Figure 6.
PTL suppresses brain tumor progression by blocking nicotine-induced M2 microglia polarization. (A) Human microglia cells (HMC3) with the Arg1 reporter plasmid were cultured in the presence or absence of compounds that were identified as the top three most effective inhibitors for Arg1 during our initial screening (see Materials and methods). After 48 h of incubation, luciferase reporter activity was measured (n = 4/group). (B) Expression of surface markers of M1/M2 microglia was examined by qRT-PCR after microglial cells were treated with or without nicotine plus PTL (n = 4/group). (C) The same set of samples in B was evaluated for quantification of Iba1+/CD11b+ (M1) cells and Iba1+/CD206+ (M2) cells by FACS (n = 4/group). (D) The same set of samples in C was examined for quantification of the protein expression of JAK2 and STAT3 by Western blot. (E) Human microglial (HMC3) cells (green) with or without nicotine treatment (1 µM) in the presence or absence of PTL (1 µM) were incubated with PKH26-labeled H2030BrM cells (red) for 24 h and photographed (left panels), followed by measurement of the microglial phagocytic activity (right panel; n = 4/group). Scale bar, 10 µm. (F) CM was prepared from human microglia (HMC3) treated with or without nicotine and PTL. The CM was added to the culture of H2030BrM, and cells were incubated for 48 h followed by evaluation of CSC population by FACS. Non-nicotine, nicotine, or Nico+PTL CM: microglia were treated with PBS, nicotine, or nicotine plus PTL for 24 h. They were then washed twice with PBS and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for a further 24 h (n = 4/group). (G) For the same set of samples as F, colony-forming ability was also measured (n = 4/group). (H) Human microglia were treated with or without nicotine (1 µM) in the presence or absence of PTL for 24 h, followed by assessment of the expression of CCL20 by qRT-PCR (n = 4/group). (I) The mouse lung cancer cells, LL/2, were intracardially injected into wild-type BALB/c mice. After 3 d of intracranial transplantation of LL/2 cells, mice received nicotine (1 mg/kg) plus PTL (1 mg/kg) by an intraperitoneal injection every 3 d for 40 d. Upper panel: BLI images of representative mice from each experimental group at day 40. Lower panel: total photon flux of ex vivo brain metastatic lesions was measured by BLI at the endpoint (day 40; n = 9/group). (J) Quantitative data of BLI in the brain regions are shown (n = 9/group). (K) Ex vivo signals in the whole brains at the end point were quantified. (L) The Kaplan–Meier analysis of brain metastasis–free survival was performed (n = 9/group). (M and N) Metastatic brain tumors in I were isolated from the brain and were examined by FACS for M2 (M) and M1 (N) microglial polarization (n = 9/group). (O) A proposed model illustrating a nicotine-induced brain metastasis (n = 9/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. Refer to the image caption for details.

PTL suppresses brain tumor progression by blocking nicotine-induced M2 microglia polarization. (A) Human microglia cells (HMC3) with the Arg1 reporter plasmid were cultured in the presence or absence of compounds that were identified as the top three most effective inhibitors for Arg1 during our initial screening (see Materials and methods). After 48 h of incubation, luciferase reporter activity was measured (n = 4/group). (B) Expression of surface markers of M1/M2 microglia was examined by qRT-PCR after microglial cells were treated with or without nicotine plus PTL (n = 4/group). (C) The same set of samples in B was evaluated for quantification of Iba1+/CD11b+ (M1) cells and Iba1+/CD206+ (M2) cells by FACS (n = 4/group). (D) The same set of samples in C was examined for quantification of the protein expression of JAK2 and STAT3 by Western blot. (E) Human microglial (HMC3) cells (green) with or without nicotine treatment (1 µM) in the presence or absence of PTL (1 µM) were incubated with PKH26-labeled H2030BrM cells (red) for 24 h and photographed (left panels), followed by measurement of the microglial phagocytic activity (right panel; n = 4/group). Scale bar, 10 µm. (F) CM was prepared from human microglia (HMC3) treated with or without nicotine and PTL. The CM was added to the culture of H2030BrM, and cells were incubated for 48 h followed by evaluation of CSC population by FACS. Non-nicotine, nicotine, or Nico+PTL CM: microglia were treated with PBS, nicotine, or nicotine plus PTL for 24 h. They were then washed twice with PBS and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for a further 24 h (n = 4/group). (G) For the same set of samples as F, colony-forming ability was also measured (n = 4/group). (H) Human microglia were treated with or without nicotine (1 µM) in the presence or absence of PTL for 24 h, followed by assessment of the expression of CCL20 by qRT-PCR (n = 4/group). (I) The mouse lung cancer cells, LL/2, were intracardially injected into wild-type BALB/c mice. After 3 d of intracranial transplantation of LL/2 cells, mice received nicotine (1 mg/kg) plus PTL (1 mg/kg) by an intraperitoneal injection every 3 d for 40 d. Upper panel: BLI images of representative mice from each experimental group at day 40. Lower panel: total photon flux of ex vivo brain metastatic lesions was measured by BLI at the endpoint (day 40; n = 9/group). (J) Quantitative data of BLI in the brain regions are shown (n = 9/group). (K) Ex vivo signals in the whole brains at the end point were quantified. (L) The Kaplan–Meier analysis of brain metastasis–free survival was performed (n = 9/group). (M and N) Metastatic brain tumors in I were isolated from the brain and were examined by FACS for M2 (M) and M1 (N) microglial polarization (n = 9/group). (O) A proposed model illustrating a nicotine-induced brain metastasis (n = 9/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

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