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Lung tumor exosomes induce a pro-inflammatory phenotype in mesenchymal stem cells via NFκB-TLR signaling pathway.

Li X, Wang S, Zhu R, Li H, Han Q, Zhao RC - J Hematol Oncol (2016)

Bottom Line: Secretion of inflammation-associated cytokines in exosome-treated MSCs were analyzed by RT-PCR and ELISA.The growth-promoting effect of exosome-treated MSCs on lung tumor cells was evaluated by in vivo mouse xenograft model.We further found that Hsp70 present on the surface of lung tumor exosomes contributed to the induction of P-MSCs by A549 exosomes.

View Article: PubMed Central - PubMed

Affiliation: Center of Excellence in Tissue Engineering, Key Laboratory of Beijing, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China.

ABSTRACT

Background: In tumor microenvironment, a continuous cross-talk between cancer cells and other cellular components is required to sustain tumor progression. Accumulating evidence suggests that exosomes, a novel way of cell communication, play an important role in such cross-talk. Exosomes could facilitate the direct intercellular transfer of proteins, lipids, and miRNA/mRNA/DNAs between cells. Since mesenchymal stem cells (MSCs) can be attracted to tumor sites and become an important component of the tumor microenvironment, there is an urgent need to reveal the effect of tumor exosomes on MSCs and to further explore the underlying molecular mechanisms.

Methods: Exosomes were harvested from lung cancer cell line A549 and added to MSCs. Secretion of inflammation-associated cytokines in exosome-treated MSCs were analyzed by RT-PCR and ELISA. The growth-promoting effect of exosome-treated MSCs on lung tumor cells was evaluated by in vivo mouse xenograft model. Signaling pathway involved in exosomes-treated MSCs was detected by PCR array of human toll-like receptor signaling pathway, RT-PCR, and Western blot.

Results: Data showed that lung tumor cell A549-derived exosomes could induce a pro-inflammatory phenotype in MSCs named P-MSCs, which have significantly elevated secretion of IL-6, IL-8, and MCP-1. P-MSCs possess a greatly enhanced ability in promoting lung tumor growth in mouse xenograft model. Analysis of the signaling pathways in P-MSCs revealed a fast triggering of NF-κB. Genetic ablation of Toll-like receptor 2 (TLR2) by siRNA and TLR2-neutralizing antibody could block NF-κB activation by exosomes. We further found that Hsp70 present on the surface of lung tumor exosomes contributed to the induction of P-MSCs by A549 exosomes.

Conclusions: Our studies suggest a novel mechanism by which lung tumor cell-derived exosomes induce pro-inflammatory activity of MSCs which in turn get tumor supportive characteristics.

No MeSH data available.


Related in: MedlinePlus

Exosomes trigger cytokine production in MSCs through TLR2. a The expression level of TLR 1 ~ 10 of MSCs. GAPDH was used as control. b mRNA expression changes of TLR2, 7, 8 were detected by RT-PCR after treatment with exosomes. c mRNA expression changes of IL-6, IL-8, and MCP-1 of MSCs. MSCs were pre-treated withTLR2 neutralizing antibody or mouse IgG2b isotype control at 37 °C for 1 h and then incubated with exosomes for 24 h. d Western blotting analysis of NF-κB activation in P-MSCs. MSCs treated with mouse IgG2b isotype control was used as control. e The interfering efficiency of three pairs of TLR2 siRNAs was measured by RT-PCR. f mRNA expression changes of IL-6, IL-8, and MCP-1. MSCs were stimulated with exosomes after knockdown of TLR2 by siRNA for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
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Fig5: Exosomes trigger cytokine production in MSCs through TLR2. a The expression level of TLR 1 ~ 10 of MSCs. GAPDH was used as control. b mRNA expression changes of TLR2, 7, 8 were detected by RT-PCR after treatment with exosomes. c mRNA expression changes of IL-6, IL-8, and MCP-1 of MSCs. MSCs were pre-treated withTLR2 neutralizing antibody or mouse IgG2b isotype control at 37 °C for 1 h and then incubated with exosomes for 24 h. d Western blotting analysis of NF-κB activation in P-MSCs. MSCs treated with mouse IgG2b isotype control was used as control. e The interfering efficiency of three pairs of TLR2 siRNAs was measured by RT-PCR. f mRNA expression changes of IL-6, IL-8, and MCP-1. MSCs were stimulated with exosomes after knockdown of TLR2 by siRNA for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)

Mentions: The effects of exosomes on NFκB prompted us to study more closely the role of TLRs in exosome-mediated signaling. RT-PCR results showed that all TLRs were detectable within 33 cycles in MSCs (Fig. 5a). To determine which TLR was specifically responsible for tumor exosome-mediated cytokine up-regulation in MSCs, we first detected TLRs changes after exosome stimulation. We found that TLR2, TLR7, and TLR8 mRNA expression was significantly increased after exosomes stimulation (Fig. 5b). Here, we focused on TLR2 for three reasons. Firstly, MSCs have higher expression of TLR2 than TLR7 or TLR8 as demonstrated in Fig. 5a, although up-regulation of TLR7 and TLR8 is more significant (Fig. 5b). Secondly, TLR2 is localized on cell plasma membrane whereas TLR7 or TLR8 resides within endosomal compartments. Lastly, TLR2 can recognize lipopeptides while both TLR7 and TLR8 were shown to sense single-stranded viral RNA [21–23]. Using a TLR2 neutralizing antibody, we found that blockade of TLR2 in MSCs decreased the expression of inflammatory factors induced by lung tumor-derived exosomes and inhibited activation of NFκB signaling pathway (Fig. 5c, d).Fig. 5


Lung tumor exosomes induce a pro-inflammatory phenotype in mesenchymal stem cells via NFκB-TLR signaling pathway.

