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Angiogenic activity of breast cancer patients' monocytes reverted by combined use of systems modeling and experimental approaches.

Guex N, Crespo I, Bron S, Ifticene-Treboux A, Faes-Van't Hull E, Kharoubi S, Liechti R, Werffeli P, Ibberson M, Majo F, Nicolas M, Laurent J, Garg A, Zaman K, Lehr HA, Stevenson BJ, Rüegg C, Coukos G, Delaloye JF, Xenarios I, Doucey MA - PLoS Comput. Biol. (2015)

Bottom Line: Angiogenesis plays a key role in tumor growth and cancer progression.In silico predicted perturbations were validated experimentally using patient TEM.In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity.

View Article: PubMed Central - PubMed

Affiliation: The Vital-IT, SIB (Swiss Institute of Bioinformatics), University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Angiogenesis plays a key role in tumor growth and cancer progression. TIE-2-expressing monocytes (TEM) have been reported to critically account for tumor vascularization and growth in mouse tumor experimental models, but the molecular basis of their pro-angiogenic activity are largely unknown. Moreover, differences in the pro-angiogenic activity between blood circulating and tumor infiltrated TEM in human patients has not been established to date, hindering the identification of specific targets for therapeutic intervention. In this work, we investigated these differences and the phenotypic reversal of breast tumor pro-angiogenic TEM to a weak pro-angiogenic phenotype by combining Boolean modelling and experimental approaches. Firstly, we show that in breast cancer patients the pro-angiogenic activity of TEM increased drastically from blood to tumor, suggesting that the tumor microenvironment shapes the highly pro-angiogenic phenotype of TEM. Secondly, we predicted in silico all minimal perturbations transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. In silico predicted perturbations were validated experimentally using patient TEM. In addition, gene expression profiling of TEM transitioned to a weak pro-angiogenic phenotype confirmed that TEM are plastic cells and can be reverted to immunological potent monocytes. Finally, the relapse-free survival analysis showed a statistically significant difference between patients with tumors with high and low expression values for genes encoding transitioning proteins detected in silico and validated on patient TEM. In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity. Results showed the successful in vitro reversion of such an activity by perturbation of in silico predicted target genes in tumor derived TEM, and indicated that targeting tumor TEM plasticity may constitute a novel valid therapeutic strategy in breast cancer.

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Synergistic and antagonistic effects of TNF-α, PlGF, ANG-2 and TGF-β on TEM pro-angiogenic phenotype.IvdTEM were exposed to different combinations of ligands and changes in the expression of receptors at their surface of was measured by flow cytometry 36 hours post-treatment and displayed as mean log2 ratios relative to untreated cells (A). Significant variations (P < 0.05, T test) in VEGFR-1 and TIE-2 expression in TEM are indicated with an asterisk in the heatmap. Changes in TEM pro-angiogenic activity in response to treatments was measures in vitro (B) and in vivo (C) using HUVEC sprouting assay and corneal vascularization assay respectively, 3 to 5 independent angiogenic assays were performed per condition. * P < 0.05, ** P < 0.01. (D) The secretion of cytokines and angiogenic factors in response to treatments was experimentally measured in the conditioned medium of the culture 36 hours post-treatments. The secretions of ivdTEM were mathematically inferred and displayed as mean log2 ratios relative to untreated cells. Angiogenic factors are boxed. Shown are cumulated data of 3 to 10 independent experiments (panels A and D), the corresponding experimental data and all P values are available in S2 and S3 Tables.
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pcbi.1004050.g002: Synergistic and antagonistic effects of TNF-α, PlGF, ANG-2 and TGF-β on TEM pro-angiogenic phenotype.IvdTEM were exposed to different combinations of ligands and changes in the expression of receptors at their surface of was measured by flow cytometry 36 hours post-treatment and displayed as mean log2 ratios relative to untreated cells (A). Significant variations (P < 0.05, T test) in VEGFR-1 and TIE-2 expression in TEM are indicated with an asterisk in the heatmap. Changes in TEM pro-angiogenic activity in response to treatments was measures in vitro (B) and in vivo (C) using HUVEC sprouting assay and corneal vascularization assay respectively, 3 to 5 independent angiogenic assays were performed per condition. * P < 0.05, ** P < 0.01. (D) The secretion of cytokines and angiogenic factors in response to treatments was experimentally measured in the conditioned medium of the culture 36 hours post-treatments. The secretions of ivdTEM were mathematically inferred and displayed as mean log2 ratios relative to untreated cells. Angiogenic factors are boxed. Shown are cumulated data of 3 to 10 independent experiments (panels A and D), the corresponding experimental data and all P values are available in S2 and S3 Tables.

