G-protein involvement in matrix-mediated motility and invasion of high and low experimental metastatic B16 melanoma clones

Membranes from a B16 murine melanoma clone of high experimental metastatic capacity show increased amounts of pertussis toxin (PT) substrate when compared to a low metastatic counterpart. Using specific antibodies, we identified Gi2 as the PT-sensitive G-protein uniquely abundant in highly metastatic cells. ADP ribosylation of a G-protein alpha subunit by PT decreased both the migration of tumor cells through Matrigel (Collaborative Research, Bedford, MA) and the fibronectin-, laminin-, and collagen type IV-mediated motility of a high metastatic clone. Treatment of cells from a low metastatic clone with PT did not alter either the relatively low invasive capacity or lower motility of these cells. While cholera toxin treatment of cells resulted in decreased invasion and motility of both high and low metastatic clones, there were significant qualitative and quantitative differences, when compared to the PT effects, which indicated that the two toxins were acting on different second messenger systems. PT treatment of B16 clones of high or low experimental metastatic capacity does not result in any alteration in cellular cyclic AMP accumulation suggesting that the PT substrate is not linked with the adenylyl cyclase enzyme complex. The data suggest that a PT-sensitive G-protein, possibly Gi2, regulates second messenger pathways that contribute to the metastatic capacity of B16 melanoma cells.     

Effects of cytokine-mediated modulation of nm23 expression on the invasion and metastatic behavior of B16F10 melanoma cells

The molecular mechanisms of tumor invasion and metastasis are yet to be fully elucidated. A potential tumor-metastasis-suppressor gene nm23 has been described in certain rodent and human tumors. In the present study, we examined the potential anti-invasive and anti-metastatic effect of nm23 gene in B16F10 cells, a malignant murine melanoma cell line. Transfection of nm23 gene into B16F10 melanoma ceils resulted in significant suppression of the invasiveness and metastatic ability of melanoma cells and significantly enhanced the survival of tumor-bearing mice. B16F10 melanoma cells transfected with nm23 produced significantly less soluble ICAM-I and were more susceptible to LAK-cell-mediated cytotoxicity. Co-culture of B16F10 melanoma cells with IL-2 had no effect on nm23 expression, whereas treatment with PGE₂, TNF-α and IFN-γ resulted in down-regulation of nm23 expression. Concomitantly, in vivo treatment with TNF-α or IFN-γ in experimental mice increased pulmonary metastases and lowered the overall survival period, as compared with IL-2 treatment alone. These results provide evidence that nm23, in addition to its anti-metastatic function, could also be involved in modulating tumor-target-structure expression, in down-regulating invasive potential and in production of soluble intracellular adhesion molecules. The down-regulation of nm23 by TNF-α, IFN-γ and particularly by PGE₂ warrants re-examination of current immunotherapeu-tic protocols and of the role played by PGE₂ in tumor progression. © 1995 Wiley-Liss, Inc.     

Isolation and metastatic properties of detachment variants of B16 melanoma cells.

Variant clones of B16 melanoma cells have been isolated that detached from plastic tissue culture dishes either more or less readily than the parent line upon treatment with EDTA or ethyleneglycol-bis(b-amino ethyl ether)N,N'-tetraacetic acid. Cells that detached more readily were spindlier in culture than parent cells, covered less surface area per cell, and formed fewer pulmonary tumors when injected iv into C57BL/6J mice. Cells that detached less readily were flatter in culture and formed more pulmonary tumors when injected iv than the parent cells. After detachment with EDTA, the cells reattached to culture dishes at the same rate.     

Cellular glycoproteins correlating with pulmonary-colonizing potential in B16 melanoma

Sublines of B16 melanoma with different pulmonary colonizing potential were examined for the relationship between cellular glycoproteins and the pulmonary colonizing potential using sodium dodecylsulfate polyacrylamide gel electrophoresis, and lectin-binding assay in nitrocellulose sheets after Western blotting. There were glycoproteins corresponding to 33,000 and 32,000, whose carbohydrate components, N-acetylgalactosamine and fucose, were positively correlated with the pulmonary colonizing potential. The protein amount of these glycoproteins was very small and showed no correlations. As regards the cell morphology and growth characteristics, there was no correlation between these parameters and the pulmonary colonizing potential.     

