Interferon-mediated enhancement of metastasis. Are MHC antigens involved?

The relationship between major histocompatibility complex (MHC) antigens and metastasis was investigated on B16 melanoma variants. B16 cell lines express low amounts of murine MHC (H-2) antigens. A high expression can be induced in line B16-A by in vitro treatment with immune interferon (IFN-gamma) or by in vivo transplant in allogeneic mice. The increase of H-2 antigens correlated with an enhancement of lung colonization in young syngeneic mice. The higher metastatic capacity of B16-A cells with induced high levels of H-2 antigens was observed also in adult mice and in young mice pretreated with cyclophosphamide. These results were confirmed investigating the behaviour of a mutant B16 clone (B78H1) which was selectively resistant to the H-2-inducing action of IFN-gamma: lung colonization ability was not increased by IFN pretreatment. The study of variants derived from individual B16-A lung colonies revealed a wide range of H-2 levels. Variants with a low expression had a low colonization ability; one out of two variants with a high H-2 expression also was poorly colonizing. IFN-gamma-mediated H-2 expression appeared to act as an enhancer, rather than a determinant of B16 metastatic capacity.     

Control of metastatic properties of BL6 melanoma cells by H-2Kb gene: immunological and nonimmunological mechanisms

The effect of class I H-2 antigen expression on the metastatic properties of BL6 melanoma cells was investigated. The BL6-8 clone isolated from the highly metastatic BL6 melanoma did not express H-2K b gene. Following transfection with the H-2K^b gene, BL6-8 cells displayed a low metastatic potential in the immunocompetent as well as immunosuppressed (X-irradiated) or triple-immunodeficient mice with impaired T, B and natural killer (NK) cells function. The expression of H-2K^b gene and the low metastatic ability of transfected BL6 melanoma cells were associated with appearance of cell membrane soybean agglutinin (SBA) and Griffonia simplicifolia 1B₄ (GS1B₄) lectin-binding carbohydrataes. These alterations in cell surface carbohydrates were found to be a result of reduction in sialylation of SBA binding sites and upregulation of the 1.3 galactosyltransferase (1.3GT) gene. To assess the importance of H-2K^b-induced alterations in cell surface carbohydrates for metastasis formation, BL6-8 melanoma cells were transfected with H-2K^b gene without neo^r gene cotransfection and selected for adherence to SBA-lectin-conjugated agarose beads. The transfected clones that expressed SBA and GS1B₄ lectin-binding carbohydrates were low metastatic. Further analysis of these clones showed that presence of SBA and GS1B₄ lectin-binding carbohydrates rather than expression of H-2K^b molecules per se might be responsible for low metastatic potentials of H-2K^b-transfected cells in the immunocompromized mice. Studies of the possible mechanisms responsible for low metastatic ability of H-2K^b-transfected melanoma cells revealed that these cells displayed a reduced ability to adhere to murine pulmonary endothelial cells as well as to laminin and collagen IV. We hypothesized that the observed nonimmunological effects of H-2K^b gene in BL6 melanoma cells is a result of an interaction between the H-2K^b gene and B16 melanoma-specific ecotropic retrovirus. It results in inhibition of this retrovirus production with consecutive alteration in the expression of cellular genes controlling cell surface glycosylation and adhesion properties essential for the metastatic phenotype of BL6 melanoma.     

