class I; Idoxuridine; Interferon-gamma; Iodine Radioisotopes; Killer Cells, Natural; Lung Neoplasms; Macrophages; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Oncogenes; Tumor Cells, Cultured

We have examined the antitumor and antimetastatic effects of native-type, glycosylated recombinant lymphotoxin (LT) on human and murine tumors transplanted in mice. The results reported here are as follows: (a) The in vivo antitumor spectrum of LT is not coincident with the in vitro study, and it has a wide antitumor spectrum and substantially inhibits the growth of human solid tumors, (b) When both syngeneic and nude mice are transplanted with Meth A tumor, the significant growth-inhibitory effect of LT is obtained in syngeneic mice, but the effect is quite small in nude mice regardless of the routes; LT attains the same degree of effectiveness as that in syngeneic mice, but at an 8 to 16 times higher dose. Furthermore, the pretreatment with anti-asialo-GM1 antibody inhibits the antitumor effects of LT in syngeneic mice, (c) In the pulmonary metastasis model induced by i.v. injection of Meth A cells, a high preventive effect of LT is obtained by systemic administration in syngeneic mice, but not in nude mice. In addition, the pretreatment with anti-asialo-GM1 antibody completely prevents the antimetastatic effect of LT, but also blocks that effect of control mice without LT treatment. In conclusion, LT appears to be a potent cytokine against tumor growth and metastasis in vivo. The differences between nude and syngeneic mice suggest the involvement of host immunity in the expression of LT function.

Topics: Animals; Female; G(M1) Ganglioside; Glycosphingolipids; Glycosylation; Humans; Immunization, Passive; Lung Neoplasms; Lymphotoxin-alpha; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Recombinant Proteins; Sarcoma, Experimental; Tumor Cells, Cultured

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

In order to investigate the antitumor effect of recombinant human interleukin-1 beta (IL-1 beta) alone and in combination with natural human tumor necrosis factor-alpha (nHuTNF-alpha), we used female BDF1 mice bearing Lewis lung carcinoma (3LL). IL-1 beta showed an antiproliferative effect against pulmonary metastatic tumors of 3LL in a dose-dependent manner. We observed 19.6 +/- 6.6, 18.6 +/- 5.3, 14.1 +/- 4.4 and 13.0 +/- 6.0 metastatic tumors at doses of 0.5, 1.0, 2.5 and 5.0 micrograms IL-1 beta/mouse/day by daily intravenous administration (the number of metastatic tumors of the control group was 26.3 +/- 8.2). Similar results were obtained by intraperitoneal administration, but in this case, mice showed a marked decrease of body weight. When IL-1 beta was administered in combination with nHuTNF-alpha, pulmonary metastatic tumors decreased much more than when IL-1 beta was administered alone. When the control group had 18.6 +/- 12.7 metastatic tumors, the nHuTNF-alpha group had 12.3 +/- 3.9 and the IL-1 beta group had 12.8 +/- 8.0, the group which was administered both cytokines had a significantly decreased number of 5.6 +/- 3.3 metastatic tumors. This antiproliferative effect of IL-1 beta in combination with nHuTNF-alpha was reduced by the intravenous administration of anti-asialo GM1 antibody and carrageenan. The number of metastatic tumors was increased from 8.9 +/- 8.0 to 18.8 +/- 11.4 by anti-asialo GM1 antibody and from 9.5 +/- 6.8 to 28.0 +/- 12.3 by carrageenan. It was suggested that asialo GM1-positive cells and macrophage were two of the most important effectors of the antiproliferative effect of IL-1 beta and TNF-alpha.

