Activation of Toll-like receptors signaling in non-small cell lung cancer cell line induced by tumor-associated macrophages.
BACKGROUND
Inflammation is often linked with the progress and poor outcome of lung cancer. The understanding of the relationship between tumor-associated macrophages (TAMs) and lung cancer cells involves in the underlying mechanism of inflammatory cytokine production. Toll-like receptors (TLRs) are engaged in promoting the production of pro-inflammatory cytokines and play an important role in tumor immunology.


METHODS
To investigate the mechanisms by which TAMs influence the production of pro-inflammatory cytokines in lung cancer cells, we established an in vitro coculture system using TAMs and human non-small cell lung cancer (NSCLC) cell line SPC-A1. Levels of interleukin (IL)-1β, IL-6 and IL-8 in SPC-A1 were evaluated by RT-PCR and cytometric bead array assay after being cocultured with TAMs. Expression changes of TLRs and TLRs signaling pathway proteins in SPC-A1 were further confirmed by RT-PCR and western blot. The level changes of IL-1β, IL-6 and IL-8 in SPC-A1 were also detected after the stimulation of TLRs agonists.


RESULTS
We found that the phenotype markers of TAMs were highly expressed after stimulating human monocyte cell line THP-1 by phorbol-12-myristate-13-acetate (PMA). Higher mRNA and supernate secretion levels of IL-1β, IL-6 and IL-8 were detected in SPC-A1 after being cocultured with TAMs. We also found that TLR1, TLR6 and TLR7 were up-regulated in SPC-A1 in the coculture system with TAMs. Meanwhile, TLRs signaling pathway proteins were also significantly activated. Moreover, pre-treatment with agonist ligands for TLR1, TLR6 and TLR7 could dramatically promote inductions of IL-1β, IL-6 and IL-8.


CONCLUSIONS
These findings demonstrated that TAMs may enhance IL-1β, IL-6 and IL-8 expressions via TLRs signaling pathway. We conclude that TAMs contribute to maintain the inflammation microenvironment and ultimately promote the development and progression of lung cancer.
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Xing
X
Ke
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Meng
M
Wu
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Jianfang
J
Lou
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Shuping
S
Zhang
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Peijun
P
Huang
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Ruihong
R
Sun
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Lei
L
Huang
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Erfu
E
Xie
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Fang
F
Wang
1 Department of Laboratory Medicine, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China ; 2 National Key Clinical Department of Laboratory Medicine, Nanjing 210029, China.
Bing
B
Gu
Toll-like receptors (TLRs); Tumor-associated macrophages (TAMs); non-small cell lung cancer (NSCLC); pro-inflammatory cytokines