Isolation and Treatment of Glial Cells

Primary microglia and astrocytes were isolated and cultured from C57BL/6J and CX3CR1^GFP/wt [51 (link),52 (link)] mice as described before [53 (link),54 (link)]. After 10–14 days microglial cells were isolated from astrocytic monolayer and used for further experiments. A375 [55 (link)] (gifted by Simon Jasinski-Bergner, University Halle-Wittenberg, Halle (Saale), Germany), BV2 microglia [56 (link)] (obtained from Ullrich, University of Zürich, Zürich, Switzerland) and primary glial cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). LN229 [57 (link)] were cultured in RPMI medium (Lonza, Basel, Switzerland) with 10% FBS and 1% penicillin/streptomycin. After two days, the medium was collected from confluent astrocytes or BV2 microglia, filtered (Sarstedt, Nümbrecht, Germany), and applied on the tumor cells in a 1:1 ratio with the respective culture medium. This medium was added 3 h before starting the imaging for both single cell and collective migration experiments.
For cannabinoid treatment cannabidiol (5 µM, Tocris Bioscience, Bristol, UK) [39 (link),58 (link),59 (link)], tetrahydrocannabinol (5 µM, Tocris) [42 (link)] or a combination of both was applied 3 h before the start of the experiments. THC and CBD were both dissolved in DMSO.