Topics: Animals; Antibodies; Antineoplastic Combined Chemotherapy Protocols; Carrageenan; Cell Division; Drug Administration Schedule; Drug Synergism; Female; G(M1) Ganglioside; Glycosphingolipids; Humans; Interleukin-1; Leukocyte Count; Lung Neoplasms; Mice; Mice, Inbred Strains; Recombinant Proteins; Time Factors; Tumor Necrosis Factor-alpha

We found that the number of pulmonary metastatic foci of spontaneously developed rat mammary carcinoma (SST-2), when transplanted subcutaneously in spontaneously hypertensive (SH) rats, decreased with aging. In the SST-2-bearing SH rats, it was observed that T cell functions progressively declined while activities of macrophages and natural killer cells were non-specifically activated by increasing age. To examine the mechanisms of the age-related decrease of pulmonary metastasis in SH rats, we treated the SST-2-bearing rats with anti-(asialo-GM1) antibody and/or carrageenan, which are known to suppress the functions of macrophages and natural killer cells, or with poly(I).poly(C), which is a stimulator to natural killer cells. The anti-(asialo-GM1) treatment significantly increased the number of pulmonary metastatic foci in both young and old SH rats, while poly(I).poly(C) significantly decreased the lung nodules in the old SH rats. These result suggest that the decrease of pulmonary metastasis in the SH rats with aging may be closely correlated with non-specifically activated natural killer cells and macrophages, though it should be also considered that non-immunological tumor-host interactions may be involved in the differences between the young and the old SH rats.

Topics: Aging; Animals; Carrageenan; Female; G(M1) Ganglioside; Glycosphingolipids; Immunity, Innate; Lung Neoplasms; Mammary Neoplasms, Experimental; Poly I-C; Rats; Rats, Inbred Strains

Using batroxobin, a thrombin-like enzyme found in snake venom, the effects of defibrinogenation on artificial lung metastasis in mice were studied. The role of natural killer (NK) cells in the inhibitory effects of defibrinogenation on metastasis was also investigated. Artificial lung metastasis experiments were performed by inoculating either B16-F10 cells or B16-BL/6 cells, highly metastatic strains of B16 melanoma cells, into C57BL/6 mice via the tail vein. The administration of batroxobin significantly inhibited lung metastasis, as did NK activity augmented by poly (I).poly (C) were administered, lung metastasis was more markedly inhibited. When NK activity was suppressed by administration of anti-(asialo GM1) antibody, lung metastasis was markedly increased. When batroxobin was administered with anti-(asialo GM1) antibody, no inhibitory effects on lung metastasis, such as those seen with batroxobin alone, were observed. The administration of batroxobin had no effect at all on spleen lymphocyte NK activity. These results indicated that defibrinogenation due to batroxobin inhibits lung metastasis, and these effects depend on NK activity of the host.

Topics: Animals; Antibodies; Batroxobin; Fibrinogen; G(M1) Ganglioside; Glycosphingolipids; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Poly I-C; Serine Endopeptidases

Prostaglandins can inhibit the generation of lymphokine-activated killer (LAK) cells by interleukin-2 (IL-2) whereas indomethacin augmented the induction of LAK cells by inhibiting prostaglandin synthesis. In the present study we demonstrate that prostaglandin E2 substantially inhibited the generation of both LAK and antibody-dependent cellular cytotoxicity (ADCC) activity by IL-2. In addition, indomethacin enhanced the induction of LAK activity and ADCC in splenocytes exposed to IL-2 in vitro. The effect of indomethacin was dose-dependent, reaching an optimal effect at 1 microM when 100-1000 units/ml IL-2 were employed. The effect of indomethacin on the generation of ADCC was seen in cells taken from both tumor-bearing mice and normal mice. ADCC induced by IL-2 was augmented by culturing cells from the spleen, liver and lungs, in the presence of indomethacin. ADCC induced in the presence of IL-2 and indomethacin was mediated by cells that were mainly plastic non-adherent cells and expressed the asialo-GM1 glycolipid. The potential of indomethacin in combined therapy with cytokines and specific anti-tumor monoclonal antibodies is discussed.

