Neural crest cell migration of mouse B16-F1 melanoma cells transplanted into the chick embryo is inhibited by the BMP-antagonist noggin

Melanoma cells are derived from the neural crest and characterized by high migratory potential and invasive growth. To test the analogies between malignant and embryonic cell migration, in previous studies we transplanted melanoma cells and non-transformed mouse neural stem cells into the neural crest compartment of the chick embryo. Human and mouse melanoma cells spontaneously migrated along the neural crest pathways while emigration of neural stem cells was dependent on pre-treatment with BMP-2 (bone morphogenetic protein-2). In the embryo neural crest cell migration is induced by BMP and inhibited by its antagonist noggin. We tested whether the spontaneous neural crest cell migration of melanoma cells was dependent on their endogenously expressed BMP and could be inhibited by noggin. Mouse B16-F1 melanoma cells transfected with GFP-VASP (vasodilator-stimulated phosphoprotein) were cultured as aggregates and treated with BMP-2 or noggin. Untreated and treated aggregates were transplanted into the neural tube of the E2 chick embryo. Untreated and BMP-2-treated melanoma cells emigrated from the neural tube along with the chick host neural crest cells. Noggin-treated aggregates showed no emigration. We conclude that spontaneous emigration of melanoma cells depends on their constitutive overexpression of BMP, and that noggin efficiently suppresses the emigration of melanoma cells in the embryonic micro-environment, thus rendering noggin a promising agent for the inhibition of melanoma cell migration in vivo.     

Non-malignant migration of B16 mouse melanoma cells in the neural crest and invasive growth in the eye cup of the chick embryo

Melanocytes originate from the neural crest. In a previous study, we observed that human SK-Mel 28 human melanoma cells resumed neural crest cell migration after transplantation into the chick embryo neural tube. Here, we used transgenic mouse B16-F1 melanoma cells transfected with green fluorescent protein-vasodilator-stimulated phosphoprotein construct to extend these observations. After the injection of a cell suspension into the trunk neural tube of E2 chick embryos, the migration of melanoma cells was followed by live fluorescence microscopy. Within 12 h, the melanoma cells formed clusters in the neural tube at the levels of the intersegmental clefts between somites. After 24 h, a segmental pattern of emigration was visible. Emigrated melanoma cells were identified in serial paraffin sections by immunohistochemistry with ab732 as a marker for melanoma cells and by in-situ hybridization of mouse-specific repetitive genomic sequence mL1. After 24 h, melanoma cells were found along the medial neural crest pathway and in the sympathetic trunk ganglia and, after 48 h, also in the lateral melanocytic pathway. During migration along the neural crest pathways, mouse melanoma cells underwent apoptosis, which was assessed by anti-caspase 3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining. To prove the ablation of malignant behavior after back-transplantation into the original embryonic neural crest environment, we injected the same cell suspension into the eye cup of the E3 embryo. In this location, invasive melanomas formed.     

Human SK-Mel 28 melanoma cells resume neural crest cell migration after transplantation into the chick embryo

Melanocytes are derived from the neural crest. We questioned whether the migratory mechanism during the invasive growth of melanoma cells is the same as that in neural crest cell migration. We transplanted human SK-Mel 28 melanoma cells into the neural tube of the chick embryo stage 11-13 and, after up to 6 days of total incubation, traced the cells by immunohistochemistry in serial paraffin sections. SK-Mel 28 cells were integrated into the host neural crest and were found in the roof plate of the neural tube, along the medial neural crest cell pathway, in the sclerotome and, finally, in developing sympathetic ganglia. At stage 21, massive segmental emigration between myotome and disintegrating dermatome was observed at the level of the upper limb bud. The melanoma cells, in contrast with the chick neural crest cells, were HNK-1-negative. They retained the premelanosome epitope HMB-45. For definite identification and exclusion of fusion with chick embryo cells, in situ hybridization with the human-specific Alu sequence was performed. The results showed that human SK-Mel 28 melanoma cells were capable of resuming neural crest cell migration in the embryo.     

