Limits...
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

P-MSCs promote tumor growth in vivo. a Tumor promoting effects of P-MSCs in a xenograft mouse model. PMSC + A549, n = 6. 2 × 106 A549 cells and 2 × 105 exosomes-treated MSCs were injected. MSC + A549, n = 4. 2 × 106 A549 cells and 2 × 105 DF12-treated MSCs were injected. A549, n = 3, only 2 × 106 A549 cells were injected. b the tumors in each group were dissected and tumor diameters were measured at 2 weeks after subcutaneous cell injection. c The macrophage infiltration was examined by F4/80 immunohistochemical staining of the tumor tissues harvested 2 weeks after A549 cell inoculation. Representatives of F4/80 staining from each group are shown (magnification 200×). d F4/80 + macrophages were quantitated and the data represent the mean number of F4/80+ macrophages per 200 × field (three fields per group). e Tumor cell proliferation was examined by Ki67 immunohistochemical staining of the tumor tissues. Representatives of Ki67 staining from each group are shown (magnification 200×). f Ki67 staining-positive cells were quantified and the data represent the mean number per 200 × field (three fields per group)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4836087&req=5

Fig3: P-MSCs promote tumor growth in vivo. a Tumor promoting effects of P-MSCs in a xenograft mouse model. PMSC + A549, n = 6. 2 × 106 A549 cells and 2 × 105 exosomes-treated MSCs were injected. MSC + A549, n = 4. 2 × 106 A549 cells and 2 × 105 DF12-treated MSCs were injected. A549, n = 3, only 2 × 106 A549 cells were injected. b the tumors in each group were dissected and tumor diameters were measured at 2 weeks after subcutaneous cell injection. c The macrophage infiltration was examined by F4/80 immunohistochemical staining of the tumor tissues harvested 2 weeks after A549 cell inoculation. Representatives of F4/80 staining from each group are shown (magnification 200×). d F4/80 + macrophages were quantitated and the data represent the mean number of F4/80+ macrophages per 200 × field (three fields per group). e Tumor cell proliferation was examined by Ki67 immunohistochemical staining of the tumor tissues. Representatives of Ki67 staining from each group are shown (magnification 200×). f Ki67 staining-positive cells were quantified and the data represent the mean number per 200 × field (three fields per group)

Mentions: We next determined whether the altered cytokine expression in P-MSCs is accompanied by changes in tumor-promoting capacity in vivo. Using a nude mouse model, we subcutaneously injected A549 cells with P-MSCs, MSCs or PBS.As shown in Fig. 3a, the size of tumors significantly increased in the presence of P-MSCs in respect to tumor alone or tumor co-injected with unstimulated MSCs. Such stimulation in tumor growth was confirmed by measurement of tumor diameter (Fig. 3b). Since MCP-1-mediated macrophage recruitment was relevant with tumor growth and angiogenesis [20], the tumors were excised for examination of immune cell infiltration. We observed that F4/80 macrophages were much more abundant in tumors receiving P-MSCs than those receiving un-stimulated MSCs (Fig. 3c, d). This tumor-promoting effect and the macrophage-recruiting function of P-MSCs corresponded well to their high expression levels of MCP-1 (Fig. 2a), the major macrophage chemokines. In addition, Ki67 staining also showed elevated cell proliferation rate of A549 cells co-administered with PMSCs (Fig. 3e, f). Thus, MSCs educated by A549 exosomes gained an enhanced capacity in recruiting macrophages and in promoting tumor growth in vivo.Fig. 3


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)

P-MSCs promote tumor growth in vivo. a Tumor promoting effects of P-MSCs in a xenograft mouse model. PMSC + A549, n = 6. 2 × 106 A549 cells and 2 × 105 exosomes-treated MSCs were injected. MSC + A549, n = 4. 2 × 106 A549 cells and 2 × 105 DF12-treated MSCs were injected. A549, n = 3, only 2 × 106 A549 cells were injected. b the tumors in each group were dissected and tumor diameters were measured at 2 weeks after subcutaneous cell injection. c The macrophage infiltration was examined by F4/80 immunohistochemical staining of the tumor tissues harvested 2 weeks after A549 cell inoculation. Representatives of F4/80 staining from each group are shown (magnification 200×). d F4/80 + macrophages were quantitated and the data represent the mean number of F4/80+ macrophages per 200 × field (three fields per group). e Tumor cell proliferation was examined by Ki67 immunohistochemical staining of the tumor tissues. Representatives of Ki67 staining from each group are shown (magnification 200×). f Ki67 staining-positive cells were quantified and the data represent the mean number per 200 × field (three fields per group)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836087&req=5

Fig3: P-MSCs promote tumor growth in vivo. a Tumor promoting effects of P-MSCs in a xenograft mouse model. PMSC + A549, n = 6. 2 × 106 A549 cells and 2 × 105 exosomes-treated MSCs were injected. MSC + A549, n = 4. 2 × 106 A549 cells and 2 × 105 DF12-treated MSCs were injected. A549, n = 3, only 2 × 106 A549 cells were injected. b the tumors in each group were dissected and tumor diameters were measured at 2 weeks after subcutaneous cell injection. c The macrophage infiltration was examined by F4/80 immunohistochemical staining of the tumor tissues harvested 2 weeks after A549 cell inoculation. Representatives of F4/80 staining from each group are shown (magnification 200×). d F4/80 + macrophages were quantitated and the data represent the mean number of F4/80+ macrophages per 200 × field (three fields per group). e Tumor cell proliferation was examined by Ki67 immunohistochemical staining of the tumor tissues. Representatives of Ki67 staining from each group are shown (magnification 200×). f Ki67 staining-positive cells were quantified and the data represent the mean number per 200 × field (three fields per group)
Mentions: We next determined whether the altered cytokine expression in P-MSCs is accompanied by changes in tumor-promoting capacity in vivo. Using a nude mouse model, we subcutaneously injected A549 cells with P-MSCs, MSCs or PBS.As shown in Fig. 3a, the size of tumors significantly increased in the presence of P-MSCs in respect to tumor alone or tumor co-injected with unstimulated MSCs. Such stimulation in tumor growth was confirmed by measurement of tumor diameter (Fig. 3b). Since MCP-1-mediated macrophage recruitment was relevant with tumor growth and angiogenesis [20], the tumors were excised for examination of immune cell infiltration. We observed that F4/80 macrophages were much more abundant in tumors receiving P-MSCs than those receiving un-stimulated MSCs (Fig. 3c, d). This tumor-promoting effect and the macrophage-recruiting function of P-MSCs corresponded well to their high expression levels of MCP-1 (Fig. 2a), the major macrophage chemokines. In addition, Ki67 staining also showed elevated cell proliferation rate of A549 cells co-administered with PMSCs (Fig. 3e, f). Thus, MSCs educated by A549 exosomes gained an enhanced capacity in recruiting macrophages and in promoting tumor growth in vivo.Fig. 3

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