asialo-gm1-ganglioside and Neoplasm-Metastasis

Tumor dormancy is a clinical phenomenon related to immune equilibrium during cancer immunoediting. The mechanisms involved in dormant metastases are poorly understood due to the lack of preclinical models. Here, we present a nontransgenic mouse model in which spontaneous metastases remain in permanent immunomediated dormancy with no additional antitumor treatment. After the injection of a GR9-B11 mouse fibrosarcoma clone into syngeneic BALB/c mice, all animals remained free of spontaneous metastases at the experimental endpoints (3-8 months) but also as long as 24 months after tumor cell injection. Strikingly, when tumor-bearing mice were immunodepleted of T lymphocytes or asialo GM1-positive cells, the restraint on dormant disseminated metastatic cells was relieved and lung metastases progressed. Immunostimulation was documented at both local and systemic levels, with results supporting the evidence that the immune system was able to restrain spontaneous metastases in permanent dormancy. Notably, the GR9-B11 tumor clone did not express MHC class I molecules on the cell surface, yet all metastases in immunodepleted mice were MHC class I-positive. This model system may be valuable for more in-depth analyses of metastatic dormancy, offering new opportunities for immunotherapeutic management of metastatic disease.

Topics: Animals; Cell Line, Tumor; Fibrosarcoma; G(M1) Ganglioside; H-2 Antigens; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; T-Lymphocytes

Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2β1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2β1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2β1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2β1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes.

Topics: Bone Neoplasms; Cell Line, Tumor; G(M1) Ganglioside; Humans; Integrin alpha2; Male; Neoplasm Metastasis; Prostatic Neoplasms; Sialic Acids

Cetuximab, a chimeric monoclonal antibody to epidermal growth factor receptor (EGFR), has been proved to have clinically significant antitumor activity against advanced colorectal cancers, but its therapeutic activity for gastric cancers remains unclear. In the present study, we investigated the antitumor effect and action mechanism of cetuximab using EGFR high-expressing (MKN-28) and EGFR low-expressing (GLM-1) gastric cancer cell lines without gene amplification. Cetuximab showed neither significant growth inhibition nor induction of apoptosis in either cell line in vitro, and only slightly inhibited ligand-induced phosphorylation of protein kinase B and extracellular signal-regulated kinase in MKN-28 cells. In contrast, cetuximab significantly inhibited subcutaneous and intraperitoneal tumor growth of MKN-28 cells, but not GLM-1 cells, in nude mice. This antitumor activity was significantly enhanced and diminished in nude mice by treatment with interleukin-2 (IL-2) and antiasialo GM1 antibody, which can expand and deplete natural killer (NK) cells, respectively. Antibody-dependent cellular cytotoxicity (ADCC) of cetuximab, as measured by (51)Cr release assay, was significantly higher in MKN-28 than in GLM-1 cells. This ADCC activity was enhanced by IL-2 and reduced by heat-aggregate of human immunoglobulin G, an inhibitor for FcR-III of NK cells. These results suggest that cetuximab in combination with IL-2 shows significant antitumor activity against EGFR high-expressing gastric cancer mainly through NK cell-mediated ADCC. Combination therapy with cetuximab and IL-2 would thus offer a new potential therapeutic approach for a subset of EGFR-overexpressing gastric cancers.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cetuximab; Drug Synergism; ErbB Receptors; G(M1) Ganglioside; Humans; Immunohistochemistry; Interleukin-2; Male; Mice; Neoplasm Metastasis; Neoplasm Transplantation; Stomach Neoplasms; Transplantation, Heterologous