Interactions between human glioma cells and fetal rat brain aggregates studied in a chemically defined medium

The in vitro invasive growth of two continuous human glioma cell lines (D-54Mg and GaMg) into aggregates of fetal rat brain cells is described. The tumor cells were first cultured as multicellular tumor spheroids and thereafter cocultured with brain aggregates in medium agar cultures. Two different types of culture media were used for the propagation of spheroids and for coculture experiments: Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, and Costar SF-X chemically defined hybridoma medium. Both cell lines showed invasive growth into brain tissue in both types of media. Apart from progressive destruction caused by the malignant cells, the brain aggregates maintained characteristics of neural tissue. One cell line (D-54Mg) showed reduced invasiveness in chemically defined medium as measured with a grading system to quantify invasion. The coculture system may represent a basis for studying invasion of human glioma cells in brain tissue under defined chemical conditions.     

The mature bone morphogenetic protein-2 is aberrantly expressed in non-small cell lung carcinomas and stimulates tumor growth of A549 cells

To help identify genes, which may regulate metastasis in lung cancer, we performed representational difference analysis between a patient-derived non-small cell lung carcinoma (NSCLC) and immortalized normal human bronchial epithelial cells. This analysis revealed that bone morphogenetic proteins-2/4 (BMP) mRNA was expressed in the lung carcinoma. BMP-2/4 are known to induce pluripotent cell differentiation, enhance cell migration and stimulate proliferation during embryonic development. Despite being powerful morphogens it is not known whether BMP-2/4 have significant biological activity in human carcinomas. Furthermore, it has not been established whether the mature active BMP-2/4 protein is aberrantly expressed in patient-derived tumors. The purpose of this study was to determine whether the expression of the mature BMP-2/4 protein is disregulated in human lung carcinomas and to establish whether it has adverse biological activity. This study reveals that the mature BMP-2 protein, but not BMP-4, is highly over-expressed in human NCSLC with little to no expression in normal lung tissue or benign lung tumors. The expression of BMP-2 localized specifically to the cancer cells. Recombinant BMP-2 stimulated in vitro, the migration and invasiveness of the A549 and H7249 human lung cancer cell lines. In vivo, recombinant BMP-2 enhanced the growth of tumors formed from A549 cells injected subcutaneously into nude mice. Furthermore, inhibition of BMP-2 activity with either recombinant noggin or anti-BMP-2 antibody resulted in a significant reduction in tumor growth. This study shows that expression of the mature BMP-2 protein is disregulated in the majority of NSCLC. BMP-2 enhancement of tumor cell migration and invasion, as well as stimulating tumor growth in vivo, suggests it has important biological activity in lung carcinomas.     

Invasion of rat neurogenic cell lines in embryonic chick heart fragments in vitro

Invasive properties of 15 continuous neurogenic rat cell lines were investigated in vitro and were compared to their tumorigenicity in inbred BD IX rats. The cell lines were obtained by treating animals with a single transplacental dose of the carcinogen N-ethyl-N-nitrosourea (ENU) and 1) subsequently transferring brain cells into cell culture shortly after treatment or 2) explanting the resultant tumors from the offspring to monolayer cultures. In addition, one continuous nonneoplastic rat fibroblast line and three samples of untreated fetal rat brain cells were investigated. Cells from monolayer cultures were suspended and allowed to form aggregates for 24 hours on a gyratory shaker. The cell aggregates were then brought into contact with and allowed to attach to fragments from 9-days embryonic chick heart and were cultured on a gyratory shaker. All tumorigenic cell lines invaded the heart fragments, as characterized by progressive replacement of heart cells by invading cells. The heart tissue degenerated irreversibly. Nontumorigenic cell lines did not show invasiveness in vitro. Confrontation of cell aggregates and heart fragments in organotypic culture appeared to be a useful method to study directly the invasive properties of malignant transformants of neurogenic cells. The method might also permit prediction of tumorigenicity in the animal.     