Topics: Animals; Antibody-Dependent Cell Cytotoxicity; Dinoprostone; Dose-Response Relationship, Drug; Female; G(M1) Ganglioside; Glycosphingolipids; Indomethacin; Interleukin-2; Killer Cells, Lymphokine-Activated; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Up-Regulation

The metastatic capacity of murine mammary tumor line 410.4 is greatly increased by treatment of the host with asialo-GM1 antiserum (5-fold), 2-chloroadenosine (4-fold) or k-carrageenan (2.5-fold). This suggests that both NK cells and macrophages contribute to control of metastatic dissemination. The metastatic potential of these cells is associated with the synthesis of high levels of prostaglandin E2 (PGE2) (1). When line 410.4 cells are cultured in vitro in the presence of the prostaglandin synthesis inhibitor indomethacin (INDO) 1 microM) their subsequent lung colonization ability (experimental metastasis) is reduced by 50-90% as compared to solvent-treated cells. The inhibitory effect of INDO is highly dependent on the presence of asGM1 positive cells, and is compromised to a lesser extent by treatments directed towards macrophages. The INDO-mediated inhibition is neither due to differential arrest of tumor cells in the lung nor does it appear to be due to shifts in the replication cycle.

Topics: 2-Chloroadenosine; Adenosine; Animals; Carrageenan; Cytotoxicity, Immunologic; Dinoprostone; Female; G(M1) Ganglioside; Glycosphingolipids; Immune Sera; Indomethacin; Killer Cells, Natural; Lung Neoplasms; Macrophages; Male; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Prostaglandins E; Tumor Cells, Cultured

Treatment of C57BL/6J mice with poly-I:C or antibodies to asialo-GM1 enhances and depresses respectively natural killer (NK) cell activity while inversely altering lung metastasis, suggesting a critical role for these cells in controlling tumor formation. We assessed the effect of these treatments on antitumor activity mediated by macrophage (M phi) populations likely to be important in lung metastasis. Alveolar and lung interstitial M phi were asialo-GM1 positive (98%) and were sensitive to in vitro treatment with the antibody plus complement; however, treatment of mice with antibodies to asialo-GM1 failed to alter their tumoricidal activity in vitro. Few blood monocytes (4%) or spleen M phi (2%) were asialo-GM1 positive and their antitumor activity was similarly unaffected. In contrast, however, this same in vivo treatment resulted in a 14-fold increase in lung metastasis. Intraperitoneal injection of poly-I:C greatly reduced metastasis formation but also failed to significantly affect in vitro cytolytic activity of the M phi populations. These findings suggest that the major metastasis altering effects of these agents result from modulation of NK rather than M phi function.

Topics: Animals; Antibodies; Complement System Proteins; Cytotoxicity, Immunologic; Female; Fibrosarcoma; G(M1) Ganglioside; Glycosphingolipids; In Vitro Techniques; Lung; Lung Neoplasms; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Poly I-C

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.

Topics: Animals; Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Antigens, Ly; Antigens, Surface; Female; G(M1) Ganglioside; Glycosphingolipids; Immunity, Cellular; Immunotherapy; Interleukin-2; Killer Cells, Natural; Lung Neoplasms; Mice; Sarcoma, Experimental; T-Lymphocytes; T-Lymphocytes, Cytotoxic

We have studied the influence of interferons (IFNs) on the metastatic potential of mouse colon adenocarcinoma, COLON 26, cells. Pre-treatment of the cells in vitro for 24 hr with recombinant murine IFN-gamma (rMuIFN-gamma) significantly increased the number of lung tumour nodules when cells were injected i.v. into immunocompetent BALB/c mice and BALB/c nude mice. However, when MuIFN-gamma-pre-treated cells were injected into beige (NK-deficient) nude mice or anti-asialoGM 1 (asGM 1)-serum-treated BALB/c mice (NK-depleted) no enhancement of metastatic potential was seen. Pre-treatment of COLON 26 cells with recombinant human IFN-alpha A/D (Bg1 I), an IFN with equal activity on human and mouse cells, did not significantly enhance their subsequent metastases in immunocompetent or immunodeficient mice. In fact, there was a small but significant decrease in the number of tumour nodules in the lungs of beige nude and asGM 1-treated mice. The effects of rMuIFN-gamma on COLON 26 cells did not appear to be related to an alteration in MHC expression. COLON 26 cells constitutively express H-2D and H-2K antigens and both IFNs had equal enhancing (approx. 2-fold) activity on the expression of these antigens at the doses used in this experiment (10(3)U/ml). We conclude that pre-treatment with rMuIFN-gamma renders COLON 26 cells resistant to in vivo NK-cell lysis via a mechanism that does not involve changes in MHC expression.

