a, Schematic of cancer cell–primary neuron co-culture system and experimental design. b, Immunofluorescence staining for MAP2 (neuronal marker, green) and GFAP (astrocyte marker, red) in primary cultures of cortical neurons, revealing the preponderance of neurons. The area circumscribed by the white box is shown at higher magnification (left). Scale bars, 1 µm (left), 10 µm (right). Three independent experiments. c, Representative bright-field (BF) and fluorescent (mKate+) images (left) and quantification (right) of MDA231 parental and MDA231-BrM cells co-cultured with primary cortical neurons for 14 days. Two-tailed Student’s t-test, mean ± s.e.m., n = 3 biological replicates over three independent experiments. Scale bar, 100 µm. d, Representative bright-field and fluorescent images (left) and quantification (right) of TS1 parental and TS1-BrM cells co-cultured with primary cortical neurons for 14 days. Data are mean ± s.e.m. Student’s t-test (two-tailed) was used. n = 3 biological replicates over three independent experiments. e–g, Quantification of MDA231 parental and MDA231-BrM cell numbers after 14 days of culture in poly-d-lysine-coated plates in three different conditions: in complete neuronal culture medium (e); in conditioned medium from primary cortical neuron cultures (f); and in a Boyden chamber with neurons in the top chamber and cancer cells in the bottom chamber (g). Mean ± s.e.m. Student’s t-test (two-tailed) was used. n = 3 biological replicates over three independent experiments. The panels below each bar graph show representative images of the cancer cells at the end of the assay, revealed by mKate fluorescence. h, Schematic inducible miR-E-based shRNA knock-down system carrying three distinct miR-E-based shRNA sequences that bind to different regions of a targeted mRNA (top vector), and tet-on inducible vector for GRIN2B open reading frame (ORF) used for rescue experiments (bottom vector). TRE, tet-on inducible promoter; rtTA, reverse tetracycline transactivator. i, Knockdown of GRIN2B in cultured MDA231-BrM cells with tet-on inducible shRNAs, as assessed by western blotting after DOX treatment (1 µg ml−1) for two days. Three independent experiments. The numbers above the blots indicate levels of pGluN2B(Y1252) and total GluN2B protein normalized to GAPDH. GAPDH was run in the same gel as pGluN2B(Y1252), and run on separate gels from GluN2B as sample processing controls. j, Representative bright-field and fluorescent images (bottom) and quantification (top) of MDA231-BrM cells transfected with inducible shCtrl and shGRIN2B (1 µg ml−1), co-cultured with primary cortical neurons for 14 days. Two-tailed Student’s t-test; mean ± s.e.m., n = 3 biological replicates over three independent experiments. Scale bar, 100 µm. k, Rescue expression of GluN2B in cultured MDA231-BrM cells with tet-on inducible shRNAs along with a GRIN2B cDNA, as assessed by western blotting after DOX treatment (1 µg ml−1) for two days. Three independent experiments. GAPDH was run in the same gel as GluN2B. l, Representative fluorescent (mKate+) images (left), and quantification (right) of MDA231-BrM shCtrl, shGRIN2B and GluN2B-rescued cells co-cultured with primary cortical neurons for 14 days. Two-tailed Student’s t-test; mean ± s.e.m., n = 3 biological replicates over three independent experiments. Scale bar, 100 µm. m, Cell proliferation in shCTRL and shGRIN2B MDA231-BrM cells as determined by MTT assays, starting with 5,000 or 10,000 cells per well, after 72 h in culture. Two-way ANOVA; mean ± s.e.m., n = 3 independent experiments. Source data