IMPAIRED H-2 EXPRESSION IN B 16 MELANOMA VARIANTS

We studied the expression of H-2^b alloantigens in three different B16 melanoma lines cultured in vitro, Cell lines were B16-F1 and two cell cultures (named B16-A and B16-B) newly derived from two different in vivo sublines of B16 melanoma. The assays used were in vivo tumour growth in allogeneic (BALB/c and B10.BR) as compared to syngeneic mice, in vitro cell-mediated cytotoxicity by anti-H-2^b immune lymphocytes and absorption of anti-H-2^b antisera activity. The B16-F1 line was able to efficiently kill allogeneic hosts, could not be lysed by anti-H-2^b cytotoxic effectors and did not express any serologically detectable amount of H-2^b alloantigens. The B16-A line was H-2 positive during the early in vitro passages, then, at the 8th–10th passages, it acquired the capacity to kill allogeneic hosts, lost the sensitivity to anti-H-2^b cytotoxic effectors and the H-2K^b antigens became undetectable. The expression of H-2D^b was reduced, although at a lower degree. Similar data were obtained with B 16-B cells, which after 10 in vitro passages grew and killed allogeneic hosts, showed a decreased sensitivity to cytotoxic anti-H-2^b effectors and a very low expression of the K region antigens. The results indicate that H-2 expression is altered in B 16 melanoma lines and this may influence the different metastatic capacity of such cells.     

Monophosphoryl lipid A (MPL) upregulates major histocompatibility complex (MHC) class I expression by increasing interferon-gamma (IFN-gamma)

Tumor immunity is primarily mediated by cells as CD8+ cytotoxic T lymphocytes (CTL) recognize tumor antigen by MHC class I molecules. But most tumors are associated with a decreased expression of MHC class I to escape the antitumor immunity of the host. Our previous data have demonstrated that MPL has an antitumor effect on metastatic lung cancer of B16 melanoma with enhancing cytotoxicity due to increase of IFN-gamma and IL-2, and decrease of IL-4, which indicates the stimulation of type 1 helper T cells (Th1). To determine the effects of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha on MHC class I expression of B16 melanoma cells, we evaluated the expression of MHC class I molecules with treatments of MPL, IFN-gamma, TNF-alpha, and IL-1 alpha by flow cytometry. The supernatant of MPL-treated spleen cells in vitro upregulated the expression of MHC class I molecules of B16 melanoma cells compared to the control supernatant of spleen cells. The MHC class I expression of B16 melanoma cells treated with IFN-gamma, but not TNF-alpha or IL-1 alpha, increased in a time-dependent manner. In conclusion, MPL upregulated MHC class I expression of B16 melanoma cells by activating spleen cells via IFN-gamma. These data suggest that increased IFN-gamma by MPL is responsible for the upregulation of MHC class I expression to augment cytotoxicity. Therefore, we suggest that MPL could play an important role in immunotherapy.     

Expression of ganglioside GM3 and H-2 antigens in clones with different metastatic and growth potentials isolated from Lewis lung carcinoma (3LL) cell line

In view of the evidence that cell expression of gangliosides in several tumors is positively involved in the metastatic phenotype, Lewis lung carcinoma (3LL) cell line, expressing GM3 as the major ganglioside, was analysed for the cell surface expression of GM3. An indirect immunofluorescence assay, using a M2590 monoclonal antibody recognizing GM3, was used for this purpose. Since the parental 3LL cells consist of heterogenous subpopulations differing in the degrees of GM3 expression, we have developed clones of this cell line with different degrees of metastatic potentials by using anin vitro non-selective procedure in order to investigate whether the expression of GM3 is associated with metastatic potential. The degree of cell surface expression of GM3 among the clones correlated well with their total cellular content of this ganglioside. However, we were unable to confirm the report of increased level of GM3 in high metastatic 3LL clones, nor did a decreased level correlate with weak metastatic ability. In our recent work, an inhibitor of glucosylceramide synthase,d-threo-l-phenyl-2-decanoylamino-3-morpholino-l-propanol (DPDMP), was found to decrease the levels of all cellular glucosphingolipids and cause the accumulation of the precursors of glucosylceramide. The present study does not, however, rule out the possible involvement of this lipid family in metastatic dissemination, since treatment of 3LL cells with D-PDMP resulted in significant inhibition of their experimental metastatic potential. Clones expressing very low GM3 grew slowly in culture dishes, suggesting that GM3 may have a regulatory role in cell proliferation. The low metastatic clones expressed high levels of H-2K^b antigen, while the expression of the same antigen on the high metastatic clones was relatively low, confirming the previous observation of this tumor system. Moreover, a clone showing the lowest tumorigenic potency revealed both a high cell surface expression of H-2K^b and a high H-2K^b/H-2D^b ratio.     

