STUDY OF EARLY MELANOMA BRAIN METASTASIS MECHANISMS USING IN VITRO AND IN VIVO MODELS OF TUMOR INVASION

Of all skin cancers, melanoma is the most fatal. Of all cancer types, melanoma is also the cancer with the highest level of brain tropism. Approximately 50% of patients with stage IV melanoma are diagnosed with melanoma brain metastases. A percentage that rises when postmortem patients are also taken into account. Following lung cancer and breast cancer, melanoma is the leading cause of malignant metastasis to the central nervous system. Of all metastatic brain tumors, melanoma represents 6-12% of cases. The overall survival rate following a diagnosis of melanoma brain metastases has been historically low. However, over the past ten years, advances in targeted therapies as well as in immunotherapies have significantly improved the survival rate of patients with advanced melanoma. Melanoma brain metastases most frequently occur at the junction between the gray and the white matter and in the frontal lobe. In order to reach the brain parenchyma, metastases must cross the brain vasculature. The specific properties of the blood vessels that perfuse the central nervous system are referred to as the blood-brain barrier. They allow these vessels to finely regulate the flow of cells, ions and molecules between the bloodstream and the brain parenchyma in order to preserve brain homeostasis for the proper functioning of neurons and the protection of the brain against toxic and pathogenic agents. Abnormalities in this functional interfacing barrier that separates the brain from the bloodstream are a critical element in the development and progression of several neurological pathologies. A poor understanding of the early mechanisms of metastasis crossing the blood-brain barrier constitutes an obstacle to the development of effective preventive therapeutic strategies as well as a particularly challenging domain of interest as it is one of the most crucial and least documented steps in the metastasizing process to the brain. Here, we focused on the ideation and consequent creation of effective in vitro and in vivo models to help identify and characterize as meticulously as possible, the players that are implicated in the crossing of melanoma metastases through the blood-brain barrier to reach the brain parenchyma. We used human immortalized cells (endothelial cells, pericytes and astrocytes) in triple coculture to recreate a blood-brain barrier in vitro and be able to investigate eventual changes in the gene expression of the tumor cells crossing the model. In parallel, we have set up an in vivo murine model to recreate the process of brain metastasis by injecting melanoma tumor cells into the left ventricle of the heart and thus be able to study the early stages of blood-brain
barrier invasion. The analysis of the murine tissues was performed by Correlative light-electron microscopy (CLEM) and the results obtained revealed the presence of cells in the brain that present artifacts that have the same appearance as melanosomes. Experiments using focused ion beam scanning electron microscopes (FIB-SEM) as well as nanoscale secondary ion mass spectrometry (NanoSIMS) may be conducted to take the investigation further.