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

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

Innovations in methods of combined administration with other BRM or chemotherapeutic drugs have been discussed. We have reported that combined administration with recombinant interleukin-2 (rIL-2) and sizofiran (SPG) is effective in prolonging survival time of C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The immunomechanisms of the combined administration were clarified investigating the intraperitoneal cell population in the primary tumor site, especially the tumor infiltrating lymphocyte (TIL) quantitatively. In the present study, to clarify the antitumor effects of combined administration with rIL-2 and SPG on the metastatic sites, the immunomechanisms of the suppressive effects of combined administration on the metastasis were studied in EL-4 lymphoma cells intraperitoneally transplanted to mice. Inasmuch as EL-4 lymphoma shows rapid hepatosplenic metastasis, we studied the metastatic foci in the liver and the spleen semiquantitatively in investigating the histopathological and immunohistochemical findings of the metastatic foci, especially the TIL. The metastatic foci were stained by hematoxylin-eosin (HE) and monoclonal antibodies (L3T4, Lyt2, asialo GM1, Mac-1, and Ia). The combined administration resulted in: 1) fewer infiltrating tumor cells, 2) more lymphocytic infiltration, and 3) more antitumor effector cells (cytotoxic T cells: Lyt2 and natural killer cells: asialo GM1), macrophages (Mac-1), helper T cells (L3T4), and cells with MHC-class-II antigen (Ia) than did administration of rIL-2 alone or SPG alone, or no administration of these two at all. Combined administration with rIL-2 and SPG appears to activate antitumor-immune response at the metastatic site more effectively than when either agent is administered alone.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Female; G(M1) Ganglioside; Immunohistochemistry; Interleukin-2; Liver; Lymphoma; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Recombinant Proteins; Sizofiran; Spleen

The murine melanoma cell line BL-6-beta m, which is a stable cell line transfected with a gene coding a unique actin subspecies called beta m to the BL-6 cell line, has low metastatic potentials as compared with those of the parent cell line. BL-6-beta m melanomas were found to be sensitive to in vivo local injection of IL-2, while BL-6 melanomas showed almost no response. Ganglioside analysis of BL-6 and BL-6-beta melanomas revealed that the main ganglioside of both melanomas was GM3, which suggested that different sensitivities between BL-6 and BL-6-beta m melanomas to the injection of IL-2 did not relate to the different compositions of main gangliosides. However, minor components of the gangliosides such as GM2 and GM1 emerged only in BL-6-beta m melanomas after treatment with IL-2. Local injection of IL-2 caused considerable infiltration of anti-asialo GM1-positive cells into the nests as well as the interstitials of BL-6-beta m melanomas. In contrast, in the BL-6 melanomas treated with IL-2, infiltration of the anti-asialo GM1-positive cells was hardly seen, although anti-Thy1,2 and anti-macrophage-positive cells were found to more or less the same extent as observed in BL-6-beta m melanomas. These results suggest that the murine metastatic variant melanoma cell lines BL-6 and BL-6-beta m have different properties in terms of sensitivity to in vivo IL-2 treatment, and a slight enhancement of the ganglioside components GM2 and GM1 expression only in BL-6-beta m after IL-2 treatment may play a role in the IL-2-mediated attraction of immune cells or may explain the different sensitivities of the two lines to treatment with IL-2.

Topics: Actins; Animals; Antigens, Neoplasm; Cell Movement; Female; G(M1) Ganglioside; Gangliosides; Immunologic Factors; Immunologic Surveillance; Injections, Intralesional; Interleukin-2; Killer Cells, Lymphokine-Activated; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Recombinant Fusion Proteins; Thy-1 Antigens; Transfection

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

In the present study, the respective roles of T cells and their subpopulations as well as of NK (natural killer) cells in antitumor immune responses were followed using the SaI (H-2a) allograft model. The development of this tumor in B10 (H-2b) mice was evaluated after pretreatment of the recipients with xenogeneic antithymocyte serum (ATS). Anti-Thy 1.2, anti-Lyt 2.2 and anti-L3T4 monoclonal antibodies were used in order to determine T lymphocyte phenotypes and to assess the frequency of TC/S and TH subpopulations at various periods of tumor development. Rabbit polyclonal anti-asialo GM1 antiserum was used for the identification of NK cells. In a previous work it was suggested that the first week following transplantation, the cells predominantly involved in the growth regulation of SaI belong to the TS subclass. Our results based on the use of anti-Lyt 2.2 monoclonal antibodies have further supported this finding. The application of anti-Thy 1.2 on the 3rd and 5th day has hampered a secondary tumor growth while anti-Lyt 2.2 was effective when given on day 5. The depletion of Lyt. 2.2+ cells on day 3 resulted in the inhibition of both primary and secondary tumor development. On the other hand, when anti-Thy 1.2 was applied on day 7 after transplantation, the primary and secondary tumor growth was strikingly enhanced. It appears that Thy 1.2+ lymphocytes display at this period effector functions and contribute, in conjunction with macrophages, to subsequent tumor regression. The depletion of L3T4 cells on days 3 and 5 after tumor inoculation has resulted in primary tumor growth enhancement. This suggests that cells of the L3T4+ phenotype display at this time helper functions contributing to CTL proliferation and maturation. A further indication, supporting the possible suppressor effect of L3T4+ cells, counts from the finding that anti-L3T4 treatment results in an inhibition of secondary tumor growth. The anti-asialo GM1 treatment has not enhanced, at least significantly, primary tumor development but has partially or totally inhibited the growth of secondary tumors. It appears that cells of the GM1+ (NK cells) phenotype do not participate in any substantial way in the early phases of SaI tumor development in ATS treated allogeneic recipients.