NIH3T3 transfectant containing human K-ras oncogene shows enhanced metastatic activity after in vivo tumor growth or co-culture with fibroblasts

A clone of NIH3T3 transformant (H-3), obtained by transfecting genomic DNA of a human colon carcinoma cell line, contains human K-ras oncogene and yields metastatic pulmonary nodules after intravenous injection of the cells into nude mice. This metastatic ability was enhanced remarkably after in vivo tumor growth (subcutaneous tumor formation in nude mice) accompanied by increased mRNA expression and gene amplification of the human-derived K-ras oncogene, while it declined gradually as the passage number increased in vitro, with corresponding decreases of gene amplification and mRNA expression. Six subclones were randomly selected from H-3 cells which had been subcultured to passage 22. All of the clones in culture showed almost the same low level of metastatic ability and exhibited little K-ras oncogene amplification with correspondingly low mRNA expression. However, after they formed tumors in nude mice, every clone acquired high metastatic ability and the gene amplification increased, with elevated mRNA expression. These experimental facts indicated that acquisition of metastatic ability coupled with the function of K-ras oncogene was conditional in nature, being strongly affected by in vivo tumor circumstances. The low metastatic and G-418-resistant H-3 cells were co-cultured with BALB/c3T3 fibroblasts for 2–4 weeks. After removal of fibroblasts by exposure to G-418, the tumor cells exhibited increased metastatic ability and human K-ras oncogene mRNA, suggesting an intimate interaction between H-3 cells and fibroblasts influencing the function of transfected human K-ras oncogene. Fibroblasts of the host animal may thus have an important role in generating enhanced metastatic activity of H-3 cells.     

Inhibition of VLA-4 and up-regulation of TIMP-1 expression in B16BL6 melanoma cells transfected with MHC class I genes

The effect of MHC class I gene transfection on the metastatic properties of B16BL6 melanoma cells was investigated. BL6-8 melanoma cells transfected with H-2K or H-2K, but not H-2D or H-2L, genes showed a dramatic reduction in their ability to generate experimental metastases in immunosuppressed CB6F1 mice. This observation suggested that some changes in the metastatic phenotype may have been induced in the H-2K- transfected melanoma cells. Analyses of adhesive and invasive properties of BL6-8 melanoma cells transfected with H-2 class I genes have been performed. We found that the loss of metastatic properties in the H-2K or H-2K gene-transfected melanoma cells was associated with reduced adherence to endothe-lial cells, laminin and collagen IV, decreased ability to form homotypic cell aggregates and with a complete loss of VLA-4 integrin expression. In addition, BL6-8 melanoma cells transfected with H-2K genes demon-strated reduced ability to invade Matrigel that paralleled up -regulation of TIMP-1 expression. Incubation of untransfected BL6-8 clone or B16F1 cells with 5-azacytidine similarly resulted in up-regulation of TIMP-1, suggesting that the changes in methylation of TIMP-1 gene could be responsible for TIMP-1 expression in the H-2K-transfected BL6-8 melanoma cells. Transfection of BL6-8 cells with the H-2D d /L d genes did not affect their adhesive and invasive properties. Previously we reported that reduction in the metastatic properties of the H-2K b transfected cells was associated with alterations in cell surface carbohydrates with appearance of a-galactosyl epitopes and reduction in cell surface sialylation. The present data indicate that, in addition to changes in cell surface carbohydrates, reduction in adhesive properties and up-regulation of TIMP-1 may be responsible for the observed loss of metastatic potential of BL6-8 cells transfected with the H-2K genes. © Rapid Science Ltd.     