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; H-2 Antigens; Immunologic Deficiency Syndromes; Interferon-gamma; Killer Cells, Natural; Lung Neoplasms; Macrophages; Mice; Mice, Inbred BALB C; Mice, Mutant Strains; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Tumor Cells, Cultured

Recombinant human interleukin-2 (rIL-2) suppressed metastatic tumour colony formation in the lungs of C57BL/6 mice bearing Lewis lung carcinoma (3LL). In tumour-bearing mice given rIL-2, non-specific killer cells that were cytotoxic not only against natural killer-sensitive YAC-1 cells but also against 3LL cells in an in vitro 51Cr-release assay were concomitantly induced as tumour metastasis was suppressed. These non-specific killer cells were mostly removed by treatment with anti-Thy 1.2 or anti-asialo GM1 antibody plus complement (C) in vitro but not with anti-Lyt 1.2 or anti-Lyt 2.2 plus C, indicating that they were positive for Thy 1 and asialo GM1 but not for Lyt 1 and Lyt 2. In order to explore the mechanism by which rIL-2 suppressed tumour metastasis, we examined the clearance of intravenously injected 51Cr-labelled 3LL cells in the lungs of mice given rIL-2. The rate of tumour cell clearance was increased. This enhanced clearance was almost completely removed by injecting anti-asialo GM1 antibody. In addition, the injection of anti-asialo GM1 antibody also depleted most of the non-specific killer cells induced by administering rIL-2. These results indicate that asialo GM1-positive cells are not only cytotoxic in vitro but also play a critical role in the clearance of 3LL cells in the lungs in vivo. Our results indicate that asialo GM1-positive cells play an important role as anti-metastatic effector cells in suppressing the metastasis of 3LL cells in mice given rIL-2.

Topics: Animals; Antigens, Surface; Female; G(M1) Ganglioside; Glycosphingolipids; Interleukin-2; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Inbred C57BL; Recombinant Proteins; Spleen

The effect of anticoagulant drugs on formation of experimental tumor metastases after i.v. inoculation of BL6 melanoma or Lewis lung carcinoma (3LL) cells was studied in mice with stimulated or depressed natural killer (NK) cell activity. When mice were treated with anticoagulants (warfarin or heparin) or when NK cell activity was stimulated by polyinosinic-polycytidylic acid, significant antimetastatic effects were observed; these effects were substantially augmented when the treatments were combined. However, when NK reactivity of mice was suppressed by anti-asialo GM1 serum or cyclophosphamide, the antimetastatic effects of warfarin and heparin were diminished or completely abrogated. In some experiments, the anticoagulants had a partial effect in mice treated with cyclophosphamide or anti-asialo GM1 serum and reduced at least to control levels the number of metastases in these mice. This limited antimetastatic effect of the anticoagulants was mostly due to the action of residual NK cells, since it was completely abrogated in mice whose NK cell activity was more completely suppressed by two injections of anti-asialo GM1 serum. In addition, the low NK reactivity of 3-week-old C57BL/6 or beige mice was sufficient to support the antimetastatic effects of the anticoagulants, effects that completely disappeared after these mice were treated with anti-asialo GM1 serum. Augmentation or abrogation of the antimetastatic effects of heparin after polyinosinic-polycytidylic acid or anti-asialo GM1 treatments, respectively, was observed in athymic nude and allogeneic BALB/c mice that received i.v. injections of B16F1 melanoma cells, indicating that the antimetastatic effects of anticoagulants depend on the presence of active NK rather than T-cells. Furthermore, adoptive transfer of NK-competent but not NK-depleted syngeneic spleen cells restored the antimetastatic effect of heparin in cyclophosphamide-treated mice. Warfarin treatment increased the elimination of radiolabeled BL6 melanoma cells from the lungs of normal mice, and the rate of tumor cell elimination was further potentiated when NK cell activity was stimulated by polyinosinic-polycytidylic acid. In contrast, after anti-asialo GM1 treatment, warfarin had no effect on the survival of i.v. administered tumor cells. Covering of YAC-1 or 3LL tumor cells with fibrin after in vitro exposure with fibrinogen and thrombin substantially protected them from the in vitro cytotoxic action of NK or lymphokine-activate

