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MDSCs mediate angiogenesis and predispose canine mammary tumor cells for metastasis via IL-28/IL-28RA (IFN-λ) signaling.

Mucha J, Majchrzak K, Taciak B, Hellmén E, Król M - PLoS ONE (2014)

Bottom Line: Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling.The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs.Thus, IL-28 may constitute an interesting target for further therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - WULS, Warsaw, Poland.

ABSTRACT

Background: Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods: Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results: In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions: We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.

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Number of MDSCs in mammary tumor-bearing dogs, their IL-28 expression and changes in canine mammary tumor cells gene/protein expression due to their co-culture with MDSCs.(A). The number of circulating MDSCs (%) within white blood cells in healthy donors, and dogs with stage I, II, III and IV mammary cancer. Results that differed significantly compared to control are marked by ‘a’ (P<0.01) or ‘b’ (P<0.001). (B). Volcano plot of gene expression in canine mammary tumor cells co-cultured with MDSCs compared to monocultured cells. Genes which expression differed significantly (P<0.05, FC>2.0) are marked by blue dots. Il-28ra is marked as a red dot. The plot was generated using BRB software. (C). Changes in expression of selected genes in canine mammary tumor cells due to co-culture with MDSCs visualized on agarose gel using UV light (ctrl, control cells grown as mono-culture; +MDSC, cells grown as co-culture with MDSCs). (D). Relative Il-28ra gene expression in canine mammary tumor control cells transfected with non-coding siRNA (ctrl), cells co-cultured with MDSCs (+MDSC), transfected with Il-28ra-specific siRNA (siRNA) and treated with IL-28 (IL28). Results that differ significantly compared to control are marked as ‘a’ (P<0.001), ‘b’ (P<0.05) or ‘c’ (P<0.01). (E). Relative gene expression of Il-28 in MDSCs isolated from blood of healthy dogs, and dogs with stage I/II or III/IV mammary cancer. (F). Expression of selected target and downstream signaling proteins in control cells mock-transfected (ctrl), cells with knockdown of IL-28RA expression (siRNA) and cells treated with IL-28 (IL28).
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pone-0103249-g001: Number of MDSCs in mammary tumor-bearing dogs, their IL-28 expression and changes in canine mammary tumor cells gene/protein expression due to their co-culture with MDSCs.(A). The number of circulating MDSCs (%) within white blood cells in healthy donors, and dogs with stage I, II, III and IV mammary cancer. Results that differed significantly compared to control are marked by ‘a’ (P<0.01) or ‘b’ (P<0.001). (B). Volcano plot of gene expression in canine mammary tumor cells co-cultured with MDSCs compared to monocultured cells. Genes which expression differed significantly (P<0.05, FC>2.0) are marked by blue dots. Il-28ra is marked as a red dot. The plot was generated using BRB software. (C). Changes in expression of selected genes in canine mammary tumor cells due to co-culture with MDSCs visualized on agarose gel using UV light (ctrl, control cells grown as mono-culture; +MDSC, cells grown as co-culture with MDSCs). (D). Relative Il-28ra gene expression in canine mammary tumor control cells transfected with non-coding siRNA (ctrl), cells co-cultured with MDSCs (+MDSC), transfected with Il-28ra-specific siRNA (siRNA) and treated with IL-28 (IL28). Results that differ significantly compared to control are marked as ‘a’ (P<0.001), ‘b’ (P<0.05) or ‘c’ (P<0.01). (E). Relative gene expression of Il-28 in MDSCs isolated from blood of healthy dogs, and dogs with stage I/II or III/IV mammary cancer. (F). Expression of selected target and downstream signaling proteins in control cells mock-transfected (ctrl), cells with knockdown of IL-28RA expression (siRNA) and cells treated with IL-28 (IL28).

Mentions: FACS analysis showed that the number of MDSCs (CD11b+/Gr1+/CD33+) in the leukocyte population in the blood of healthy dogs was 0.35 (SD = 0.48). In dogs with stage I or II mammary cancer, MDSC number was significantly higher (P<0.01): 5.94 (SD = 2.99) and 8.13 (SD = 0.54), respectively. However, the number of MDSCs in dogs with stage III and IV of mammary cancer was the highest (P<0.001): 15.63 (SD = 3.91) and 15.44 (SD = 2.92), respectively (Fig. 1A).