Tumorigenicity of mouse T lymphoma cells is controlled by the level of major histocompatibility complex class I H-2Kk antigens

We have previously found that an increased tumorigenicity and spontaneous metastatic potential of BW5147-derived T lymphoma cells was associated with a decrease in major histocompatibility complex (MHC) class I H-2K^k antigen expression. This suggested that H-2K^k antigens may control the tumorigenic potential of BW T lymphoma cells. Our current experiments aimed to prove this association by specifically altering H-2K^k expression by gene transfection. Transfected cells expressing a high level of H-2K^k antigens were significantly less tumorigenic and metastatic after subcutaneous inoculation. However, there was selectionin vivo for cells expressing a reduced level of H-2K^k antigens, which concomitantly led to an increased tumorigenicity. These data further confirmed the strong association between H-2K^k expression and tumorigenicity. We subsequently tested whether the immune system is implicated in this phenomenon by inoculating the H-2K^k transfectants into irradiated, immunocompromised recipients. Our results indicate that the reduced tumorigenicity of the BW H-2K^k transfectants is due to an immune rejection mechanism, mediated by CD8⁺ immune effector cells, as revealed byin vivo depletion experiments with anti-CD8 antibodies. Hence, we hereby demonstrated that H-2K^k antigens increased the immunogenicity of BW cells, via a CD8-dependent mechanism, which consequently reduced their tumorigenicity.     

Up-regulation of TIMP-1 expression in B16-F10 melanoma cells suppresses their metastatic ability in chick embryo

Clonal B16-F10 cell lines with increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) have been generated by transfection with a TIMP-1-containing expression vector. The parental B16-F10 and control 1–2 cells, and two TIMP-1 up-regulated clones (2–10, 6–5), were studied for their growth characteristics in tissue culture and their experimental metastatic ability in the chick embryo. Both of the TIMP-1 up-regulated clones showed slower in vitro growth and had lower saturation densities. Both clones were also less metastatic following intravenous injection into chick embryos, and formed significantly fewer metastatic tumors in the chorioallantoic membrane and in the liver than did parental B16-F10 and control cells. Furthermore, the size of tumors formed by TIMP-1 up-regulated cells was significantly reduced in comparison to the tumors produced by B16-F10 or control cells. Our results show that malignant cell lines genetically modified to express increased levels of TIMP-1 exhibit a suppressed experimental metastatic ability in vivo. We propose that TIMP-1 suppresses metastatic ability by decreasing both invasive and growth abilities.     

DMSO-induced changes in the procoagulant and fibrinolytic activity of B16 melanoma cells: Influence on lung colony formation

In this study DMSO (dimethylsulphoxide) was used as a tool to test the significance ofin vitro modifications of procoagulant and fibrinolytic activity of tumor cells for theirin vivo metastatic ability. Bl6 melanoma cells were chosen as the experimental model. After four days' treatment DMSO increased both the procoagulant and fibrinolytic (plasminogen activator) activity of B16 melanoma cells in a dose-related manner. DMSO treated cells showed significantly greater lung colonizing ability than untreated cells. Our results indicate that DMSO treatmentin vitro can modulate procoagulant and fibrinolytic activity and the metastatic ability of B16 melanoma cells; however a direct causal relationship between thesein vitro andin vivo effects remains to be established.     

In vivo activation of NK cells induces inhibition of lung colonization of H-2 positive and H-2 negative fibrosarcoma tumor clones

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 betweenin vivo andin vitro results.     

Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on fibroblast-like synoviocytes: Paradoxical induction of IFN-gamma and TNF-alpha receptor expression

