Project description:We established a model of human melanoma metastasis to identify differentially expressed genes in brain metastasis, compared to cutaneous melanoma from which they were developed. Such genes may control brain metastasis. The identification and characterization of these genes would advance the understanding of the metastatic process and may lead to new diagnostics and therapeutic approach. Brain metastases occur in almost 40% of melanoma patients. The median survival of such patients does not exceed a few months. Very little information is available on mechanisms underlying the progression of melanoma towards brain metastasis. The function, and significance of the various factors involved in melanoma progression must be deciphered using relevant models. Currently, most human melanoma brain metastasis models consist of xenografted cells inoculated into immune-deficient mice mainly by intracarotid or intra-cardiac administration. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, sub-dermal variants that originate from the same melanomas thus sharing a common genetic background. The brain metastasizing variants were obtained by intra-cardiac inoculation. One of the brain metastasizing variants when inoculated sub-dermally yielded spontaneous brain dormant micrometastasis. Cells from the spontaneous brain micrometastasis when removed from the brain microenvironment proliferate very well in vitro and generate tumors in the skin being the orthotopic organ site. The brain metastasis and micro-metastasis cells expressed higher levels of ANGPTL4, COX-2, MMP1, MMP2 and PRAME and lower levels of CLDN1, CYR61 and IL-6R than the cutaneous variants. These gene products may be involved in melanoma brain metastasis and may serve as novel brain metastasis biomarkers and targets for therapy. 8 Samples (arrays) were analyzed. We generated pairwise comparisons between cutaneous and brain metastatic variants of the same genetic background, using Partek Genomics Suite, in the three melanoma models. Genes with pM-bM-^IM-$5% and a fold-change difference of M-bM-^IM-%1.25 or <-1.25 were selected.

Project description:The present study deals with functional interactions of cutaneous and brain-metastasizing human melanoma cells with brain-derived molecules. In this study we employed the unique melanoma xenograft model developed by Izraely and described in Int J Cancer. 2011 Oct 25. doi: 10.1002/ijc.27324. The present study aims to determine if brain-derived soluble factors regulate malignancy-associated functions of cutaneous and brain-metastasizing melanoma cells and identify which functions are regulated by such factors. The working hypothesis of this study is that the interactions between the brain microenvironment and melanoma cells determine metastasis formation at this organ site. The aim of the study was to evaluate the contribution of such interactions to the formation of brain metastasis in nude mice xenografted with human melanoma cells. An insight into these interactions is an essential pre-requisite for the development of effective targeted therapy for melanoma brain metastasis. We assessed the effects of soluble factors present in supernatants of short-term cultures of normal mouse brain (referred here after as brain-derived soluble factors) on several characteristics linked to melanoma brain metastasis. It was found that brain-derived soluble factors affect differentially cutaneous and brain-metastasizing melanoma cells variants in-vitro. Such factors enhanced the viability of cutaneous melanoma cells but caused an S phase arrest followed by apoptosis of brain-metastasizing cells. Brain-derived soluble factors enhanced migration of melanoma cells metastasizing to the brain, but did not affect the migration of the cutaneous variants. Such factors up-regulated the expression of the chemokine receptor CCR4 in both cutaneous and brain metastasizing melanoma cells. It is not unlikely that CCR4 ligands expressed in the brain interact with the CCR4-expressing melanoma cells thereby directing them to the brain. Brain-derived soluble factors enhanced the transmigration, across human brain endothelial cells of cutaneous but not of brain metastasizing melanoma variants. This activity could promote the capacity of the cutaneous cells to metastasize to the brain. 4 Samples (arrays) were analyzed. There is 1 replicate for each variant and each treatment. We generated pairwise comparisons between cutaneous and brain metastatic variants of the same genetic background, using Partek Genomics Suite, in the three melanoma models. Genes with p≤5% and a fold-change difference of ≥2 or <-2 were selected.

Project description:The present study deals with functional interactions of cutaneous and brain-metastasizing human melanoma cells with brain-derived molecules. In this study we employed the unique melanoma xenograft model developed by Izraely and described in Int J Cancer. 2011 Oct 25. doi: 10.1002/ijc.27324. The present study aims to determine if brain-derived soluble factors regulate malignancy-associated functions of cutaneous and brain-metastasizing melanoma cells and identify which functions are regulated by such factors. The working hypothesis of this study is that the interactions between the brain microenvironment and melanoma cells determine metastasis formation at this organ site. The aim of the study was to evaluate the contribution of such interactions to the formation of brain metastasis in nude mice xenografted with human melanoma cells. An insight into these interactions is an essential pre-requisite for the development of effective targeted therapy for melanoma brain metastasis. We assessed the effects of soluble factors present in supernatants of short-term cultures of normal mouse brain (referred here after as brain-derived soluble factors) on several characteristics linked to melanoma brain metastasis. It was found that brain-derived soluble factors affect differentially cutaneous and brain-metastasizing melanoma cells variants in-vitro. Such factors enhanced the viability of cutaneous melanoma cells but caused an S phase arrest followed by apoptosis of brain-metastasizing cells. Brain-derived soluble factors enhanced migration of melanoma cells metastasizing to the brain, but did not affect the migration of the cutaneous variants. Such factors up-regulated the expression of the chemokine receptor CCR4 in both cutaneous and brain metastasizing melanoma cells. It is not unlikely that CCR4 ligands expressed in the brain interact with the CCR4-expressing melanoma cells thereby directing them to the brain. Brain-derived soluble factors enhanced the transmigration, across human brain endothelial cells of cutaneous but not of brain metastasizing melanoma variants. This activity could promote the capacity of the cutaneous cells to metastasize to the brain.

Project description:Brain metastases of melanoma are associated with therapy resistance and poor prognosis. It is not fully understood whether and how the selection of cells capable of metastasizing into the brain is accompanied by the establishment of specific features. For the investigation of these questions, we made use of previously described xenograft mouse models for primary human melanoma cells distinguishing cutaneous from cerebellar metastases from the same genetic background. Previous experiments suggested that cultured cells derived from these xenografts still maintain properties characteristic for the microenvironment of the originating metastases. Such corresponding pairs of metastatic cells were obtained from four individual donors, resulting in eight cell-lines presently investigated with regard to molecular properties characteristic for metastasis. Label free proteome profiling revealed significant alterations when comparing corresponding pairs of cutaneous and cerebellar metastases from the same donor. Molecules previously associated with metastasis such as cell adhesion molecules, immune regulators, epithelial mesenchymal transition markers, stem cell markers, redox regulators and cytokines were found differently expressed. This was also observed with regard to eicosanoids considered relevant for metastasis such as PGE2 and 12-HETE. However, no commonalities in the molecular characteristics of cerebellar metastases were identified in all four donors. Multiparametric morphological analysis of cells also revealed alterations associated with the kind of metastases, while lacking uniformity. In conclusion, here we describe that xenografted melanoma cells derived from two different microenvironments, i.e. cutaneous and cerebellar metastases, display significant alterations in the expression of molecules associated with metastastic properties. The observed lack of uniformity suggests that metastatic cells may find individual strategies to adapt to their microenvironmental challenges accompanied by the establishment of individual cell characteristics.

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A Molecular Signature Associated with Human Melanoma Brain Metastasis and Spontaneous Micrometastasis

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