Establishment and characterization of human uveal malignant melanoma xenografts in nude mice

The purpose of this study was to develop a suitable animal model for the investigation of the pathogenesis and therapy of uveal malignant melanoma. Eight choroidal malignant melanomas from eight patients were transplanted into nude mice in an attempt to establish a serially transplantable tumour model. Tumour tissue blocks (2 x 2 x 2 mm) from enucleated eyes with choroidal malignant melanoma were transplanted subcutaneously into the flanks of nude mice. The growing tumours were measured and serially transplanted. The tumour samples were investigated by histology, immunohistochemistry and electron microscopy. Only one of the eight transplanted primary tumours (13%) was established as a xenograft in nude mice. Furthermore, the take rate of the transplantable tumour was low (13%). The growth of the tumour fitted a Gompertz function, and the calculated tumour volume doubling time was 54 days. The transplanted tumour cells were epithelioid and slightly larger than the primary tumour cells and had prominent nucleoli. However, the transplanted tumour retained a morphological appearance similar to that of the primary tumour. Immunohistochemical examinations demonstrated that the cells preserved the characteristic properties of malignant melanoma. However, the transplanted cells demonstrated vimentin reactivity, whereas the primary tumour cells were negative for vimentin. It can be concluded that a new experimental model of malignant uveal melanoma with tumours that were easy to observe and access was established in nude mice.     

Development of invasive and growth factor-independent cell variants from primary human melanomas

Growth autonomy and high levels of invasiveness are characteristics of human melanoma cells that are metastatic in vivo. By consecutive passage through a reconstructed basement membrane, we have selected from 5 of 6 primary melanoma cell lines variants which show an up to 10-fold increase in invasiveness. The invasive variants grew more rapidly than the parental, noninvasive cells in serum- and growth factor-free medium and one of the 3 variant cell lines with the highest invasive capacity in vitro metastasized to the lungs when injected s.c. into nude mice. In a second approach, variants of 6 primary melanoma cell lines were clonally selected in medium without exogenous growth factors (protein-free medium). These selected cells showed higher invasive properties in vitro and in vivo than the parental cells. Clones of invasive and growth factor-independent cell variants were heterogenous and changed over time in the absence of selected pressure to a phenotype similar to that of parental nonselected cells. These results indicate that primary melanoma cells contain subpopulations of cells that have the phenotype of an advanced (metastatic) stage of tumor progression, but this phenotype is not stable without selective pressure.     

Resveratrol modulates the malignant properties of cutaneous melanoma through changes in the activation and attenuation of the antiapoptotic protooncogenic protein Akt/PKB

Resveratrol, a nontoxic natural product, exhibits multifaceted biological effects including antimutagenic and anticancer properties. We examined the effect of resveratrol on the expression and activation of Akt/protein kinase B and its impact on melanoma cell migration and invasiveness. We also explored the use of resveratrol as an antimalignant treatment option against skin melanoma in mouse models of the disease. Akt expression and activity were determined by a combination of real-time PCR and western blot analysis. Cell lines stably expressing Akt or a dominant negative variant were used to further establish the role of Akt during the response to resveratrol. Wound healing and transwell assays were used as in-vitro correlates of melanoma cell migration and invasiveness. The efficacy of resveratrol in the treatment of melanoma was assessed in two syngeneic mouse models. Resveratrol downregulated and inactivated Akt in B16F10 and B16BL6 melanoma cells. Resveratrol also inhibited the migratory and invasive properties of these highly malignant cells. The reduction of cell migration and invasion, however, was reversed in cell lines overexpressing Akt or after cotreatment with pharmacological inhibitors that blocked Akt degradation. Dominant-negative Akt cells were more sensitive to resveratrol and had diminished migratory properties. Oral treatment with resveratrol reduced primary tumor volume, Akt expression, and the propensity for metastasis in syngeneic mouse models of melanoma. These results suggest that resveratrol can reduce the malignant properties of highly invasive melanoma cells by inactivating Akt. The nontoxic targeting of Akt by resveratrol makes it an attractive treatment option for melanoma.     

Overexpression of lysosomal-type sialidase leads to suppression of metastasis associated with reversion of malignant phenotype in murine B16 melanoma cells