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; Combined Modality Therapy; G(M1) Ganglioside; Glycosphingolipids; Heparin; Immune Sera; Immunotherapy; Killer Cells, Natural; Lung Neoplasms; Lymphoma; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Warfarin

Murine fibrosarcoma cells were examined for sensitivity to killing by natural killer (NK) and natural cytotoxic lymphocytes from mouse spleens. These tumor cell lines were sensitive to killing by effector cells which were nonadherent to plastic or nylon wool, Thy-1 negative, asialo-GM1 negative, and present in the spleens of beige mice, nude mice, and A/J mice, as well as in the spleens of normal syngeneic and allogeneic control mice. This indicates that the cytotoxic effects were due to natural cytotoxic lymphocytes rather than to NK lymphocytes, T-cells, or macrophages. Although the fibrosarcoma cells were not killed in vitro by endogenous NK cells, these tumor cells were able to "cold target" compete for Yac-1 (an NK-sensitive target) killing and to bind to asialo-GM1-positive, nonadherent spleen lymphocytes in a target cell binding assay. This suggests that the fibrosarcoma cells were recognized by NK cells. In addition, these cell lines were killed in a 4-h NK cytotoxicity assay by polyinosinic-polycytidylic acid-activated effector lymphocytes. The interaction between NK cells and the murine fibrosarcoma cells may have in vivo significance. When syngeneic mice were treated with anti-asialo-GM1 serum to eliminate NK activity and then given i.v. injections of the fibrosarcoma cells, many more lung tumors developed than in control animals. The structural basis for the recognition of the murine fibrosarcoma cells by the NK effector cells is not known. However, laminin may be involved. When the fibrosarcoma cells, which have receptors for the laminin molecule, were preincubated with laminin, they were reduced in their ability to compete for the killing of Yac-1 cells by the NK effectors and had reduced capacity to bind to NK cells in a target cell binding assay.

Topics: Animals; Antibodies, Monoclonal; Cell Line; Cytotoxicity, Immunologic; Fibrosarcoma; G(M1) Ganglioside; Glycosphingolipids; Immunity, Innate; Killer Cells, Natural; Laminin; Lung Neoplasms; Mice; Poly I-C; Receptors, Immunologic; Receptors, Laminin

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

Both macrophages and natural killer cells have been implicated in the antimetastatic activity of maleic anhydride-divinyl ether (MVE-5). In the present study, we attempted to utilize anti-asialo-GM1 antibody and 2-chloroadenosine, agents that kill natural killer (NK) cells and macrophages, respectively, to determine the relative contribution of each effector cell type to the overall host defense. These agents were tested in the M109 lung metastasis model in syngeneic BALB/c mice, and the cytotoxic activities of both peritoneal macrophages and splenic NK cells were followed. The most profound antitumor effect was observed when MVE-5 was given before rather than after i.v. tumor inoculation. Treatment i.p. with MVE-5 at 20 mg/kg produced greater than 98% inhibition of subsequent lung metastases when given 2 days prior to tumor. Anti-asialo-GM1 antibody (25 mg/kg, i.p.) and 2-chloroadenosine (50 mg/kg, i.p.) were administered concurrently with MVE-5. Although each agent exhibited greater selectivity for its respective target, the early (Day 2) inhibitory response was nonspecific. By Day 5 after MVE-5 treatment, 2-chloroadenosine only inhibited macrophage tumoricidal activity, and conversely, anti-asialo-GM1 antibody only inhibited NK reactivity. Despite the ability of these agents to increase survival of metastases in control animals, they only slightly abrogated the antimetastatic activity of MVE-5. Our data suggest that caution should be exercised in using these agents to discriminate macrophage and NK responses.

