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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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Phenotypical signature of pro-angiogenic TEM.(A) In vivo corneal vascularization assay to assess the pro-angiogenic activity of TEM isolated from peripheral blood and tumor of breast cancer patients. Bright field pictures of the eyes and fluorescent microscopy images of sagittal sections of the eyes stained with CD31 (stains specifically blood vessel endothelial cells) and Dapi (stains cell nucleus) are shown. Double-head and single arrows depict cornea and iris respectively. Corneas of control eyes were injected with buffer alone and show similar vascularization to uninjected eyes. Note the presence of blood vessels in the cornea injected with tumor TEM (double-head arrow) and its absence in the corneas injected with buffer (control) or blood TEM. Shown are representative data of 10 experiments. Bars in bright field and fluorescent images are 500 and 100 μm respectively. Bar graph represents a quantification of the vascular network of the cornea and the iris. (B) Secretion profile of cytokines and angiogenic factors in TEM isolated from patient blood and tumor. Angiogenic factors are boxed. Shown are cumulated data of 5 experiments, significant variations (P < 0.05) are indicated with an asterisk. (C) Workflow diagram of the strategy combining experimental and computational approaches to discover anti-angiogenic therapies. Green: experiments using ivdTEM. Blue, experiments using patient TEM; red: computational approach.
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pcbi.1004050.g001: Phenotypical signature of pro-angiogenic TEM.(A) In vivo corneal vascularization assay to assess the pro-angiogenic activity of TEM isolated from peripheral blood and tumor of breast cancer patients. Bright field pictures of the eyes and fluorescent microscopy images of sagittal sections of the eyes stained with CD31 (stains specifically blood vessel endothelial cells) and Dapi (stains cell nucleus) are shown. Double-head and single arrows depict cornea and iris respectively. Corneas of control eyes were injected with buffer alone and show similar vascularization to uninjected eyes. Note the presence of blood vessels in the cornea injected with tumor TEM (double-head arrow) and its absence in the corneas injected with buffer (control) or blood TEM. Shown are representative data of 10 experiments. Bars in bright field and fluorescent images are 500 and 100 μm respectively. Bar graph represents a quantification of the vascular network of the cornea and the iris. (B) Secretion profile of cytokines and angiogenic factors in TEM isolated from patient blood and tumor. Angiogenic factors are boxed. Shown are cumulated data of 5 experiments, significant variations (P < 0.05) are indicated with an asterisk. (C) Workflow diagram of the strategy combining experimental and computational approaches to discover anti-angiogenic therapies. Green: experiments using ivdTEM. Blue, experiments using patient TEM; red: computational approach.

Mentions: The angigoenic profile of TEM was investigated in a group of 40 newly diagnosed breast cancer patients (Table 1). We characterized by flow cytometry the phenotype of TEM from patient peripheral blood and freshly dissociated tumor specimens obtained at time of surgery (see Material and Methods). Based on our immunostaining and flow cytometry protocol we observed that TEM did not constitute a distinct subset of monocytes. In contrast, all monocytes showed expression of TIE-2, which was particularly low in patient blood and substantially higher on monocytes isolated from tumor tissue (S1 Fig. and Table 2). Thus, CD11b+, CD14+ monocytes from patient blood and tumor tissue were referred to as “TEM” and compared with respect to receptor and cytokine expression. However, tumor TEM co-expressed VEGFR-1 and TGFR-1 at significantly higher levels compared to peripheral blood TEM (Table 2). We next assess the pro-angiogenic activity of TEM using the in vivo corneal vascularization assay [39]. The cornea itself is avascular and was injected with TEM isolated from patient peripheral blood and tumor tissue. Thus, any growth of new vessels from the peripheral limbal vasculature must be due to injected TEM and reflect their pro-angiogenic activity. Tumor TEM showed a heterogeneous and consistently high pro-angiogenic activity inducing cornea and iris vascularization. By contrast, blood TEM were unable to induce de novo vascularization of the cornea but did increase the pre-existing vascular network of the iris (Fig. 1A). Thus, tumor and blood TEM show distinct pro-angiogenic phenotypes with the expression levels of TIE-2, VEGFR-1 and TGFR-1 mirroring their pro-angiogenic activity (Fig. 1A and Table 2). Finally, secretions were profiled in the conditioned medium of patient-isolated TEM and revealed that tumor TEM are paracrine inducers of tumor angiogenesis by releasing high levels of angiogenic factors (i.e. VEGF, bFGF, and ANG-1) and MMP9 (matrix metalloproteinase 9) (Fig. 1B). Blood and tumor TEM display a mixed M1-like (tumor-associated macrophages releasing inflammatory molecules) and M2-like (immunosuppressive macrophages polarized by anti-inflammatory molecules) phenotype, with secretion of both the pro- and anti-inflammatory cytokines IL-12 and IL-10, respectively (Fig. 1B). Given that TEM circulating in the blood infiltrate tumor tissue where they further differentiate [34], our data suggest that the tumor microenvironment shapes their highly pro-angiogenic phenotype.


