Figure S3.
Nicotine treatment of microglia increases secretion of CCL20 and IGF-1 followed by promotion of tumor cell stemness.(A) Human microglial (HMC3) cells were treated with PBS or nicotine (1 µM) for 24 h. CM was collected and analyzed by using the cytokine/growth factor array (Raybio). (B) Human microglial (HMC3) cells were treated with or without nicotine (1 µM) plus STATTIC (0.5 µM) for 24 h. Cells were washed with PBS twice and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for additional 24 h. CM was collected and analyzed by IGF-1 and CCL20 ELISA (n = 4/group). (C) Human microglial (HMC3) cells and macrophage (THP-1) were treated with or without nicotine (1 µM) for 24 h. Cells were washed with PBS twice and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for an additional 24 h. CM was collected and analyzed by IGF-1 and CCL20 ELISA (n = 4/group). (D and E) Kaplan–Meier analysis was performed for examining relapse-free survival of lung cancer patients in relation to CCL20 (P = 0.02; D) and IGF-1 (P = 0.36; E) expression using the TCGA dataset. (F and G) Expressions of CCL20 (F) and CCR6 (G) were examined in lung tumor tissues from healthy individual and lung cancer patients using the GEO dataset (GSE22220). (H and I) Expression of CCL20 in human microglia and tumor cells (H) or mouse microglia and mouse tumor cells (I) were examined by qRT-PCR (n = 4/group). (J–L) H2030BrM cells were treated with or without nicotine or CM extracted from human microglia that were treated with nicotine for 24 h. These cells were examined for their colony-forming ability (J), CD44+/ESA+ CSC population (K), and mRNA expression of stemness-related genes (L; n = 4/group). (M) Effect of CM extracted from nicotine-treated mouse microglial cells on LL/2 mouse lung CSC population was examined by FACS. (N) Colony-forming ability of LL/2 was measured in the presence or absence of the CM derived from nicotine-treated microglia by qRT-PCR (n = 4/group). (O) LL/2 cells were incubated with the CM derived from nicotine-treated microglia for 24 h. They were then examined for cell viability (n = 4/group). (P and Q) Mouse microglia cells were treated with or without nicotine in the presence or absence of STAT3 inhibitor for 24 h. Cells were then examined for the expression of IGF-1 (P) or CCL20 (Q) by qRT-PCR. LL/2 cells were treated with CM derived from microglia that were treated with or without nicotine and/or STATTIC for 24 h (n = 4/group). (R and S) The cells were then examined for CD44+/ESA+ CSC population (R) and for cell viability (S; n = 4/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Nicotine treatment of microglia increases secretion of CCL20 and IGF-1 followed by promotion of tumor cell stemness. (A) Human microglial (HMC3) cells were treated with PBS or nicotine (1 µM) for 24 h. CM was collected and analyzed by using the cytokine/growth factor array (Raybio). (B) Human microglial (HMC3) cells were treated with or without nicotine (1 µM) plus STATTIC (0.5 µM) for 24 h. Cells were washed with PBS twice and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for additional 24 h. CM was collected and analyzed by IGF-1 and CCL20 ELISA (n = 4/group). (C) Human microglial (HMC3) cells and macrophage (THP-1) were treated with or without nicotine (1 µM) for 24 h. Cells were washed with PBS twice and incubated in the fresh DMEM/F12 medium supplemented with 2% FBS for an additional 24 h. CM was collected and analyzed by IGF-1 and CCL20 ELISA (n = 4/group). (D and E) Kaplan–Meier analysis was performed for examining relapse-free survival of lung cancer patients in relation to CCL20 (P = 0.02; D) and IGF-1 (P = 0.36; E) expression using the TCGA dataset. (F and G) Expressions of CCL20 (F) and CCR6 (G) were examined in lung tumor tissues from healthy individual and lung cancer patients using the GEO dataset (GSE22220). (H and I) Expression of CCL20 in human microglia and tumor cells (H) or mouse microglia and mouse tumor cells (I) were examined by qRT-PCR (n = 4/group). (J–L) H2030BrM cells were treated with or without nicotine or CM extracted from human microglia that were treated with nicotine for 24 h. These cells were examined for their colony-forming ability (J), CD44+/ESA+ CSC population (K), and mRNA expression of stemness-related genes (L; n = 4/group). (M) Effect of CM extracted from nicotine-treated mouse microglial cells on LL/2 mouse lung CSC population was examined by FACS. (N) Colony-forming ability of LL/2 was measured in the presence or absence of the CM derived from nicotine-treated microglia by qRT-PCR (n = 4/group). (O) LL/2 cells were incubated with the CM derived from nicotine-treated microglia for 24 h. They were then examined for cell viability (n = 4/group). (P and Q) Mouse microglia cells were treated with or without nicotine in the presence or absence of STAT3 inhibitor for 24 h. Cells were then examined for the expression of IGF-1 (P) or CCL20 (Q) by qRT-PCR. LL/2 cells were treated with CM derived from microglia that were treated with or without nicotine and/or STATTIC for 24 h (n = 4/group). (R and S) The cells were then examined for CD44+/ESA+ CSC population (R) and for cell viability (S; n = 4/group). The data are presented as the mean ± SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

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