Interactions between cancer cells and glial cells play a crucial role in the formation of brain metastases; however, there are few methods to stably analyse these events in vitro over a long period of time. Here, we develop a simple and stable culture method, termed mixed-glial culture on/in soft substrate (MGS), which favourably mimics the brain microenvironment for various types of cancer. Utilizing this method, we discover that lung cancer cells become overly dependent on metabotropic glutamate receptor 1 (mGluR1) signalling in the brain microenvironment. Mechanistically, interactions with reactive astrocytes induce mGluR1 in cancer cells through the Wnt-5a / prickle planar cell polarity protein 1 (PRICKLE1) / RE1-silencing transcription factor (REST) axis. Induced mGluR1 interacts directly with epidermal growth factor receptor (EGFR), which activates extracellular signal-regulated kinase (ERK) in a glutamate-dependent manner. Notably, mGluR1 may also be an alternative target for osimertinib-resistant brain metastatic lung cancer. Our results demonstrate that MGS is a useful platform for investigating cancer-glia interactions, and that increased dependence on mGluR1 signalling may be one of the adaptation strategies, but also a vulnerability of brain metastatic lung cancer cells.