We recently described mutual antagonism between IFN-gamma and TNF-alpha on human fibroblast-like synoviocytes (FLS). TNF-alpha inhibits IFN-gamma-induced HLA-DR expression and IFN-gamma blocks TNF-alpha-dependent synoviocyte proliferation, collagenase production, and GM-CSF secretion. To study the mechanism of antagonism we have analyzed the effect these factors on the expression of cytokine surface receptors.¹²⁵I-Labeled cytokine binding was measured on cultured FLS and the results were analyzed by Scatchard plots. Unstimulated synoviocytes expressed 9300 ± 1560 IFN-gamma binding sites per cell. A single class of high-affinity receptor was observed (K _d=4.5±2.5×10^–10 M). TNF-alpha did not competitively inhibit¹²⁵I-IFN-gamma binding. When FLS were incubated with TNF-alpha (100 ng/ml), there was a paradoxical 49.5 ± 5.6% increase in the number of binding sites for IFN-gamma (P=0.001), with no change in theK _d. Unstimulated FLS also expressed 2850 ± 700 TNF-alpha receptors per cells, with a singleK _d consistent with the lower-affinity TNF-alpha receptor (7.4±0.2×10^–10 M). IFN-gamma did not directly interfere with TNF-alpha binding. Preincubation of FLS with 100 U/ml of IFN-gamma resulted in a 28.9 ± 9.0% increase in TNF-alpha receptor expression (P<0.008), with no change in theK _d. Low levels of the soluble 55-kD TNF receptor were detected in FLS supernatants. IFN-gamma did not effect soluble TNF receptor production. These data are the first demonstration of IFN-gamma and TNF-alpha receptors on FLS and show that TNF-alpha and IFN-gamma increase the expression of each other's receptor. Therefore the mutual antagonism between these two cytokines must occur through a postreceptor mechanism.     

Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells

Cytotoxic T lymphocyte (CTL) peptide epitopes can be used for immunization of mice against lethal virus infection. To study whether this approach can be successful against virus-induced tumors we generated a B6 (H-2^b) tumorigenic cell line transformed by human papillomavirus (HPV). This virus is detected in over 90% of all human cervical cancers. To identify vaccine candidates, we generated a set of 240 overlapping peptides derived from the HPV type 16 (HPV16) oncogenes E6 and E7. These peptides were tested for their ability to bind H-2K^b and H-2D^b MHC class I molecules. Binding peptides were compared with the presently known peptide-binding motifs for H-2K^b and H-2D^b and the predictive value of these motifs is shortly discussed. The high-affinity H-2D^b-binding peptide and putative CTL epitope E₇ 49-57 (RAHYNIVTF) was used in vaccination studies against HPV 16-transformed tumor cells. Immunization with peptide E₇ 49-57 rendered mice insensitive to a subsequent challenge with HPV 16-transformed tumor cells in vivo, and induced a CTL response which lysed the tumor cells in vitro.     

Biological properties associated with the enhanced lung-colonizing potential in a B16 murine melanoma line grown in a medium conditioned by syngeneic Corynebacterium parvum-elicited macrophages

A previous study by our laboratory showed that the peritoneal murine Corynebacterium parvum-elicited macrophages released into their growth medium an activity which enhanced the ability of B16-F10 melanoma cells to form experimental metastases in the lung of syngeneic mice. In the present study, we used a clone of B16-F10 line (F10-M3 cells) to investigate whether the increase in lung-colonizing potential due to the pro-clonogenic activity released by C. parvum-elicited macrophages was associated with biological properties characteristic of a metastatic phenotype. We have found that the pulmonary retention, growth rate in lung parenchyma, invasiveness through Matrigel, adhesiveness to IL-1-activated endothelium and MHC class I expression were increased in F10-M3 cells stimulated by the macrophage pro-clonogenic activity. By using an in vitro experimental protocol, the enhancement of lung-colonizing potential in the stimulated melanoma cells turned out to be a transient phenomenon as was the increase of invasiveness through Matrigel and the higher expression of MHC class I antigens. In conclusion, the melanoma cells stimulated by the pro-clonogenic activity released by C. parvum-elicited macrophages showed changes in biological parameters which are relevant to metastatic diffusion. These changes appeared as a temporary phenomenon which sustains the view that the metastatic phenotype represents a transient biological character influenced by host factors. This revised version was published online in July 2006 with corrections to the Cover Date.     

H-2Kb transfection of B16 melanoma cells results in reduced tumourigenicity and metastatic competence

The metastatic B16 mouse melanoma shows a low cell surface expression of H-2Kb and H-2Db class I antigens on cells of both the high-metastatic line B16-F10 and the low-metastatic line B16-F1. Similarly, newly generated clones of these lines, having different metastatic properties, all express low levels of major histocompatibility antigens. One of these clones, the high-metastatic F10.9, was transfected with H-2Kb genes to generate H-2Kb-expressing transfectants. The resulting clones showed reduced tumourigenicity and a low metastatic phenotype. Unlike the parental cells, H-2Kb-positive transfectants are potent inducers and sensitive targets of H-2Kb-restricted syngeneic cytotoxic T cells. Immunization of mice with H-2Kb-positive transfectants conferred protection against a subsequent challenge with Kb-positive transfectants but had only a small effect on growth and metastatic spread of parental cells.     