Topics: Animals; Antibodies, Monoclonal; Female; Fibrosarcoma; G(M1) Ganglioside; Immunity, Cellular; Immunophenotyping; Isoantibodies; Killer Cells, Natural; Mice; Mice, Inbred A; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; T-Lymphocytes; Time Factors; Transplantation, Homologous

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

The effect of the streptococcal preparation OK-432, which is one of the biological response modifiers, was examined in BALB/c mice using a transplantable murine renal cell carcinoma (Renca) of spontaneous origin, and an analysis of effector cells was performed. The tumor grew progressively and metastasized consistently to the abdominal lymph nodes and then to distant organs following the inoculation of Renca cells in the left renal subcapsular site in BALB/c mice, and the survival time of the mice was under 42 days. In this tumor model, i.p. administration of OK-432 after tumor inoculation significantly extended the survival time and significantly inhibited the formation of the inoculated tumor itself. Removal of the left kidney on the 7th day after tumor inoculation neither extended the survival time nor augmented the effect of OK-432. Splenic cells obtained on the 7th day after tumor inoculation from Renca-bearing mice treated with OK-432 were capable of lysing syngeneic Renca cells, NK-sensitive allogenic YAC-1 cells, and LAK-sensitive EL-4 cells in a 4-hour 51Cr-release assay in vitro. Those obtained from healthy mice treated with OK-432 also showed cytotoxic activity against Renca cells. The cytotoxicity of splenic cells from Renca-bearing mice treated with OK-432 was lost almost completely for both Renca and YAC-1 cells after in vitro treatment with anti-asialo GM1 antibody, and was partially lost after in vitro treatment with anti-Thy-1,2 antibody. Additionally, in vivo i.p. administration of anti-asialo GM1 antibody significantly counteracted the effect of OK-432 on survival. These findings demonstrated that Renca cells were NK-sensitive and that the i.p. administration of OK-432 was beneficial for the prevention of the spontaneous metastasis of Renca carcinoma. As the effectors, NK cells played a dominant role and activated T cells were also involved.

Topics: Animals; Carcinoma, Renal Cell; Female; G(M1) Ganglioside; Glycosphingolipids; Injections, Intraperitoneal; Kidney Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Picibanil

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 investigated the effect of recombinant polypeptides with cell-binding domain (C-274) or with heparin-binding domain (H-271) and their fusion polypeptide (CH-271) on liver metastasis of murine lymphoid tumor. The polypeptides containing heparin-binding domain, H-271 and CH-271, were able to inhibit liver metastasis when co-injected i.v. with L5178Y-ML25 T-lymphoma cells, while C-274 with cell-binding domain showed much weaker antimetastatic activity. Treatment with H-271 or CH-271 substantially prolonged the survival time of mice injected i.v. with L5178Y-ML25 cells. CH-271, containing cell- and heparin-binding domains, was more antimetastatic than H-271. The reason why CH-271 was more effective in inhibiting liver metastasis than H-271 can not be explained in terms of a difference in the stability in the circulation or in the molecular size of the polypeptide. The polypeptides used in this study did not affect the tumor cell growth or viability in vitro. CH-271 was found to be still active in inhibiting liver metastasis even when natural killer cells or macrophages were removed from this system. Furthermore, multiple administrations of CH-271 after tumor implantation effectively inhibited liver metastasis and enhanced the survival rate as compared with H-271, C-274 and untreated control. Thus, the fusion of H-271 with C-274 (i.e. CH-271) augments the antimetastatic property of H-271, possibly through the interaction between tumor cells and the heparin-binding domain of fibronectin.