Increased sialylation in cell surface glycoproteins is one characteristic feature of cancer cells, particularly related to their metastatic potential and invasiveness. Expression of lysosomal-type sialidase, which plays a major role in hydrolysis of such sialo-glycoproteins, is therefore considered to have a great influence on malignant properties of cancer cells. To investigate whether the sialidase expression level is linked to the malignant phenotype, we transfected B16-BL6 murine melanoma cells, a highly invasive and metastatic line, with an expression vector harboring a rat lysosomal sialidase cDNA; then clones were isolated and examined for changes in biological character. Sialidase-overexpressing cells showed suppression of experimental pulmonary metastasis and tumor progression. The transfectants exhibited diminished cell growth, anchorage-independent growth and increased sensitivity to apoptosis induced by suspension culture or serum depletion in vitro, but no significant alterations in invasiveness, cell motility and cell attachment to fibronectin, collagen IV and laminin. Flow cytometric analysis with either peanut agglutinin (PNA) or Ricinus communis agglutinin (RCA) lectin revealed that desialylated forms of glycoproteins on the cell surfaces were increased. In particular, a desialylated form of a cell surface glycoprotein of 83 kDa was prominent in the transfectants, as determined by galactose oxidase labeling. These observations indicate that sialidase expression is inversely associated with metastatic potential and tumor growth in cancer cells, probably through a regulation mechanism that suppresses cell growth and anchorage-independent growth and promotes apoptosis with deprivation of cell anchorage.     

Co-expression of urokinase-type plasminogen activator and its receptor in human gastric-cancer cell lines correlates with their invasiveness and tumorigenicity

The expression of urokinase-type plasminogen activator (u-PA), its receptor (u-PAR) and metalloproteases activity were analyzed in 4 human gastric-cancer cell lines (AGS, Hs746T, SNU-1, and SNU-5), in an attempt to relate these activities to their invasive potential and tumorigenicity on the modified chorioallantoic membranes (CAM) of chick embryos. Only 1 of the 4 cell lines tested, Hs746T, expressed both u-PA and u-PAR as well as MMP-2, but not MMP-9. This cell line was both tumorigenic and highly invasive (51.3 ± 13.1%) on a modified CAM. Its invasive capacity was comparable with that of a highly malignant human epidermoid-carcinoma cell line (HEp3), which usually showed 40 to 50% invasiveness. The 3 other cell lines all produced MMP-2 and MMP-9, but only AGS showed moderate invasiveness (24.2 ± 8.8%). While antibodies to u-PA were significantly effective in reducing CAM invasiveness of Hs746T cells by approximately 40%, the invasiveness of the t-PA-expressing AGS cell line was not affected by anti-t-PA antibodies. These results suggest that when one of the components of the u-PA/u-PAR system (the enzyme and/or the receptor) is not produced and u-PA/u-PAR-dependent cell-surface proteolytic activity is thereby diminished, the malignant phenotype that can be determined by tumorigenicity and invasion of connective tissue on a CAM is compromised. Production of both type-IV collagenases (MMP-2 and MMP-9) cannot offset this deficiency.Int. J. Cancer 71: 867-873, 1997. © 1997 Wiley-Liss Inc.     

Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal-regulated kinase signaling in melanoma cell lines

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.     

Cytokine-dependent invasiveness in B16 murine melanoma cells: role of uPA system and MMP-9

Proteases are crucial for the spread of cancer cells from a primary tumor to the site of secondary growth. This study examined the ability of IFNgamma and TNFalpha to stimulate a better invasiveness in B16 murine melanoma cells, and investigated whether this enhanced ability was related to a higher expression of protease activities, such as urokinase plasminogen activator (uPA) and its receptor (uPAR), and matrix metalloproteinases 2 and 9 (MMP-2, MMP-9). We found that murine melanoma cells enhanced their lung-colonizing potential in vivo and invasiveness through Matrigel-coated filters upon costimulation with IFNgamma and TNFalpha; neither IFNgamma nor TNFalpha alone, at the dose used in the experiments, was able to elicit a change in the invasive/metastatic efficiency of melanoma cells. The invasive phenotype of murine melanoma cells stimulated with IFNgamma and TNFalpha was characterized by an enhanced uPA/uPAR and MMP-9 expression: TNFalpha promoted MMP-9 mRNA expression and pro-MMP-9 protein secretion, and the costimulation with IFNgamma and TNFalpha was required to potentiate the expression of mRNA and protein for uPAR, and to induce a redistribution of uPA from the soluble to the cell body-associated form. Both monoclonal antibodies, anti-uPAR and anti-MMP-9, caused a significant reduction of invasiveness in IFNgamma/TNFalpha-stimulated melanoma cells. These results indicate that invasiveness in B16 murine melanoma cells can be regulated in a cytokine-specific fashion and is dependent on the synergism between the uPA/uPAR system and MMP-9.     