Lowered superoxide dismutase in highly metastatic B16 melanoma cells

Superoxide dismutases (SOD), which are enzymes scavenging the superoxide radical, were studied in two variant lines of the B16 melanoma: B16F1 with low metastatic potential and B16F10 with high metastatic potential. SOD activity was measured by a method utilizing reduction in the chemiluminescence of luminol. Using cell free extracts it was shown that the highly metastatic B16F10 cell line has a SOD activity lower (20.70 +/- 3.07) units/mg protein, n = 8, than that of the less metastatic B16F1 cell line (81.38 +/- 6.78) units/mg protein, n = 8. Acrylamide gel electrophoresis suggested that Mn-SOD activity is higher in B16F1 cells.     

Preferential organ attachment and invasion in vitro by B16 melanoma cells selected for differing metastatic colonization and invasive properties

Murine B16 melanoma sublines have been sequentially selected in vivo for low (B16-F1) or high (B16-F10) lung, high ovary (B16-O10) or high brain (B16-B15b) colonization, and in vitro for enhanced tissue invasion (B16-BL6). These B16 sublines were tested in vitro in a syngeneic organ adhesion/invasion assay to determine differences in tumor and/or host tissue properties that might account for preferential metastasis to certain sites. Tissues used were murine C57BL/6 lung, ovary and heart. In 8 independent experiments high lung-colonizing B16-F10 cells bound to and infiltrated into lung tissue better than ovary or heart tissue, while high ovary-colonizing B16-O10 cells attached to and invaded into ovary tissue at higher rates than lung or heart tissue. Only highly tissue-invasive B16-BL6 cells were able to invade heart tissue within 18 h in the experiments. The results suggest that organ metastatic colonization preferences by malignant cells may be determined, in part, by differences in the abilities of metastatic tumor cells to attach to and invade target tissue.     

Oligodendroglial tumors. An immunohistochemical and electron microscopic study

Fifty-five cases of oligodendrogliomas and mixed oligoastrocytomas were evaluated using immunohistochemical (IH) study for glial fibrillary acidic protein (GFAP) and electron microscopic (EM) study. Most of the tumors in both of these groups showed many neoplastic oligodendroglial cells with GFAP-positive staining in their cytoplasm by IH study. By EM study too, many tumor cells showing features of oligodendroglial cells contained intermediate filaments. Our observations suggested the presence of a transitional form of cells in these tumors. The current study supports the contention that both oligodendrogliomas and oligoastrocytomas arise from a common progenitor cell capable of differentiation into both oligodendrocyte and astrocyte. The nature and degree of differentiation depends probably on gene expression and/or some microenvironmental factors.     

Malignant fibrous histiocytoma. An electron microscopic study.

Sixteen cases of malignant fibrous histiocytoma are presented. Electron microscopy of 15 cases demonstrated fibroblast-like and mononuclear and multinucleated histiocyte-like cells. A small capillary was at the center of all storiform areas examined. Ultrastructural examination can be diagnostically useful within the context of a narrow differential diagnosis by conventional microscopy and the ability, by electron microscopy, to eliminate other mesenchymal cell types. In 13 cases, follow-up information was available from 18 months to 9 years following histological diagnosis. Five patients are alive and 8 patients have died, including two non-tumor related deaths. In 3 cases follow-up was less than 4 months. The biologic behavior of the tumor in this series was generally not related to histopathological parameters. The issue of histogenesis is largely unresolvable. Ultrastructural studies of various types of fibrous histiocytomas, suggesting cells of origin other than histiocytes, give credence to the concept that the histiocyte may represent a morphologic state of a given mesenchymal cell rather than a particular cell type.     