Topics: 2-Chloroadenosine; Adenosine; Animals; Antibodies; Antigen-Antibody Reactions; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Immunity, Cellular; Immunologic Surveillance; Killer Cells, Natural; Lung Neoplasms; Macrophages; Mice; Mice, Inbred BALB C; Polymers; Pyran Copolymer

The role of NK cells in the control of the metastatic spread of tumor cells was studied. Rats pretreated with rabbit anti-asialo GM1 (anti-asGM1) serum exhibited a diminished ability to destroy circulating MADB106 mammary adenocarcinoma cells, which in turn caused an increased incidence of experimental pulmonary metastasis. The anti-asGM1 treatment caused a selective inhibition of NK activity without detectable effect on T cell-mediated immunity, and overall had no effect on the cytotoxic activity or numbers of alveolar macrophages (alv.M phi) or monocytes. The suggestion of a role for NK cells in resistance to metastases from the MADB106 tumor cells was confirmed by the adoptive transfer of 5 X 10(6) highly purified large granular lymphocytes (LGL) into NK-depressed animals 2 hr before tumor challenge. This transfer of LGL, highly enriched in NK activity, partially or fully restored the ability of these rats to inhibit the development of pulmonary metastases. This ability to adoptively transfer resistance to metastases appeared to be confined to the LGL population, because transfer of the same number of mature peripheral blood T cells had no effect on tumor development. These results provide the first unequivocal evidence that LGL, with high NK activity, are involved in in vivo resistance to tumors, particularly in the elimination of potentially metastatic tumor cells from the circulation and capillary beds.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Cytotoxicity, Immunologic; Female; G(M1) Ganglioside; Glycosphingolipids; Immune Tolerance; Immunity, Innate; Immunization, Passive; Killer Cells, Natural; Lung Neoplasms; Macrophages; Mammary Neoplasms, Experimental; Rats; Rats, Inbred F344; T-Lymphocytes

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

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

We have studied the role of natural killer activity during the growth and dissemination of a transplantable renal adenocarcinoma (Renca) of spontaneous origin in BALB/c mice. The pattern of growth of this tumor accurately mimics that of adult human renal cell carcinoma in terms of clinical stages I-IV, particularly with regard to spontaneous metastasis to lung and liver. Renca is moderately sensitive to lysis by natural killer cells from normal mice and is more efficiently lysed by natural killer cells from mice treated with the biological response modifier maleic anhydride divinyl ether, a pyran copolymer. Our studies demonstrate that selective depression of natural killer activity by administration of antiserum specific for the neutral glycosphingolipid asialo GM1 correlated with increased formation of spontaneous metastases in the lungs, liver, and lymph nodes. Conversely, augmentation of natural killer activity by the biological response modifier decreased the formation of spontaneous metastases in lungs, liver and lymph nodes. Further, the suppression of natural killer activity and subsequent increased formation of metastases were accompanied by a significantly reduced survival time, whereas the augmented natural killer activity and decreased incidence of metastases in biological response modifier-treated mice were accompanied by an increase in time of survival. These results demonstrate a significant role for natural killer cells in the control of spontaneous metastasis during growth of this murine renal cancer.

Topics: Animals; Carcinoma, Renal Cell; Cell Line; Female; G(M1) Ganglioside; Glycosphingolipids; Immune Sera; Kidney Neoplasms; Killer Cells, Natural; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Pyran Copolymer

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