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)

Phenotypical signature of pro-angiogenic TEM.(A) In vivo corneal vascularization assay to assess the pro-angiogenic activity of TEM isolated from peripheral blood and tumor of breast cancer patients. Bright field pictures of the eyes and fluorescent microscopy images of sagittal sections of the eyes stained with CD31 (stains specifically blood vessel endothelial cells) and Dapi (stains cell nucleus) are shown. Double-head and single arrows depict cornea and iris respectively. Corneas of control eyes were injected with buffer alone and show similar vascularization to uninjected eyes. Note the presence of blood vessels in the cornea injected with tumor TEM (double-head arrow) and its absence in the corneas injected with buffer (control) or blood TEM. Shown are representative data of 10 experiments. Bars in bright field and fluorescent images are 500 and 100 μm respectively. Bar graph represents a quantification of the vascular network of the cornea and the iris. (B) Secretion profile of cytokines and angiogenic factors in TEM isolated from patient blood and tumor. Angiogenic factors are boxed. Shown are cumulated data of 5 experiments, significant variations (P < 0.05) are indicated with an asterisk. (C) Workflow diagram of the strategy combining experimental and computational approaches to discover anti-angiogenic therapies. Green: experiments using ivdTEM. Blue, experiments using patient TEM; red: computational approach.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359163&req=5

pcbi.1004050.g001: Phenotypical signature of pro-angiogenic TEM.(A) In vivo corneal vascularization assay to assess the pro-angiogenic activity of TEM isolated from peripheral blood and tumor of breast cancer patients. Bright field pictures of the eyes and fluorescent microscopy images of sagittal sections of the eyes stained with CD31 (stains specifically blood vessel endothelial cells) and Dapi (stains cell nucleus) are shown. Double-head and single arrows depict cornea and iris respectively. Corneas of control eyes were injected with buffer alone and show similar vascularization to uninjected eyes. Note the presence of blood vessels in the cornea injected with tumor TEM (double-head arrow) and its absence in the corneas injected with buffer (control) or blood TEM. Shown are representative data of 10 experiments. Bars in bright field and fluorescent images are 500 and 100 μm respectively. Bar graph represents a quantification of the vascular network of the cornea and the iris. (B) Secretion profile of cytokines and angiogenic factors in TEM isolated from patient blood and tumor. Angiogenic factors are boxed. Shown are cumulated data of 5 experiments, significant variations (P < 0.05) are indicated with an asterisk. (C) Workflow diagram of the strategy combining experimental and computational approaches to discover anti-angiogenic therapies. Green: experiments using ivdTEM. Blue, experiments using patient TEM; red: computational approach.
Mentions: The angigoenic profile of TEM was investigated in a group of 40 newly diagnosed breast cancer patients (Table 1). We characterized by flow cytometry the phenotype of TEM from patient peripheral blood and freshly dissociated tumor specimens obtained at time of surgery (see Material and Methods). Based on our immunostaining and flow cytometry protocol we observed that TEM did not constitute a distinct subset of monocytes. In contrast, all monocytes showed expression of TIE-2, which was particularly low in patient blood and substantially higher on monocytes isolated from tumor tissue (S1 Fig. and Table 2). Thus, CD11b+, CD14+ monocytes from patient blood and tumor tissue were referred to as “TEM” and compared with respect to receptor and cytokine expression. However, tumor TEM co-expressed VEGFR-1 and TGFR-1 at significantly higher levels compared to peripheral blood TEM (Table 2). We next assess the pro-angiogenic activity of TEM using the in vivo corneal vascularization assay [39]. The cornea itself is avascular and was injected with TEM isolated from patient peripheral blood and tumor tissue. Thus, any growth of new vessels from the peripheral limbal vasculature must be due to injected TEM and reflect their pro-angiogenic activity. Tumor TEM showed a heterogeneous and consistently high pro-angiogenic activity inducing cornea and iris vascularization. By contrast, blood TEM were unable to induce de novo vascularization of the cornea but did increase the pre-existing vascular network of the iris (Fig. 1A). Thus, tumor and blood TEM show distinct pro-angiogenic phenotypes with the expression levels of TIE-2, VEGFR-1 and TGFR-1 mirroring their pro-angiogenic activity (Fig. 1A and Table 2). Finally, secretions were profiled in the conditioned medium of patient-isolated TEM and revealed that tumor TEM are paracrine inducers of tumor angiogenesis by releasing high levels of angiogenic factors (i.e. VEGF, bFGF, and ANG-1) and MMP9 (matrix metalloproteinase 9) (Fig. 1B). Blood and tumor TEM display a mixed M1-like (tumor-associated macrophages releasing inflammatory molecules) and M2-like (immunosuppressive macrophages polarized by anti-inflammatory molecules) phenotype, with secretion of both the pro- and anti-inflammatory cytokines IL-12 and IL-10, respectively (Fig. 1B). Given that TEM circulating in the blood infiltrate tumor tissue where they further differentiate [34], our data suggest that the tumor microenvironment shapes their highly pro-angiogenic phenotype.

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