Mentions: Our strategy was to expose TEM to several treatments to identify the ligands and pathways critically controlling their pro-angiogenic activity. TEM differentiated in vitro were exposed to angiogenic factors (VEGF, PlGF and ANG-1, ANG-2 which are the ligands of VEGFR-1 and TIE-2 respectively) in combination with either TGF-β or TNF-α and the changes in their phenotype, angiogenic activity and paracrine secretion profile were examined. These experimental results were used as the foundations for a computational model that would allow predicting treatments increasing or dampening TEM proangiogenic activity. First, changes in TEM phenotype were evaluated by flow cytometry 36h post treatment. Globally, treatments combined with TGF-β or TNF-α displayed a stronger impact on TEM phenotype than single treatments with however, the exception of TGF-β. Overall, CD11b, CD14, VEGFR-1 and TIE-2 expression displayed larger changes in response to treatment than CCR5, TNF-R1 and TGFBR-1 (Fig. 2A). A hallmark of TGF-β treatments was a strong decrease in VEGFR-1 and CD11b expression and an increase in TIE-2 expression (Fig. 2A). By contrast, TNF-α treatments had no impact on VEGFR-1 expression and TNF-α increased TIE-2 expression when combined with PlGF or ANG-2 (Fig. 2A).


Angiogenic activity of breast cancer patients' monocytes reverted by combined use of systems modeling and experimental approaches.

Guex N, Crespo I, Bron S, Ifticene-Treboux A, Faes-Van't Hull E, Kharoubi S, Liechti R, Werffeli P, Ibberson M, Majo F, Nicolas M, Laurent J, Garg A, Zaman K, Lehr HA, Stevenson BJ, Rüegg C, Coukos G, Delaloye JF, Xenarios I, Doucey MA - PLoS Comput. Biol. (2015)

Synergistic and antagonistic effects of TNF-α, PlGF, ANG-2 and TGF-β on TEM pro-angiogenic phenotype.IvdTEM were exposed to different combinations of ligands and changes in the expression of receptors at their surface of was measured by flow cytometry 36 hours post-treatment and displayed as mean log2 ratios relative to untreated cells (A). Significant variations (P < 0.05, T test) in VEGFR-1 and TIE-2 expression in TEM are indicated with an asterisk in the heatmap. Changes in TEM pro-angiogenic activity in response to treatments was measures in vitro (B) and in vivo (C) using HUVEC sprouting assay and corneal vascularization assay respectively, 3 to 5 independent angiogenic assays were performed per condition. * P < 0.05, ** P < 0.01. (D) The secretion of cytokines and angiogenic factors in response to treatments was experimentally measured in the conditioned medium of the culture 36 hours post-treatments. The secretions of ivdTEM were mathematically inferred and displayed as mean log2 ratios relative to untreated cells. Angiogenic factors are boxed. Shown are cumulated data of 3 to 10 independent experiments (panels A and D), the corresponding experimental data and all P values are available in S2 and S3 Tables.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359163&req=5