MDSCs mediate angiogenesis and predispose canine mammary tumor cells for metastasis via IL-28/IL-28RA (IFN-λ) signaling.

Mucha J, Majchrzak K, Taciak B, Hellmén E, Król M - PLoS ONE (2014)

Number of MDSCs in mammary tumor-bearing dogs, their IL-28 expression and changes in canine mammary tumor cells gene/protein expression due to their co-culture with MDSCs.(A). The number of circulating MDSCs (%) within white blood cells in healthy donors, and dogs with stage I, II, III and IV mammary cancer. Results that differed significantly compared to control are marked by ‘a’ (P<0.01) or ‘b’ (P<0.001). (B). Volcano plot of gene expression in canine mammary tumor cells co-cultured with MDSCs compared to monocultured cells. Genes which expression differed significantly (P<0.05, FC>2.0) are marked by blue dots. Il-28ra is marked as a red dot. The plot was generated using BRB software. (C). Changes in expression of selected genes in canine mammary tumor cells due to co-culture with MDSCs visualized on agarose gel using UV light (ctrl, control cells grown as mono-culture; +MDSC, cells grown as co-culture with MDSCs). (D). Relative Il-28ra gene expression in canine mammary tumor control cells transfected with non-coding siRNA (ctrl), cells co-cultured with MDSCs (+MDSC), transfected with Il-28ra-specific siRNA (siRNA) and treated with IL-28 (IL28). Results that differ significantly compared to control are marked as ‘a’ (P<0.001), ‘b’ (P<0.05) or ‘c’ (P<0.01). (E). Relative gene expression of Il-28 in MDSCs isolated from blood of healthy dogs, and dogs with stage I/II or III/IV mammary cancer. (F). Expression of selected target and downstream signaling proteins in control cells mock-transfected (ctrl), cells with knockdown of IL-28RA expression (siRNA) and cells treated with IL-28 (IL28).
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pone-0103249-g001: Number of MDSCs in mammary tumor-bearing dogs, their IL-28 expression and changes in canine mammary tumor cells gene/protein expression due to their co-culture with MDSCs.(A). The number of circulating MDSCs (%) within white blood cells in healthy donors, and dogs with stage I, II, III and IV mammary cancer. Results that differed significantly compared to control are marked by ‘a’ (P<0.01) or ‘b’ (P<0.001). (B). Volcano plot of gene expression in canine mammary tumor cells co-cultured with MDSCs compared to monocultured cells. Genes which expression differed significantly (P<0.05, FC>2.0) are marked by blue dots. Il-28ra is marked as a red dot. The plot was generated using BRB software. (C). Changes in expression of selected genes in canine mammary tumor cells due to co-culture with MDSCs visualized on agarose gel using UV light (ctrl, control cells grown as mono-culture; +MDSC, cells grown as co-culture with MDSCs). (D). Relative Il-28ra gene expression in canine mammary tumor control cells transfected with non-coding siRNA (ctrl), cells co-cultured with MDSCs (+MDSC), transfected with Il-28ra-specific siRNA (siRNA) and treated with IL-28 (IL28). Results that differ significantly compared to control are marked as ‘a’ (P<0.001), ‘b’ (P<0.05) or ‘c’ (P<0.01). (E). Relative gene expression of Il-28 in MDSCs isolated from blood of healthy dogs, and dogs with stage I/II or III/IV mammary cancer. (F). Expression of selected target and downstream signaling proteins in control cells mock-transfected (ctrl), cells with knockdown of IL-28RA expression (siRNA) and cells treated with IL-28 (IL28).
Mentions: FACS analysis showed that the number of MDSCs (CD11b+/Gr1+/CD33+) in the leukocyte population in the blood of healthy dogs was 0.35 (SD = 0.48). In dogs with stage I or II mammary cancer, MDSC number was significantly higher (P<0.01): 5.94 (SD = 2.99) and 8.13 (SD = 0.54), respectively. However, the number of MDSCs in dogs with stage III and IV of mammary cancer was the highest (P<0.001): 15.63 (SD = 3.91) and 15.44 (SD = 2.92), respectively (Fig. 1A).

Bottom Line: Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling.The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs.Thus, IL-28 may constitute an interesting target for further therapies.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences - WULS, Warsaw, Poland.

ABSTRACT

Background: Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods: Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results: In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions: We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.

Show MeSH
Related in: MedlinePlus