Fig. 2.

Exogenous and astrocyte-released brain-derived neurotrophic factor (BDNF) binds Tropomyosin-related kinase B (TrkB) leading to increased tumor cell proliferation. a Real-time PCR and western blot analysis of TrkB knockdown with two different short hairpin (sh)RNAs in BBM1 cells. Control was non-transduced BBM1 cells. b Effect of exogenous BDNF (25 ng/mL) on BBM1 (BDNF) and TrkB knockdown BBM1 (shTrkB) cell proliferation over 12 days in vitro. Control was non-treated cells (n = 3; ****p < 0.0001; bars indicate SEM). c Western blot analysis of PI3K pathway activation by exogenous BDNF (25 ng/mL) over 24 h in BBM1 and TrkB knockdown BBM1 (BBM1-KD) cells. d Immunohistochemical staining of glial fibrillary acidic proteins (GFAP) and BDNF in the peritumoral region in a human breast cancer brain metastasis (BBM) specimen. GFAP (green), BDNF (red), DAPI (blue). e ELISA quantification of BDNF released from BBM1 cells, astrocytes, and fibroblasts (nc medium only; ****p < 0.0001). f Proliferation of BBM1 (left) and TrkB knockdown BBM1 (BBM1-KD) (right) cultured with control DMEM (Control), astrocyte-conditioned medium (Astrocyte CM), or fibroblast-conditioned medium (Fibroblast CM) for 12 days in vitro (n = 3; **p < 0.01, ****p < 0.0001; bars indicate SEM). g Phosphorylation of TrkB in BBM1 and TrkB knockdown BBM1 (BBM1-KD) cells grown in astrocyte-conditioned medium (CM)