Li X, Wang S, Zhu R, Li H, Han Q, Zhao RC - J Hematol Oncol (2016)

Exosomes trigger cytokine production in MSCs through TLR2. a The expression level of TLR 1 ~ 10 of MSCs. GAPDH was used as control. b mRNA expression changes of TLR2, 7, 8 were detected by RT-PCR after treatment with exosomes. c mRNA expression changes of IL-6, IL-8, and MCP-1 of MSCs. MSCs were pre-treated withTLR2 neutralizing antibody or mouse IgG2b isotype control at 37 °C for 1 h and then incubated with exosomes for 24 h. d Western blotting analysis of NF-κB activation in P-MSCs. MSCs treated with mouse IgG2b isotype control was used as control. e The interfering efficiency of three pairs of TLR2 siRNAs was measured by RT-PCR. f mRNA expression changes of IL-6, IL-8, and MCP-1. MSCs were stimulated with exosomes after knockdown of TLR2 by siRNA for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836087&req=5

Fig5: Exosomes trigger cytokine production in MSCs through TLR2. a The expression level of TLR 1 ~ 10 of MSCs. GAPDH was used as control. b mRNA expression changes of TLR2, 7, 8 were detected by RT-PCR after treatment with exosomes. c mRNA expression changes of IL-6, IL-8, and MCP-1 of MSCs. MSCs were pre-treated withTLR2 neutralizing antibody or mouse IgG2b isotype control at 37 °C for 1 h and then incubated with exosomes for 24 h. d Western blotting analysis of NF-κB activation in P-MSCs. MSCs treated with mouse IgG2b isotype control was used as control. e The interfering efficiency of three pairs of TLR2 siRNAs was measured by RT-PCR. f mRNA expression changes of IL-6, IL-8, and MCP-1. MSCs were stimulated with exosomes after knockdown of TLR2 by siRNA for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
Mentions: The effects of exosomes on NFκB prompted us to study more closely the role of TLRs in exosome-mediated signaling. RT-PCR results showed that all TLRs were detectable within 33 cycles in MSCs (Fig. 5a). To determine which TLR was specifically responsible for tumor exosome-mediated cytokine up-regulation in MSCs, we first detected TLRs changes after exosome stimulation. We found that TLR2, TLR7, and TLR8 mRNA expression was significantly increased after exosomes stimulation (Fig. 5b). Here, we focused on TLR2 for three reasons. Firstly, MSCs have higher expression of TLR2 than TLR7 or TLR8 as demonstrated in Fig. 5a, although up-regulation of TLR7 and TLR8 is more significant (Fig. 5b). Secondly, TLR2 is localized on cell plasma membrane whereas TLR7 or TLR8 resides within endosomal compartments. Lastly, TLR2 can recognize lipopeptides while both TLR7 and TLR8 were shown to sense single-stranded viral RNA [21–23]. Using a TLR2 neutralizing antibody, we found that blockade of TLR2 in MSCs decreased the expression of inflammatory factors induced by lung tumor-derived exosomes and inhibited activation of NFκB signaling pathway (Fig. 5c, d).Fig. 5

Bottom Line: Secretion of inflammation-associated cytokines in exosome-treated MSCs were analyzed by RT-PCR and ELISA.The growth-promoting effect of exosome-treated MSCs on lung tumor cells was evaluated by in vivo mouse xenograft model.We further found that Hsp70 present on the surface of lung tumor exosomes contributed to the induction of P-MSCs by A549 exosomes.

View Article: PubMed Central - PubMed

Affiliation: Center of Excellence in Tissue Engineering, Key Laboratory of Beijing, Institute of Basic Medical Sciences and School of Basic Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China.

ABSTRACT

Background: In tumor microenvironment, a continuous cross-talk between cancer cells and other cellular components is required to sustain tumor progression. Accumulating evidence suggests that exosomes, a novel way of cell communication, play an important role in such cross-talk. Exosomes could facilitate the direct intercellular transfer of proteins, lipids, and miRNA/mRNA/DNAs between cells. Since mesenchymal stem cells (MSCs) can be attracted to tumor sites and become an important component of the tumor microenvironment, there is an urgent need to reveal the effect of tumor exosomes on MSCs and to further explore the underlying molecular mechanisms.

Methods: Exosomes were harvested from lung cancer cell line A549 and added to MSCs. Secretion of inflammation-associated cytokines in exosome-treated MSCs were analyzed by RT-PCR and ELISA. The growth-promoting effect of exosome-treated MSCs on lung tumor cells was evaluated by in vivo mouse xenograft model. Signaling pathway involved in exosomes-treated MSCs was detected by PCR array of human toll-like receptor signaling pathway, RT-PCR, and Western blot.

Results: Data showed that lung tumor cell A549-derived exosomes could induce a pro-inflammatory phenotype in MSCs named P-MSCs, which have significantly elevated secretion of IL-6, IL-8, and MCP-1. P-MSCs possess a greatly enhanced ability in promoting lung tumor growth in mouse xenograft model. Analysis of the signaling pathways in P-MSCs revealed a fast triggering of NF-κB. Genetic ablation of Toll-like receptor 2 (TLR2) by siRNA and TLR2-neutralizing antibody could block NF-κB activation by exosomes. We further found that Hsp70 present on the surface of lung tumor exosomes contributed to the induction of P-MSCs by A549 exosomes.

Conclusions: Our studies suggest a novel mechanism by which lung tumor cell-derived exosomes induce pro-inflammatory activity of MSCs which in turn get tumor supportive characteristics.

No MeSH data available.


Related in: MedlinePlus