Figure 4.
Nicotine-mediated microglial polarization promotes tumor stemness.(A) Effect of human microglial cells (HMC3) treated with or without nicotine (1 µM), and collection of CM. Brain metastatic lung cancer cells (H2030BrM and PC9BrM) were treated with the non-nicotine and nicotine CM for 24 h, and their sphere-forming ability was measured. Left panel: representative images. Arrowheads indicate sphere formation. Right panel: quantification of number of spheres. Non-nicotine or nicotine CM: microglia cells were treated with PBS or nicotine (1 µM) for 24 h, and they were washed with PBS and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for 24 h (n = 4/group). Scale bar, 10 µm. (B) Effect of the human microglial CM on CSC markers, ESA+/CD44+, and ALDH activity were examined using FACS (n = 4/group). (C and D) Expressions of stemness genes in H2030BrM (C) and in PC9BrM cells (D) were examined after the treatment with or without nicotine-derived human microglial CM by qRT-PCR (n = 6/group). (E) Colony-forming abilities of H2030BrM and PC9BrM cells were measured in the presence or absence of the CM derived from nicotine-treated human microglia. Left panel: representative images. Right panel: quantification of number of colonies (n = 4/group). (F) Expressions of IGF-1 and CCL20 receptors in H2030BrM cells were examined after the treatment of cells with or without nicotine-derived human microglial CM by qRT-PCR (n = 4/group). (G) Effect of recombinant proteins, CCL20 and IGF-1, on sphere formation ability of H2030BrM was measured (n = 3/group). (H) H2030BrM cells were treated with CCL20 or IGF-1 for 24 h followed by measuring the ESA+/CD44+ stem cell population by FACS (n = 4/group). (I and J) H2030BrM cells were treated with or without 20 µM of CCL20 (I) or 1 µM of IGF-1 (J) for 24 h, and the expressions of stemness genes were examined by qRT-PCR and Western blot (n = 4/group). (K) Colony-forming assay was performed for the same set of samples as in G and H (n = 4/group). (L) Human microglia were treated with or without nicotine (1 µM) for 24 h, and their CM was prepared. The CM was then added with anti-CCL20 antibody to deplete CCL20. H2030BrM cells were then treated with the CCL20-depleted CM followed by assaying the ESA+/CD44+ stem cell population by FACS (n = 4/group). (M and N) Metastatic lesions in the brain and serum were prepared from the mice used in Fig. 2 E. The protein expression of CCL20 in the brain (M) and in the serum (N) in each group was measured by ELISA (n = 6/group). (O) The protein expression of CCL20 in the serum of lung cancer patients with or without smoking history were measured by ELISA (n = 10/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Nicotine-mediated microglial polarization promotes tumor stemness. (A) Effect of human microglial cells (HMC3) treated with or without nicotine (1 µM), and collection of CM. Brain metastatic lung cancer cells (H2030BrM and PC9BrM) were treated with the non-nicotine and nicotine CM for 24 h, and their sphere-forming ability was measured. Left panel: representative images. Arrowheads indicate sphere formation. Right panel: quantification of number of spheres. Non-nicotine or nicotine CM: microglia cells were treated with PBS or nicotine (1 µM) for 24 h, and they were washed with PBS and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for 24 h (n = 4/group). Scale bar, 10 µm. (B) Effect of the human microglial CM on CSC markers, ESA+/CD44+, and ALDH activity were examined using FACS (n = 4/group). (C and D) Expressions of stemness genes in H2030BrM (C) and in PC9BrM cells (D) were examined after the treatment with or without nicotine-derived human microglial CM by qRT-PCR (n = 6/group). (E) Colony-forming abilities of H2030BrM and PC9BrM cells were measured in the presence or absence of the CM derived from nicotine-treated human microglia. Left panel: representative images. Right panel: quantification of number of colonies (n = 4/group). (F) Expressions of IGF-1 and CCL20 receptors in H2030BrM cells were examined after the treatment of cells with or without nicotine-derived human microglial CM by qRT-PCR (n = 4/group). (G) Effect of recombinant proteins, CCL20 and IGF-1, on sphere formation ability of H2030BrM was measured (n = 3/group). (H) H2030BrM cells were treated with CCL20 or IGF-1 for 24 h followed by measuring the ESA+/CD44+ stem cell population by FACS (n = 4/group). (I and J) H2030BrM cells were treated with or without 20 µM of CCL20 (I) or 1 µM of IGF-1 (J) for 24 h, and the expressions of stemness genes were examined by qRT-PCR and Western blot (n = 4/group). (K) Colony-forming assay was performed for the same set of samples as in G and H (n = 4/group). (L) Human microglia were treated with or without nicotine (1 µM) for 24 h, and their CM was prepared. The CM was then added with anti-CCL20 antibody to deplete CCL20. H2030BrM cells were then treated with the CCL20-depleted CM followed by assaying the ESA+/CD44+ stem cell population by FACS (n = 4/group). (M and N) Metastatic lesions in the brain and serum were prepared from the mice used in Fig. 2 E. The protein expression of CCL20 in the brain (M) and in the serum (N) in each group was measured by ELISA (n = 6/group). (O) The protein expression of CCL20 in the serum of lung cancer patients with or without smoking history were measured by ELISA (n = 10/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

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