Nicotine promotes brain metastasis by activating microglia in vivo. (A) Expression of α4β2 nicotine receptor on human microglia (HMC3), mouse microglia (SIM-A9), human lung cancer cells (H2030BrM), and mouse lung cancer cells (LL/2) were evaluated by qRT-PCR (n = 3/group). (B) LL/2 (5×10⁴ cells) were intracardially injected into immune-competent BALB/c mice (n = 9). After 3 d of intracardiac transplantation of LL/2 cells, mice received nicotine treatment (1 mg/kg) by intraperitoneal injection every 3 d for 60 d. Upper panel: BLI images of brain metastatic lesions of representative mice from each experimental group (vehicle alone or nicotine treatment). Lower panel: total photon flux of ex vivo image of brain metastatic lesions was measured by BLI at the end point (day 60). Quantitative data of bone and brain metastasis of lung cancer are shown in the right panel. (C) At the end point, the brain sections from mice with or without treatment with nicotine were examined for Iba1⁺ signal (brown) on microglia. Scale bar, 20 µm. (D) Representative images of immunohistochemical analysis for CD206⁺ and Iba1⁺ microglia in the metastatic brain lesions of mice that were treated with or without nicotine (n = 9/group). Scale bar, 100 µm. (E) Mouse lung cancer LL/2 cells were intracranially injected into wild-type BALB/c mice (n = 9/group) followed by administering PLX3397 by intracranial injection. Upper panels are BLI images of brain metastatic lesions of representative mice from each experimental group at day 40. Lower panels represent the total photon flux of ex vivo brain metastatic lesions as measured by BLI at the end point (day 40). (F) Quantitative data of in vivo brain metastasis of lung cancer (n = 9/group). (G) Ex vivo BLI signals in the brain at the end point (n = 9/group). (H) Kaplan–Meier analysis of brain metastasis–free survival was performed (n = 9/group). (I and J) Metastatic brain tumors in E were isolated from the brain and were examined by FACS for M2 (I) and M1 (J) microglial polarization (n = 4 or 5/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus respective nicotine group. ##, P < 0.01 versus respective PLX3397 or Nico+PLX3397 group.