Simple SummaryAn estimated 60% of melanoma patients develop melanoma brain metastases (MBMs). However, the molecular factors that govern the growth of MBMs are still unknown. The excitatory neurotransmitter glutamate has been shown to control the proliferation of various types of cancer cells within the brain parenchyma, but the cellular sources and molecular mechanisms involved in this process remain unclear. By their well-known role in inhibiting synaptic glutamate release, cannabinoid CB1receptors (CB1Rs) located on glutamatergic nerve terminals are conceivably well-positioned to control the growth of MBMs. In silico data mining in cancer-genome atlases and in vitro studies with melanoma cell lines supported that a glutamate-NMDA receptor axis drives melanoma cell proliferation. Strikingly, grafting melanoma cells into the brain of mice lacking CB1Rs selectively in glutamatergic neurons increased tumour size and concomitantly activated NMDA receptors on tumour cells. Altogether, our findings reveal an unprecedented role of neuronal CB1Rs in controlling MBMs.AbstractMelanoma is one of the deadliest forms of cancer. Most melanoma deaths are caused by distant metastases in several organs, especially the brain, the so-called melanoma brain metastases (MBMs). However, the precise mechanisms that sustain the growth of MBMs remain elusive. Recently, the excitatory neurotransmitter glutamate has been proposed as a brain-specific, pro-tumorigenic signal for various types of cancers, but how neuronal glutamate shuttling onto metastases is regulated remains unknown. Here, we show that the cannabinoid CB1receptor (CB1R), a master regulator of glutamate output from nerve terminals, controls MBM proliferation. First, in silico transcriptomic analysis of cancer-genome atlases indicated an aberrant expression of glutamate receptors in human metastatic melanoma samples. Second, in vitro experiments conducted on three different melanoma cell lines showed that the selective blockade of glutamatergic NMDA receptors, but not AMPA or metabotropic receptors, reduces cell proliferation. Third, in vivo grafting of melanoma cells in the brain of mice selectively devoid of CB1Rs in glutamatergic neurons increased tumour cell proliferation in concert with NMDA receptor activation, whereas melanoma cell growth in other tissue locations was not affected. Taken together, our findings demonstrate an unprecedented regulatory role of neuronal CB1Rs in the MBM tumour microenvironment.