Zebrafish embryo extracts promote sphere-forming abilities of human melanoma cell line

Sphere-forming abilities in culture condition are considered a hallmark of cancer stem-like cells, which represents tumor cell invasiveness and stem-like characteristics. We aimed to show that the sphere-forming subpopulation of human malignant melanoma cell line WM-266-4 acts differently to zebrafish embryo extracts compared with their bulk counterpart. Spheres were maintained in neural stem cell culture conditions. The embryos of zebrafish at specific developmental stages were collected and the extracts were purified under 100 kDa. Spheres were treated with embyo extracts and proliferation assay and immunocytochemistry were conducted. Spheroid cells expressed nestin and epidermal growth factor receptor (EGFR) but not melanoma antigen recognized by T-cells (MART)1, indicating their stem-like character. Zebrafish embryo extracts at 50% epiboly stage inhibited melanoma bulk cell proliferation in a dose-dependent manner. However, sphere-forming abilities were significantly enhanced under 1 µg/mL concentration of 50% epiboly stage embryo extract treatment. Our findings implicate that we should consider cell subsets of a different character from the tumor origin that can respond differently to exogenous substances or tumor microenvironments. We suggest that cancer research should consider both minor stem-like subpopulations and the other major bulk tumor cells. ( Cancer Sci 2009)     

Biophysical measurement of brain tumor cohesion

An advantage of using 3D multicellular spheres to study tumor biology is that they better approximate the interactions encountered by cells in vivo. Our previous studies have shown that the process of spheroid formation is governed by the same thermodynamic principles driving the formation of liquid droplets. This liquid-like behavior enables us to measure a key property influencing tumor behavior, namely, intercellular cohesion. We have developed a method, tissue surface tensiometry (TST), to measure the cohesivity, expressible as surface tension (sigma), of tissue aggregates under physiologic conditions. This study utilizes TST to measure the cohesivity of 3 widely used malignant astrocytoma cell lines of different in vitro invasive potentials. We compare invasiveness with aggregate cohesivity and with the expression of N-cadherin, a key mediator of cell-cell cohesion in neural tissues. We show that the cell lines exhibit liquid-like behavior since they form spheroids whose surface tension is both force- and volume-independent; that aggregates from each cell line have a distinct surface tension that correlates with their in vitro invasive capacity; that dexamethasone (Dex), a widely used therapeutic agent for the treatment of tumor-related cerebral edema, increases aggregate cohesivity and decreases invasiveness; that dexamethasone treatment decreases invasion in a dose-dependent manner but only when cells are in direct contact with one another; and that dex-mediated decreased invasiveness correlates with increased aggregate cohesivity as measured by TST but not with N-cadherin expression or function. Our results demonstrate that for these cell lines, cohesivity is an excellent predictor of in vitro invasiveness.     

Effect of ultraviolet-B radiation on the in vivo growth of murine melanoma cells

The role of UV radiation in the development of malignant melanoma has yet to be clearly defined. The purpose of these studies was to determine whether UV irradiation of mice produces local or systemic alterations that increase the in vivo growth of transplanted melanoma cells. K-1735 melanoma cells were injected into the external ears of syngeneic C3H mice. UV irradiation of the mice before or at the time of injection of the melanoma cells accelerated the appearance of the tumors. The effect was observed when melanoma cells were transplanted directly into the site of UV irradiation, but not when they were injected into an unirradiated site. The initial survival of radiolabeled melanoma cells at the site of inoculation was not altered by UV irradiation of the host, suggesting that the accelerated appearance of tumors was due to an increase in the clonogenic potential of cells injected into UV-irradiated skin. The effect of UV irradiation on the development of other syngeneic tumors was also investigated. The outgrowth of a second melanoma was also accelerated in UV-irradiated mice, whereas the growth of a UV-induced fibrosarcoma, a methylcholanthrene-induced fibrosarcoma, and a spontaneous hepatocarcinoma was not affected. These results suggest that, in addition to its carcinogenic activity, UV radiation may contribute to the incidence of cutaneous melanoma because of a local effect on the skin that stimulates melanoma development.     

Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through a dual mechanism

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.