C-met activation is necessary but not sufficient for liver colonization by B16 murine melanoma cells

Metastasis to the liver is a frequent event in clinical oncology, the molecular mechanisms of which are not fully understood. We have recently reported a consistent overexpression of c-met in B16 melanoma cells selected in vivo for enhanced liver metastatic ability. In this study we address the question as to whether constitutive activation of c-met is a necessary and sufficient condition for enhanced liver colonization B16 melanoma cells. Different levels of c-met expression and/or activation in B16 cells were achieved subcloning, or by c-DNA transfection with either HGF/SF or the oncogenic form of c-met (tpr-met). Metastatic ability of the different populations was then evaluated in vivo by the lung colonization (experimental metastasis) assay. Results indicate that c-met (but not tpr-met) activation in B16 melanoma cells may increase their liver colonizing potential, probably by enhancing motility and invasion in response paracrine interactions with its ligand. C-met expres sion per se, however, is not able to change the organ specificity of the cells. C-met activation appears instead to be required at later stages of liver colonization by B16 melanoma cells, in order to enhance their site-specific metastatic ability. © Rapid Science 1998     

Lewis lung carcinoma (3LL) cells treated in vitro with ultraviolet radiation show reduced metastatic ability due to an augmented immunogenicity

The metastatic ability of 3LL tumor following in vitro irradiation with ultraviolet (u.v.) light was studied. Tumor cells were exposed to two courses of u.v.-irradiation (3LL × 2u.v. cells) and after two weeks of culture they were inoculated intravenously (i.v.) into syngeneic mice. These cells produced significantly fewer pulmonary metastases than the untreated population. In addition, intrafootpad (i.f.p.) injections of 3LL × 2u.v. cells into immunocompetent animals induced tumors only in 40 per cent of recipients. Interestingly, in normal mice with progressively growing 3LL × 2u.v. tumors, the formation of spontaneous pulmonary metastases was prevented, whereas metastatic foci were observed in 70 per cent of the nude recipients. The metastatic properties of u.v.-treated tumor cells were further analysed by using individual clones with varying immunogenicity. We found that variants with augmented immunogenicity also showed a parallel decrease in metastatic potential. Studies on H-2 antigen expression in different clones revealed that immunogenic and low metastatic variants expressed levels of H-2 antigens higher than the tumorigenic and metastatic clones. Finally, by using cyclophosphamide (Cy) treatment and adoptive transfer of immune spleen cells we were able to eradicate macroscopic 3LL pulmonary metastasis. These results demonstrate that the decrease of metastatic ability in u.v.-treated cells was mainly due to an increase in their immunogenicity and H-2 antigen expression.     

TNF Impairs In Vivo and In Vitro Natural Killer (NK) Susceptibility of B16 Melanoma Cells

Tumour necrosis factor alpha (TNF) is a multipotent cytokine which affects many biological properties of both normal and neoplastic cells. Here we show that treatment with TNF reduces B16-A melanoma cell susceptibility to normal and in vivo- and in vitro-activated NK cell-mediated killing. This resistance is associated with an enhancement of B16-A metastatic potential in normal syngeneic mice, but not in anti-asialo GM1-treated animals, further supporting the NK dependence of TNF-induced enhancement of metastatic ability. A significant increase of MHC class I expression on B16-A murine melanoma cells is observed after TNF treatment. In all these effects TNF interacts positively with interferon gamma (IFN gamma). Taken together, these results indicate that TNF treatment negatively affects the susceptibility of B16-A murine melanoma to NK effectors in vivo and in vitro. This decreased susceptibility may be related, at least in part, to enhanced expression of MHC class I antigens on tumour cells.