Topics: 2-Chloroadenosine; Animals; Binding Sites; Cell Adhesion; Fibronectins; G(M1) Ganglioside; Glycosphingolipids; Heparin; Leukemia L5178; Liver Neoplasms; Mice; Neoplasm Metastasis; Recombinant Fusion Proteins; Structure-Activity Relationship; Survival Analysis

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

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

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

This investigation was undertaken in order to assess both the preventive and antiproliferative effects of tumor necrosis factor (TNF) in a hepatic metastasis model, by means of inoculation of mouse colon-26 tumor cells into the portal vein via the superior mesenteric vein in male CDF1 mice, aged 5 weeks. Continuous 10-day administration of natural human TNF-alpha (nHuTNF-alpha) following the tumor cell inoculation caused no reduction but rather an increase in the number of hepatic metastases. However, pretreatment with this preparation daily for 10 days before the inoculation caused a remarkable decrease in the number of hepatic metastases. This prophylactic effect was reversed by the intravenous administration of anti-asialo GM1 antibody 24 h before the inoculation. The result of immunoperoxidase staining of liver specimens suggested that organ-associated natural killer cells might play a role in the metastatic inhibition. An apparent antiproliferative effect on metastatic liver tumors was also recognized following injection of nHuTNF-alpha from the 10th day after the inoculation. Thus, TNF appears to have important effects upon the host immune system, acting against liver metastases.

Topics: Animals; Antigen-Antibody Reactions; Cell Division; Colonic Neoplasms; G(M1) Ganglioside; Glycosphingolipids; Immunoenzyme Techniques; Liver Neoplasms; Mice; Neoplasm Metastasis; Tumor Necrosis Factor-alpha

Metastasis can be inhibited by asialo-GM1-positive spleen cells, and in this paper we show that there are two such spleen cell populations. One population is adherent and non-cytotoxic to YAC cells, whereas the other population is non-adherent and cytotoxic to YAC cells. Both cell populations exert an antimetastatic activity in cyclophosphamide-treated mice that are inoculated with LL2 Lewis lung carcinoma cells. We conclude that the antimetastatic activity is not only exerted by cytotoxic asialo-GM1-positive cells (apparently natural killer cells), but also by adherent, non-cytotoxic asialo-GM1+, Thy1.2-, IgG- cells. This means that the latter exert their antimetastatic activity by a non-cytotoxic mechanism.

Topics: Animals; Cyclophosphamide; Female; G(M1) Ganglioside; Glycosphingolipids; Immunoglobulin G; Killer Cells, Natural; Mice; Neoplasm Metastasis; Spleen; T-Lymphocytes

Tissue infiltrating lymphocytes isolated from the preneoplastic mouse mammary hyperneoplastic alveolar nodule (HAN) tissue line C4 express high levels of natural killer (NK) activity, which gradually wanes as spontaneous tumors develop (W. Z. Wei and G. Heppner. Br. J. Cancer, 55: 589-594, 1987). Experiments were performed to test whether modulation of NK cell activity would be associated with altered progression of HAN to tumor. Administration of polyinosinic-polycytidylic acid, which activates NK activity but does not directly affect mammary epithelial cell growth, to HAN-bearing mice enhanced tumor progression, as measured by a decrease in the latency period and increase in the incidence of mammary adenocarcinomas developing in the HAN implants. Antiasialo GM1, which reduces NK activity, reduced tumor progression. The net effect of indomethacin, which may inhibit mammary epithelial cell growth but enhances NK cell function, was to prolong the latency period of tumor development. However, this effect was reversed by interleukin 2, which activates NK cells. These findings suggest that NK activity may provide positive signals for progression of preneoplastic mammary lesions to frank neoplasia.

Topics: Animals; Female; G(M1) Ganglioside; Glycosphingolipids; Indomethacin; Interleukin-2; Killer Cells, Natural; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Poly I-C; Precancerous Conditions

Swainsonine, an indolizidine alkaloid, has been found to inhibit the experimental metastasis of B16-F10 melanoma cells when administered systemically to syngeneic C57BL/6 mice. The inhibition was both potent and dose dependent with greater than or equal to 80% reduction in pulmonary colonization being observed after only 24-h exposure to 3 micrograms/ml of swainsonine in drinking water. In contrast, the inhibitory activity of swainsonine was completely abrogated when assays were performed in mice depleted of their natural killer (NK) cell activity either experimentally (anti-asialo-GM1 antibody- or cyclophosphamide-treated C57BL/6 mice) or as a result of genetic mutation (homozygous C57BL/6bg/bg beige mice). Swainsonine elicited a 32.0% increase in spleen cell number 2 days after administration and induced a concomitant 2- to 3-fold increase in splenic NK cell activity. These results indicate (a) an absolute requirement for a functional NK cell population in order for swainsonine to exert its inhibitory effects on experimental metastasis, and (b) that the antimetastatic activity of swainsonine is mediated primarily through the ability of the drug to augment NK cell reactivity. On the basis of these findings, swainsonine can be classified as a new immunomodulator that has the ability, at least in a prophylactic setting, to block tumor metastasis.

