Figure 5.
Nicotine suppresses innate anti-tumor immune function of microglia.(A) Human microglia cells (HMC3) were treated with or without nicotine (1 µM) for 24 h, and the expression of SIRPα was examined by qRT-PCR and Western blot (n = 4/group). (B) H2030BrM and PC9BrM cells were treated with or without nicotine (1 µM) for 24 h, and the expression of CD47 was examined by qRT-PCR and Western blot (n = 4/group). (C) Representative images of immunohistochemical analysis for CD47+ cells in the metastatic brain lesions of lung cancer patients with or without smoking history. Arrows indicate CD47+ cells. Non-smoker (n = 4) and smoker (n = 7). Scale bar, 50 µm. (D) Phagocytic activities of HMC3 human microglial cells (green) with or without nicotine treatment were examined using PKH26-labeled H2030BrM cells (red). Both cells were incubated together for 0 or 24 h and photographed (left panels), and the number of microglia that phagocytosed tumor cells was counted to quantify microglial phagocytic activity as shown in the right panel (n = 4/group). Scale bar, 10 µm. (E) A parallel set of cells in D was analyzed by FACS to quantify phagocytic abilities of microglia after nicotine or STATTIC treatment. Upper panels: representative FACS data. Lower panel: statistical analysis of the FACS data (n = 4/group). (F) Mouse microglia (SIM-A9; green) were incubated with PKH26-labeled LL/2 mouse lung cancer cells (red) for 24 h. They were then photographed under a fluorescent microscope (n = 4/group). Scale bar, 100 μm. (G) Phagocytic abilities of microglia with or without nicotine treatment were examined by incubating mouse microglia with PKH26-labeled LL/2 mouse lung cancer cells for 24 h, followed by analysis of the cells by FACS (n = 4). (H) The metastatic lesions in the brain of mice in Fig. 2 E were sectioned, and they were subjected to immunohistochemical analysis for SIRPα and Iba1. Representative immunohistochemistry images of the microglia are shown (n = 9/group). Scale bar, 100 µm. (I and J) The metastatic lesions in the brain of mice in Fig. 2 E were subjected to FACS analysis for CD47+ tumor cell (I) and SIRPα/CD11b+ (J) microglia cells (n = 9/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Nicotine suppresses innate anti-tumor immune function of microglia. (A) Human microglia cells (HMC3) were treated with or without nicotine (1 µM) for 24 h, and the expression of SIRPα was examined by qRT-PCR and Western blot (n = 4/group). (B) H2030BrM and PC9BrM cells were treated with or without nicotine (1 µM) for 24 h, and the expression of CD47 was examined by qRT-PCR and Western blot (n = 4/group). (C) Representative images of immunohistochemical analysis for CD47+ cells in the metastatic brain lesions of lung cancer patients with or without smoking history. Arrows indicate CD47+ cells. Non-smoker (n = 4) and smoker (n = 7). Scale bar, 50 µm. (D) Phagocytic activities of HMC3 human microglial cells (green) with or without nicotine treatment were examined using PKH26-labeled H2030BrM cells (red). Both cells were incubated together for 0 or 24 h and photographed (left panels), and the number of microglia that phagocytosed tumor cells was counted to quantify microglial phagocytic activity as shown in the right panel (n = 4/group). Scale bar, 10 µm. (E) A parallel set of cells in D was analyzed by FACS to quantify phagocytic abilities of microglia after nicotine or STATTIC treatment. Upper panels: representative FACS data. Lower panel: statistical analysis of the FACS data (n = 4/group). (F) Mouse microglia (SIM-A9; green) were incubated with PKH26-labeled LL/2 mouse lung cancer cells (red) for 24 h. They were then photographed under a fluorescent microscope (n = 4/group). Scale bar, 100 μm. (G) Phagocytic abilities of microglia with or without nicotine treatment were examined by incubating mouse microglia with PKH26-labeled LL/2 mouse lung cancer cells for 24 h, followed by analysis of the cells by FACS (n = 4). (H) The metastatic lesions in the brain of mice in Fig. 2 E were sectioned, and they were subjected to immunohistochemical analysis for SIRPα and Iba1. Representative immunohistochemistry images of the microglia are shown (n = 9/group). Scale bar, 100 µm. (I and J) The metastatic lesions in the brain of mice in Fig. 2 E were subjected to FACS analysis for CD47+ tumor cell (I) and SIRPα/CD11b+ (J) microglia cells (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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