pcbi.1004050.g002: Synergistic and antagonistic effects of TNF-α, PlGF, ANG-2 and TGF-β on TEM pro-angiogenic phenotype.IvdTEM were exposed to different combinations of ligands and changes in the expression of receptors at their surface of was measured by flow cytometry 36 hours post-treatment and displayed as mean log2 ratios relative to untreated cells (A). Significant variations (P < 0.05, T test) in VEGFR-1 and TIE-2 expression in TEM are indicated with an asterisk in the heatmap. Changes in TEM pro-angiogenic activity in response to treatments was measures in vitro (B) and in vivo (C) using HUVEC sprouting assay and corneal vascularization assay respectively, 3 to 5 independent angiogenic assays were performed per condition. * P < 0.05, ** P < 0.01. (D) The secretion of cytokines and angiogenic factors in response to treatments was experimentally measured in the conditioned medium of the culture 36 hours post-treatments. The secretions of ivdTEM were mathematically inferred and displayed as mean log2 ratios relative to untreated cells. Angiogenic factors are boxed. Shown are cumulated data of 3 to 10 independent experiments (panels A and D), the corresponding experimental data and all P values are available in S2 and S3 Tables.
Mentions: Our strategy was to expose TEM to several treatments to identify the ligands and pathways critically controlling their pro-angiogenic activity. TEM differentiated in vitro were exposed to angiogenic factors (VEGF, PlGF and ANG-1, ANG-2 which are the ligands of VEGFR-1 and TIE-2 respectively) in combination with either TGF-β or TNF-α and the changes in their phenotype, angiogenic activity and paracrine secretion profile were examined. These experimental results were used as the foundations for a computational model that would allow predicting treatments increasing or dampening TEM proangiogenic activity. First, changes in TEM phenotype were evaluated by flow cytometry 36h post treatment. Globally, treatments combined with TGF-β or TNF-α displayed a stronger impact on TEM phenotype than single treatments with however, the exception of TGF-β. Overall, CD11b, CD14, VEGFR-1 and TIE-2 expression displayed larger changes in response to treatment than CCR5, TNF-R1 and TGFBR-1 (Fig. 2A). A hallmark of TGF-β treatments was a strong decrease in VEGFR-1 and CD11b expression and an increase in TIE-2 expression (Fig. 2A). By contrast, TNF-α treatments had no impact on VEGFR-1 expression and TNF-α increased TIE-2 expression when combined with PlGF or ANG-2 (Fig. 2A).

Bottom Line: Angiogenesis plays a key role in tumor growth and cancer progression.In silico predicted perturbations were validated experimentally using patient TEM.In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity.

View Article: PubMed Central - PubMed

Affiliation: The Vital-IT, SIB (Swiss Institute of Bioinformatics), University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Angiogenesis plays a key role in tumor growth and cancer progression. TIE-2-expressing monocytes (TEM) have been reported to critically account for tumor vascularization and growth in mouse tumor experimental models, but the molecular basis of their pro-angiogenic activity are largely unknown. Moreover, differences in the pro-angiogenic activity between blood circulating and tumor infiltrated TEM in human patients has not been established to date, hindering the identification of specific targets for therapeutic intervention. In this work, we investigated these differences and the phenotypic reversal of breast tumor pro-angiogenic TEM to a weak pro-angiogenic phenotype by combining Boolean modelling and experimental approaches. Firstly, we show that in breast cancer patients the pro-angiogenic activity of TEM increased drastically from blood to tumor, suggesting that the tumor microenvironment shapes the highly pro-angiogenic phenotype of TEM. Secondly, we predicted in silico all minimal perturbations transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. In silico predicted perturbations were validated experimentally using patient TEM. In addition, gene expression profiling of TEM transitioned to a weak pro-angiogenic phenotype confirmed that TEM are plastic cells and can be reverted to immunological potent monocytes. Finally, the relapse-free survival analysis showed a statistically significant difference between patients with tumors with high and low expression values for genes encoding transitioning proteins detected in silico and validated on patient TEM. In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity. Results showed the successful in vitro reversion of such an activity by perturbation of in silico predicted target genes in tumor derived TEM, and indicated that targeting tumor TEM plasticity may constitute a novel valid therapeutic strategy in breast cancer.

Show MeSH
Related in: MedlinePlus