Fig. 5. NMDAR activity mediates colonization but not seeding of brain metastases.

a, Bio-luminescent imaging (BLI) and quantification of brain metastatic lesions formed by MDA231-BrM cells transfected with doxycycline (DOX) inducible shCTRL or shGluN2B. DOX food was supplied concomitantly with ICD injections and metastasis burden was assessed 4 weeks later by BLI. Two-tailed Student t-test, mean ± s.e.m., n = 10 mice per group, two independent experiments.

b, Brain metastasis-free survival of mice seeded by intracardiac inoculation with MDA231-BrM cells transfected with shCTRL or shGluN2B. DOX food was supplied concomitantly with ICD injection. Two-sided log-rank test.

c, Evaluation of brain seeding capability of luciferase⁺ shGluN2B vs. shCTRL MDA231-BrM. DOX food was supplied concomitantly with ICD injection; mice were sacrificed at day 7. Every 10^th brain section was stained with luciferase antibody, and all luciferase⁺ cells were counted. Representative images show single luciferase⁺ B2BM cells (brown staining; black arrowhead). Two-tailed Student t-test, mean ± s.e.m., n = 3 mouse brains per group. Scale bar, 100 μm.

d, Evaluation of Luciferase⁺/Ki67⁺ shGluN2B vs. shCTRL MDA231-BrM cells after initial colonization of brain-metastatic lesions. DOX food was supplied 7 days after ICD injection; mice were sacrificed at day 14. Every 10^th brain section was stained with luciferase and Ki67 antibody, and all luciferase⁺ and Ki67⁺ cells were counted. Representative images show luciferase (green) and Ki67 (red) staining. DAPI; nuclear staining. Two-tailed Student t-test, mean ± s.e.m., n = 3 mouse brains per group. Scale bar, 100 μm.

e, Bio-luminescent imaging (BLI) and quantification of brain metastatic growth by MDA231-BrM cells transfected with inducible shGluN2B or shCTRL. DOX food was supplied at day 7 after ICD injection, and continued for 3 weeks, until day 28. Two-tailed Student t-test, mean ± s.e.m., n = 9 mice for shCTRL, and 8 mice for shGluN2B.