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Melanoma has the highest propensity to metastasize to the brain compared to other cancers, as brain metastases are found frequently high in patients who have prolonged survival with visceral metastasis. Once disseminated in the brain, melanoma cells communicate with brain resident cells that include astrocytes and microglia. Microglia cells are the resident macrophages of the brain and are the main immunological cells in the CNS involved in neuroinflammation. Data on the interactions between brain metastatic melanoma cells and microglia and on the role of microglia-mediated neuroinflammation in facilitating melanoma brain metastasis are lacking. To elucidate the role of microglia in melanoma brain metastasis progression, we examined the bidirectional interactions between microglia and melanoma cells in the tumor microenvironment. We identified the molecular and functional modifications occurring in brain-metastasizing melanoma cells and microglia cells after the treatment of each cell type with supernatants of the counter cell type. Both cells induced alteration in gene expression programs, cell signaling, and cytokine secretion in the counter cell type. Moreover, melanoma cells exerted significant morphological changes on microglia cells, enhanced proliferation, induced matrix metalloproteinase-2 (MMP-2) activation, and cell migration. Microglia cells induced phenotypic changes in melanoma cells increasing their malignant phenotype: increased melanoma proliferation, MMP-2 activity, cell migration, brain endothelial penetration, and tumor cells ability to grow as spheroids in 3D cultures. Our work provides a novel insight into the bidirectional interactions between melanoma and micoglia cells, suggesting the contribution of microglia to melanoma brain metastasis formation.

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.

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.