Effects of tertiary amine local anesthetics on the blood-borne implantation and cell surface properties of metastatic mouse melanoma cells

Tertiary amine local anesthetics, such as dibucaine and tetracaine, have been found to reversibly modify the blood-borne implantation and experimental metastatic properties of B16 melanoma cells in syngeneic C57BL/6 mice. Local anesthetic treatment in vitro of low (B16-F1) and high (B16-F10) lung-colonizing melanoma variants reduced significantly their abilities to form lung tumor colonies, but the cells recovered their original in vivo colonizing properties within several hours after drug removal. Low drug concentrations (0.5 mM tetracaine) that altered the metastatic properties of these cells did not modify cell plating efficiencies or tumor growth rates at subcutaneous sites. With the use of [125]-5-iodo-2'-deoxyuridine-labeled B16 cells, the kinetic distributions of viable tumor cells in mice were also shown to be altered. Fewer cells were initially localized and retained in the lungs, and more cells reached extrapulmonary sites. Local anesthetics modified B16 cell morphology, inducing cell rounding and loss of microvilli. These effects occurred concomitant with alterations in microfilament organization. Anesthetic-treated cells also exhibited decreased cell-adhesion characteristics in both homotypic and heterotypic (endothelial and subendothelial matrix) cell-adhesion assays. Despite these changes, cell surface lactoperoxidase-catalyzed iodination of external proteins and labeling of cellular glycoproteins on polyacrylamide gels with 125I-labeled lectins did not reveal differences in the displays or quantities of cell surface glycoproteins after drug treatment. The data are consistent with the idea that reversible disruptions in transmembrane cytoskeletal control induced by local anesthetics result in alterations in cell shape, loss of stable adhesive interactions, and a decrease in blood-borne arrest and metastatic properties.     

Generation of drug-resistant variants in metastatic B16 mouse melanoma cell lines

Genetic instability is recognized as an important aspect of the development of tumor heterogeneity and malignancy. In a previous study [Hill et al. Science (Wash. DC), 244:998-1001, 1984], we demonstrated that metastatic variants are generated at a more rapid rate in the highly metastatic B16F10 mouse melanoma cell line than in the less metastatic B16F1 cell line. The metastatic variants were phenotypically unstable, being generated and lost at high rates; consequently, we proposed a dynamic heterogeneity model of tumor metastasis which describes these properties quantitatively. As an extension of this work, we have examined the ability of these two melanoma cell lines to generate variants resistant to the drugs methotrexate and N-(phosphonacetyl)-L-aspartate. We observed that the highly metastatic B16F10 cell line generated variants resistant to a given concentration of methotrexate or N-(phosphonacetyl)-L-aspartate at higher rates than the B16F1 cell line. We conclude that B16F10 cells are genetically less stable than B16F1 cells and since resistance to methotrexate and N-(phosphonacetyl)-L-asparate usually results from gene amplification that B16F10 cells possess increased ability to amplify DNA. This higher rate of generation of drug-resistant variants corresponds to the higher rate of generation of metastatic variants we observed previously and suggests that a gene amplification mechanism may be involved in the generation of a metastic phenotype in B16 melanoma cells.     

Association between pterostilbene and quercetin inhibits metastatic activity of B16 melanoma

Inhibition of cancer growth by resveratrol (trans-3,5,4'-trihydroxystilbene; RESV), a phytoalexin present in many plant species, is limited by its low bioavailability. Pterostilbene (3,5-dimethoxy-4'-hydroxystilbene; PTER) and quercetin (3,3',4',5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols, show longer half-life in vivo. In vitro growth of highly malignant B16 melanoma F10 cells (B16M-F10) is inhibited (56%) by short-time exposure (60 min/day) to PTER (40 microm) and QUER (20 microm) (approximate mean values of plasma concentrations measured within the first hour after intravenous administration of 20 mg/kg each polyphenol). Intravenous administration of PTER and QUER (20 mg/kg per day) to mice inhibits (73%) metastatic growth of B16M-F10 cell in the liver, a common site for metastasis development. The anti-metastatic mechanism involves: 1) a PTER-induced inhibition of vascular adhesion molecule 1 expression in the hepatic sinusoidal endothelium, which consequently decreases B16M-F10 cell adhesion to the endothelium through very late activation antigen 4; and 2) a QUER- and PTER-induced inhibition of Bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. Our findings demonstrate that the association of PTER and QUER inhibits metastatic melanoma growth and extends host survival.     

