Nicotine promotes brain metastasis in mouse model. (A) Expression of the α4β2 nicotine receptor on human microglia (HMC3) and human monocyte/macrophage (THP-1) were evaluated before and after nicotine treatment by qRT-PCR (n = 4/group). (B) Mouse lung cancer LL/2 cells were intracardially injected into wild-type C57BL/6 mice (n = 9/group). After 3 d of intracardiac transplantation of LL/2 cells, mice received nicotine (1 mg/kg) treatment by intraperitoneal injection every 3 d. Upper panels are BLI images of brain metastatic lesions of representative mice from each experimental group at day 40. The lower panels represent the total photon flux of ex vivo brain metastatic lesions as measured by BLI at the endpoint (day 40). (C) Quantitative data of in vivo brain metastasis of lung cancer (n = 9/group). (D) Percentage of brain metastasis of lung cancer in C57BL/6 mice with or without nicotine treatment (n = 9/group). (E and F) Quantitative data of in vivo brain metastasis of lung cancer (left panel) and ex vivo signals in the brain at the end point (right panel) of the experiment conducted in Fig. 2 B (n = 9/group). (G and H) Metastatic brain tumor tissues in Fig. 2 B were dissociated, and the effect of nicotine was examined on Iba1+/CD206+ (G) or Iba1+/F4/80+ cells (H) by FACS (n = 9/group). (I) Metastatic brain tumor tissues in Fig. 2 B were dissociated, and the Iba1+/CD45− (microglia) and Iba1+/CD45+ (macrophage) were measured and plotted in relation to the nicotine treatment (n = 9/group). (J) Scheme of the experimental design. After 3 d of intracranial transplantation of LL/2 cells, the mice received nicotine (1 mg/kg) by intraperitoneal injection every 3 d. After 1 wk of tumor transplantation, PLX3397 (20 mg/kg in 7 µl PBS) was directly injected into the right brain of the mice every 2 wk. (K) Upper panels are BLI images of brain metastatic lesions of representative mice from each experimental group at day 40. The lower panels represent the total photon flux of ex vivo brain metastatic lesions as measured by BLI at the endpoint (day 40; n = 9/group). (L) Quantitative data of ex vivo brain metastasis of lung cancer at the end point. (M and N) Metastatic brain tumors in K were isolated from the brain and examined by FACS for M2 (M) and M1 (N) microglial polarization (n = 9/group). (O) H2030BrM (2 × 105 cells) were intracardially injected into nude mice (n = 9/group) followed by administering them with nicotine plus PLX3397 (1 mg/kg) by intraperitoneal injection every 3 d. Upper and middle panels are BLI images of brain metastatic lesions of representative mice from each experimental group at day 0 and day 60, respectively. Lower panels represent the total photon flux of ex vivo brain metastatic lesions as measured by BLI at the endpoint (day 60; n = 9/group). (P and Q) Quantitative data of in vivo brain metastasis of lung cancer and (Q) ex vivo signals in the brain at the end point (day 60; n = 9/group). (R) Kaplan–Meier analysis of brain metastasis–free survival was performed. (S) Serum of mice in Fig. 2 E was collected, and an aspartate aminotransferase test (AST) was measured using the AST activity assay kit (Sigma-Aldrich) (n = 9/group). (T) H2030BrM cells were incubated with the indicated concentration of nicotine for 24 h. They were then examined for cell viability (n = 9/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 and Nico+PLX3397 groups.