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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

NFκB signaling pathway is strikingly activated in P-MSCs. a 3D profile of RT2 Profiler™ PCR Array of Human Toll-Like Receptor Signaling Pathway. b Activation of NF-κB in P-MSC was determined by Western blotting. The fold difference in band intensities was quantified and indicated under the image using the software of AlphaView. c Representative pictures of cellular immunofluorescence staining for nuclear p65 in MSCs or P-MSC at 2 h. Immunocytochemistry staining was performed using an anti-p65 antibody (red) and Hoechst (blue) for nuclear staining. d Relative mRNA expression of IL-6, IL-8, and MCP-1. MSCs were pre-treatment with or without the NF-κB inhibitor (PDTC, 10 μM) at 37 °C for 1 h and then incubated with 200 μg/ml A549 cell-derived exosomes at 37 °C for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
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Fig4: NFκB signaling pathway is strikingly activated in P-MSCs. a 3D profile of RT2 Profiler™ PCR Array of Human Toll-Like Receptor Signaling Pathway. b Activation of NF-κB in P-MSC was determined by Western blotting. The fold difference in band intensities was quantified and indicated under the image using the software of AlphaView. c Representative pictures of cellular immunofluorescence staining for nuclear p65 in MSCs or P-MSC at 2 h. Immunocytochemistry staining was performed using an anti-p65 antibody (red) and Hoechst (blue) for nuclear staining. d Relative mRNA expression of IL-6, IL-8, and MCP-1. MSCs were pre-treatment with or without the NF-κB inhibitor (PDTC, 10 μM) at 37 °C for 1 h and then incubated with 200 μg/ml A549 cell-derived exosomes at 37 °C for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)

Mentions: To further investigate the signaling pathways involved in induction of PMSCs by A549 exosomes, we focused on TLR pathway as it has been reported to be closely related to inflammation. We analyzed gene-expression profiles of MSCs activated by or not by A549 exosomes through the use of a human toll-like receptor signaling pathway PCR array (see the Gene list at Table 1). According to the PCR array analysis (Fig. 4a), inflammation-related pathways including NFκB, JNK, and p38 signaling, were actually activated in P-MSCs. We then investigated the signaling events triggered by exosomes through Western blotting. As shown in Fig. 4b, a rapid activation of NFκB was detected by phosphorylation of Ikkα/β and the p65 subunit that peaked at 0.5–2 h after exosome exposure. Rapid activation of JNK, ERK, and p38 signaling was not detected (Fig. 4b). NFκB signaling activation involves phosphorylation of the p65 protein and translocation of the p65 protein to the cell nucleus. Therefore, we determined the localization of p65 in MSCs before and after exposure to A549 exosomes. As expected, A549 exosome stimulation resulted in a significant translocation of p65 from a cytoplasmic to a nuclear localization in Fig. 4c. Moreover, we demonstrated that NFκB inhibitor PDTC remarkably suppressed the enhanced expression of IL-6, IL-8, and MCP-1 induced by A549 exosomes in MSCs (Fig. 4d). Together, these results suggest that lung tumor-derived exosomes induce the generation of P-MSCs through activation of NFκB signaling pathway.Table 1


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)

NFκB signaling pathway is strikingly activated in P-MSCs. a 3D profile of RT2 Profiler™ PCR Array of Human Toll-Like Receptor Signaling Pathway. b Activation of NF-κB in P-MSC was determined by Western blotting. The fold difference in band intensities was quantified and indicated under the image using the software of AlphaView. c Representative pictures of cellular immunofluorescence staining for nuclear p65 in MSCs or P-MSC at 2 h. Immunocytochemistry staining was performed using an anti-p65 antibody (red) and Hoechst (blue) for nuclear staining. d Relative mRNA expression of IL-6, IL-8, and MCP-1. MSCs were pre-treatment with or without the NF-κB inhibitor (PDTC, 10 μM) at 37 °C for 1 h and then incubated with 200 μg/ml A549 cell-derived exosomes at 37 °C for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
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Fig4: NFκB signaling pathway is strikingly activated in P-MSCs. a 3D profile of RT2 Profiler™ PCR Array of Human Toll-Like Receptor Signaling Pathway. b Activation of NF-κB in P-MSC was determined by Western blotting. The fold difference in band intensities was quantified and indicated under the image using the software of AlphaView. c Representative pictures of cellular immunofluorescence staining for nuclear p65 in MSCs or P-MSC at 2 h. Immunocytochemistry staining was performed using an anti-p65 antibody (red) and Hoechst (blue) for nuclear staining. d Relative mRNA expression of IL-6, IL-8, and MCP-1. MSCs were pre-treatment with or without the NF-κB inhibitor (PDTC, 10 μM) at 37 °C for 1 h and then incubated with 200 μg/ml A549 cell-derived exosomes at 37 °C for 24 h (*P < 0.05, **P < 0.01, ***P < 0.001)
Mentions: To further investigate the signaling pathways involved in induction of PMSCs by A549 exosomes, we focused on TLR pathway as it has been reported to be closely related to inflammation. We analyzed gene-expression profiles of MSCs activated by or not by A549 exosomes through the use of a human toll-like receptor signaling pathway PCR array (see the Gene list at Table 1). According to the PCR array analysis (Fig. 4a), inflammation-related pathways including NFκB, JNK, and p38 signaling, were actually activated in P-MSCs. We then investigated the signaling events triggered by exosomes through Western blotting. As shown in Fig. 4b, a rapid activation of NFκB was detected by phosphorylation of Ikkα/β and the p65 subunit that peaked at 0.5–2 h after exosome exposure. Rapid activation of JNK, ERK, and p38 signaling was not detected (Fig. 4b). NFκB signaling activation involves phosphorylation of the p65 protein and translocation of the p65 protein to the cell nucleus. Therefore, we determined the localization of p65 in MSCs before and after exposure to A549 exosomes. As expected, A549 exosome stimulation resulted in a significant translocation of p65 from a cytoplasmic to a nuclear localization in Fig. 4c. Moreover, we demonstrated that NFκB inhibitor PDTC remarkably suppressed the enhanced expression of IL-6, IL-8, and MCP-1 induced by A549 exosomes in MSCs (Fig. 4d). Together, these results suggest that lung tumor-derived exosomes induce the generation of P-MSCs through activation of NFκB signaling pathway.Table 1

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