Melanoma antigen expression and metastatic ability of mutant B16 melanoma clones

The biological functions of murine melanoma-associated antigens recognized by monoclonal antibodies (MAbs) (M562, M622 and M2590) were examined by using mutant clones which differed in their degree of expression of these antigens. Four clones of high expressors of 3 types of antigen (MEA group), 5 clones of low or non-expressors of M562- and M622-recognizing antigens (MEB group) and 4 clones of non-expressor of GM3 recognized by M2590 (MEC group) were used. Attachment of these clones to components of extracellular matrix was different between the groups. Two clones of the MEA group showed the highest ability to adhere to laminin and type-IV collagen, whereas the clones of the MEB and MEC groups significantly lost their ability to attach to laminin and type-IV collagen. In experimental lung metastasis, metastasizing ability of MEA-group cells was higher than that of MEB- and MEC-group cells. Our results suggest that these antigens play some functional role in metastasis mediated by increasing capacity for attachment to laminin and type-IV collagen.     

Desmoplastic malignant melanoma: A light and electron microscopic study of two cases.

This light and electron microscopic study of two recent cases of desmoplastic malignant melanoma (DMM) attempts to resolve the conflict in views regarding the nature of the cells responsible for the desmoplasia associated with this clinicopathologic entity. On the basis of evidence presented, it is concluded that the cells are dedifferentiated tumor cells with fibroblastic features and probably functions, rather than host engendered fibroblasts in response to invasive melanoma. The evidence includes: observation of macular desmosomes between tumor cells, an unheralded feature previously noted in amelanotic and melanotic melanomas; electron microscopic observation of fibroblast-like cells by others in spindle cell squamous carcinomas; and light microscopic features of malignancy including vascular invasion in one of the two cases. A reproducible light microscopic pattern diagnosis of this variant of malignant melanoma is reaffirmed in both cases.     

Effect of lactose derivatives on metastatic potential of B16 melanoma cells

The effects of various sugars and sugar derivatives on lung colonization (i.e., metastatic deposition) of the highly metastatic BL6 clone of B16 mouse melanoma cells in syngeneic mice were studied, based on the assumption that carbohydrate structures, particularly those with a Gal terminus, play a crucial role in defining the metastatic potential of B16 cells. After incubation with sugar compounds (usually at 0.1 M concentration), tumor cells were injected via the tail vein into 8-week old female mice. Mice were sacrificed after 18-21 days, and tumor cell colonies in lung were counted under a dissecting microscope. Only methyl beta-D-lactoside and lacto-N-tetraose caused significant reduction (35-45% and 36%, respectively) of metastatic deposition compared to controls. Methyl beta-D-lactoside did not exhibit a growth inhibitory effect on BL6 tumor cells, as determined by several methods: in vitro [3H]thymidine incorporation assay, in vitro plating in RPMI-1640 medium culture under physiological conditions followed by cell counting, and in vivo subcutaneous inoculation of age-matched C57/BL mice followed by tumor measurement. These results indicate that the inhibitory effect of methyl beta-D-lactoside on tumor deposition was not related to its effect on tumor cell growth.     

TNF-receptor-1 adaptor protein FAN mediates TNF-induced B16 melanoma motility and invasion

Background:

Locomotion of cancer cells can be induced by TNF and other motogenic factors secreted by cells of the tumour microenvironment such as macrophages. Based on our recent findings that the TNF receptor adaptor protein FAN mediates TNF-induced actin reorganisation and regulates the directed migration of immune cells responding to chemotactic cues, we addressed the role of FAN in cancer cell motility and the formation of invadopodia, a crucial feature in tumour invasion.

Methods:

In B16 mouse melanoma cells, FAN was downregulated and the impact on FAN on cell motility and invasion was determined using in vitro assays and in vivo animal models.

Results:

Like FAN^−/− murine embryonic fibroblasts, FAN-deficient B16 melanoma cells showed defective motility responses to TNF in vitro. In vivo FAN-deficient B16 melanoma cells produced significantly less disseminated tumours after i.v. injection into mice. Danio rerio used as a second in vivo model also revealed impaired spreading of FAN-deficient B16 melanoma cells. Furthermore, FAN mediated TNF-induced paxillin phosphorylation, metalloproteinase activation and increased extracellular matrix degradation, the hallmarks of functionally active invadopodia.

Conclusion:

The results of our study suggest that FAN through promoting melanoma cellular motility and tumour invasiveness is critical for the tumour-promoting action of TNF.