Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain-metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain-metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietinlike 4, prostaglandin-synthesizing enzyme cyclooxygenase-2, matrix metalloproteinase-1 and preferentially expressed antigen in melanoma and lower levels of claudin-1 and cysteine-rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.Brain metastasis represents a significant cause of death in melanoma patients, and its frequency is increasing, 1 possibly as a result of new therapies prolonging patient survival. 2 Of all human solid tumors, malignant cutaneous melanoma has one of the highest risks to develop brain metastasis. More than 40% of advance stage melanoma patients are treated for complications due to brain metastasis. 1,3 Treatment options for melanoma patients with cerebral brain metastasis are limited and not effective to date. 4 Tumor cells with the potential to metastasize and colonize the brain may express distinctive molecular determinants that promote metastasis formation in this organ. They may also be able to respond to brain-derived growth factors or to deliver signals that alter the brain microenvironment, making it more supportive to metastasis development. 3 Prevention strategies for brain metastasis could be used if cells expressing such molecules could be identified in the primary melanoma. Currently, such molecular biomarkers are unknown.Human to mouse melanoma xenograft models that recapitulate the phenotypes seen in the clinic provide a valuable resource of cells for translational research and can accelerate drug discovery processes for this disease. 5 Current human melanoma brain metastasis models consist of xenografted cells inoculated into immune-deficient mice mainly by intracarotid or intracardiac administration. 6 Although these types of injections bypass the initial steps of brain metastasis

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood-brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain-metastasizing melanoma cells to express pro-inflammatory factors, including the cytokine IL-23, which was highly expressed by metastases-associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up-regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain-metastasizing melanoma cells. IL-23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL-23 could block this enhanced migration, implying a functional role for astrocyte-derived IL-23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL-23-induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro-inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells.

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We previously identified the chemokine receptor CCR4 as part of the molecular signature of melanoma brain metastasis. The aim of this study was to determine the functional significance of CCR4 in melanoma brain metastasis. We show that CCR4 is more highly expressed by brain metastasizing melanoma cells than by local cutaneous cells from the same melanoma. Moreover, we found that the expression of CCR4 is significantly higher in paired clinical specimens of melanoma metastases than in samples of primary tumors from the same patients. Notably, the expression of the CCR4 ligands, Ccl22 and Ccl17 is upregulated at the earliest stages of brain metastasis, and precedes the infiltration of melanoma cells to the brain. In-vitro, CCL17 induced migration and transendothelial migration of melanoma cells. Functionally, human melanoma cells over-expressing CCR4 were more tumorigenic and produced a higher load of spontaneous brain micrometastasis than control cells. Blocking CCR4 with a small molecule CCR4 antagonist in-vivo, reduced the tumorigenicity and micrometastasis formation of melanoma cells. Taken together, these findings implicate CCR4 as a driver of melanoma brain metastasis.

Brain metastases occur frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. Identification of molecular determinants regulating melanoma brain metastasis would advance the development of prevention and therapy strategies for this disease. Gene expression profiles of cutaneous and brain-metastasizing melanoma variants from three xenograft tumor models established in our laboratory revealed that expression of tight junction component CLDN1 was lower in the brain-metastasizing variants than in cutaneous variants from the same melanoma. The objective of our study was to determine the significance of CLDN1 downregulation/loss in metastatic melanoma and its role in melanoma brain metastasis. An immunohistochemical analysis of human cells of the melanocyte lineage indicated a significant CLDN1 downregulation in metastatic melanomas. Transduction of melanoma brain metastatic cells expressing low levels of CLDN1 with a CLDN1 retrovirus suppressed their metastatic phenotype. CLDN1-overexpressing melanoma cells expressed a lower ability to migrate and adhere to extracellular matrix, reduced tumor aggressiveness in nude mice and, most importantly, eliminated the formation of micrometastases in the brain. In sharp contrast, the ability of the CLDN1-overexpressing cells to form lung micrometastases was not impaired. CLDN1-mediated interactions between these cells and brain endothelial cells constitute the mechanism underlying these results. Taken together, we demonstrated that downregulation or loss of CLDN1 supports the formation of melanoma brain metastasis, and that CLDN1 expression could be a useful prognostic predictor for melanoma patients with a high risk of brain metastasis.Malignant melanoma has a high tendency to develop brain metastasis. Almost 40% of melanoma patients are treated for complications due to brain metastasis, and in additional 30-40% of patients CNS lesions are detected at autopsy. 1,2 Treatment options for melanoma patients with cerebral metastasis are limited and depend on the number and size of the lesions. In view of the poor survival rates of these patients, it is necessary to develop novel treatment strategies for the therapy of melanoma brain metastasis.

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