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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

Hsp70 on exosomes contribute to cytokine production through TLR2. a mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with proteinase K (5 μg/ml) at 37 °C for 1 h and then treated at 95 °C for 10 min after proteinase K treatment to abolish its activity. Proteinase K treatment alone was used as another control. b mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with or without anti-HSP70 antibody at 37 °C for 1 h. 10 μg/ml anti-HSP70 was used for blocking. c mRNA expression changes of IL-6,IL-8, and MCP-1 in MSCs treated with different concentrations of rHSP70 proteins for 24 h. d mRNA expression changes of IL-6, IL-8, and MCP-1 in MSCs treated with rhHSP70 after knockdown of TLR2 by siRNA for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
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Fig6: Hsp70 on exosomes contribute to cytokine production through TLR2. a mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with proteinase K (5 μg/ml) at 37 °C for 1 h and then treated at 95 °C for 10 min after proteinase K treatment to abolish its activity. Proteinase K treatment alone was used as another control. b mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with or without anti-HSP70 antibody at 37 °C for 1 h. 10 μg/ml anti-HSP70 was used for blocking. c mRNA expression changes of IL-6,IL-8, and MCP-1 in MSCs treated with different concentrations of rHSP70 proteins for 24 h. d mRNA expression changes of IL-6, IL-8, and MCP-1 in MSCs treated with rhHSP70 after knockdown of TLR2 by siRNA for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)

Mentions: To find out whether proteins on exosomes were responsible for PMSCs induction, we treated exosomes with proteinase K before exposure to MSCs. Interestingly, diminished expression of IL-6, IL-8, andMCP-1 could be observed after treatments with proteinase K (Fig. 6a), suggesting that proteins on exosomes were related with MSCs cytokine production. A variety of molecules have been classified as TLR2 ligands including Versican, HMGB1, and heat shock proteins (HSP). HSP70 is highly expressed on A549 exosomes and it was reported to be a TLR2 ligand [24]. We then evaluated whether A549 exosomes induced generation of P-MSCs through HSP70 or not. Neutralizing antibody of HSP70 could partially suppress the enhanced expression of IL-6, IL-8, and MCP-1 induced by A549 exosomes in MSCs (Fig. 6b). Recombinant human HSP70 (rHSP70) added to MSCs culture mimicked the effect of A549 exosomes in a dose-dependent manner (Fig. 6c). Moreover, interfering TLR2 expression with siRNA could attenuate the effects caused by rHSP70 (Fig. 6d). Altogether, these results indicate that Hsp70 expressed on the surface of exosomes could trigger TLR2 signaling in MSCs.Fig. 6


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)

Hsp70 on exosomes contribute to cytokine production through TLR2. a mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with proteinase K (5 μg/ml) at 37 °C for 1 h and then treated at 95 °C for 10 min after proteinase K treatment to abolish its activity. Proteinase K treatment alone was used as another control. b mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with or without anti-HSP70 antibody at 37 °C for 1 h. 10 μg/ml anti-HSP70 was used for blocking. c mRNA expression changes of IL-6,IL-8, and MCP-1 in MSCs treated with different concentrations of rHSP70 proteins for 24 h. d mRNA expression changes of IL-6, IL-8, and MCP-1 in MSCs treated with rhHSP70 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

Fig6: Hsp70 on exosomes contribute to cytokine production through TLR2. a mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with proteinase K (5 μg/ml) at 37 °C for 1 h and then treated at 95 °C for 10 min after proteinase K treatment to abolish its activity. Proteinase K treatment alone was used as another control. b mRNA expression changes of IL-6, IL-8, and MCP-1. Exosomes were pre-treated with or without anti-HSP70 antibody at 37 °C for 1 h. 10 μg/ml anti-HSP70 was used for blocking. c mRNA expression changes of IL-6,IL-8, and MCP-1 in MSCs treated with different concentrations of rHSP70 proteins for 24 h. d mRNA expression changes of IL-6, IL-8, and MCP-1 in MSCs treated with rhHSP70 after knockdown of TLR2 by siRNA for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
Mentions: To find out whether proteins on exosomes were responsible for PMSCs induction, we treated exosomes with proteinase K before exposure to MSCs. Interestingly, diminished expression of IL-6, IL-8, andMCP-1 could be observed after treatments with proteinase K (Fig. 6a), suggesting that proteins on exosomes were related with MSCs cytokine production. A variety of molecules have been classified as TLR2 ligands including Versican, HMGB1, and heat shock proteins (HSP). HSP70 is highly expressed on A549 exosomes and it was reported to be a TLR2 ligand [24]. We then evaluated whether A549 exosomes induced generation of P-MSCs through HSP70 or not. Neutralizing antibody of HSP70 could partially suppress the enhanced expression of IL-6, IL-8, and MCP-1 induced by A549 exosomes in MSCs (Fig. 6b). Recombinant human HSP70 (rHSP70) added to MSCs culture mimicked the effect of A549 exosomes in a dose-dependent manner (Fig. 6c). Moreover, interfering TLR2 expression with siRNA could attenuate the effects caused by rHSP70 (Fig. 6d). Altogether, these results indicate that Hsp70 expressed on the surface of exosomes could trigger TLR2 signaling in MSCs.Fig. 6

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