Topics: Adjuvants, Immunologic; Alkaloids; Animals; Antineoplastic Agents; Cyclophosphamide; G(M1) Ganglioside; Glycosphingolipids; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Swainsonine; Time Factors

Treatment of Wistar/AG rats with a single i.p. injection of 1 mg/kg of synthetic double-stranded polynucleotides, either polyadenylic-polyuridylic acid (rAn.rUn), or a mismatched analogue of polyinosinic-polycytidylic acid (rIn.r(C12U)n), enhanced the cytotoxicity of natural killer (NK) cells among peripheral blood leukocytes and lung intracapillary leukocytes (LICL). The enhancement reached a peak 24 h after treatment and returned to control values after 4 days. In rats given repeated injections of double-stranded polynucleotides (2 per week), the NK cytotoxicity expressed by LICL reached more than ten times (in lytic units) the control levels between day 8, after 3 injections, and day 360, after 100 injections. No hyporesponsiveness was observed. Moreover, NK activity was frequently and significantly higher in rats given multiple injections than in those given a single injection. In rats with experimentally induced P77 lung fibrohistiocytoma colonies, repeated injections of rIn.r(C12U)n stimulated NK activity and reduced the number of metastatic nodules from 172 to 19. The same significant reduction (from 172 to 27) was also observed in animals given repeated injections of rAn.rUn. However, with two models of spontaneous metastases, significant reduction in lung metastases (M37 bronchioloalveolar carcinoma) or lack of effect (S4T19 rhabdomyosarcoma) were observed.

Topics: Animals; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Killer Cells, Natural; Male; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms, Experimental; Polynucleotides; Rats; Rats, Inbred Strains

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

The highly malignant and metastatic RAW117-H10 cell line was developed by in vivo selection from the Abelson leukemia virus induced parental RAW117-P lymphoma. In this study we have characterized these cell lines with regard to their expression of lymphocyte and macrophage differentiation antigens, adherence, phagocytic properties, binding of various lectins, binding of antibodies to glycolipid asialo-monoganglioside, and the role of butanol extractable cell surface molecules to determine if any of these cell surface properties are associated with the malignant potential of RAW117-H10 cells. The only major difference in immunological phenotypes between RAW117-P and RAW117-H10 cells was an increased expression of Thy-1 molecules by the latter. However, the highly malignant RAW117-H10 cells bound significantly less concanavalin A, Ricinia communis agglutinin, succinylated wheat germ agglutinin, and particularly anti-asialomonoganglioside than their parental counterpart and were resistant to natural killer cell mediated cytolysis. Removal of butanol extractable cell surface molecules significantly decreased the malignancy of RAW117-H10 cells and increased their susceptibility to natural killer cell mediated cytolysis. The butanol treated RAW117-H10 cells regained high in vivo malignancy when recultured for 3 days to permit regeneration of their cell surface components. The butanol extracted RAW117-H10 cells still expressed high levels of Thy-1 indicating that this most probably represented "inappropriate" antigen expression. Since the expression of lymphocyte differentiation antigens did not correlate with the malignant behavior of the cells, we postulate that these antigenic differences merely represent phenotypic variation. The decreased malignant potential of the butanol treated RAW117-H10 cells did correlate with increased cell surface anti-asialomonoganglioside binding (glycolipid) and increased natural killer cell susceptibility.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Butanols; Cell Line; Cytotoxicity, Immunologic; G(M1) Ganglioside; Glycosphingolipids; Killer Cells, Natural; Lymphocytes; Lymphoma; Mice; Neoplasm Metastasis; Phagocytosis; Receptors, Antigen, B-Cell; Receptors, Mitogen; Rosette Formation; Surface Properties

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

Topics: Animals; G(M1) Ganglioside; Glycosphingolipids; Humans; Immune Sera; Immunity, Innate; Killer Cells, Natural; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Transplantation, Heterologous