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. 2015 Mar 13;11(3):e1004050.
doi: 10.1371/journal.pcbi.1004050. eCollection 2015 Mar.

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

Nicolas Guex  1 , Isaac Crespo  1 , Sylvian Bron  2 , Assia Ifticene-Treboux  3 , Eveline Faes-Van't Hull  2 , Solange Kharoubi  2 , Robin Liechti  1 , Patricia Werffeli  4 , Mark Ibberson  1 , Francois Majo  5 , Michäel Nicolas  5 , Julien Laurent  6 , Abhishek Garg  7 , Khalil Zaman  8 , Hans-Anton Lehr  9 , Brian J Stevenson  1 , Curzio Rüegg  10 , George Coukos  2 , Jean-François Delaloye  3 , Ioannis Xenarios  1 , Marie-Agnès Doucey  2
Affiliations

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

Nicolas Guex et al. PLoS Comput Biol. .

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. 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.
Fig 2
Fig 2. 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.
Fig 3
Fig 3. ivdTEM network topology.
Dynamical models of treatments/receptors/cytokines interactions in ivdTEM. Inputs (treatments) and output (receptor and secreted soluble factors) are depicted in yellow and red respectively. Factors that were used as treatment and measured as output are depicted in orange. Combined treatments (network nodes i.e. AND) are represented as small pink circles. Stimulatory and inhibitory effects of single or combined treatments are depicted by black arrow-headed edges and green edges respectively. Circles and diamonds represent soluble factors and receptors respectively. All the links presented are provided in S4 Table. Boolean equations used for representing ivdTEM regulatory networks are provided in S6 Table.
Fig 4
Fig 4. Experimental validation of in silico predicted treatments using ivdTEM.
TEM differentiated in vitro were exposed to the treatments predicted in silico and their changes in TIE-2 and VEGFR-1 expression (A) and pro-angiogenic activity (B) measured by flow cytometry and in vitro angiogenesis sprouting assay respectively. The impact of inhibitory treatments was examined on ivdTEM previously treated with TNF-α/PlGF/ANG-2 (panels A and B). Significant variations (P < 0.05) are indicated with an asterisk. In panel A, small and large asterisks referred to VEGFR-1 and TIE-2 expression respectively.
Fig 5
Fig 5. Gene expression changes in monocytes treated with TGF-β/ANG-2 and TGF-β/PlGF relative to untreated and VEGF/TNF-α cells.
398 significantly (P ≤ 0.05) differentially expressed genes were manually annotated and classified in categories (S5 Table). In each category the percentage of up- and down-regulated genes are displayed as well as the total number of genes (under brackets). 50 genes could not be assigned to these categories.
Fig 6
Fig 6. Controlling the pro-angiogenic activity of TEM from breast cancer patients.
(A and B) In vivo corneal vascularization assay, as described in Fig. 1A, showing the variations in the pro-angiogenic activity of patient TEM in response to in silico predicted treatments. TIE-2 kinase inhibitor/TGF-β/VEGF treatment decreased tumor TEM pro-angiogenic activity while TNF-α/PlGF/ANG-2 treatment increased the pro-angiogenic activity of blood TEM. Bars are 500 and 250 μm in A and B respectively. Cornea and iris are depicted by double-head and single arrows respectively. Bar graph represents a quantification of the vascular network of the cornea and the iris. (C) Variations of patient blood and tumor TEM secretion profiles in response to TNF-α/PLGF/ANG-2 and TIE-2 kinase inhibitor/TGF-β/VEGF treatments. Angiogenic factors are boxed. No significant variations were detected for IL-4 and TNF-α. (D) Variations of patient blood and tumor TEM expression of TIE-2 and VEGFR-1 in response to the same treatments. Fold increase to isotype control antibody is indicated, similarly as in Table 2. Shown are cumulated data (C, D) or representative results (A and B) of 3 to 5 experiments. Significant variations (* P < 0.05, T test).
Fig 7
Fig 7. Tie-2 and VEGFR-1, and Ang-2 and PIGF represent attractive targets in breast cancer (A) Correlation of Ang-2 and PlGF and CD14 protein expression levels in the tumor with tumor size in 17 patients.
The significance of their linear correlation is shown by Pearson r and p values. (B-F) Survival analysis. The good prognostic effect of the lower expression of CD14, ANG-2 and PIGF (B) is reflected by the clear separation from the over expression group on the Kaplan-Meier plot, with a P value of 0.0257 from a Log-rank test (F), whereas only ANG-2 (C) or PIGF (D) combined with CD14 separate worse lower and over expression groups, suggesting a synergistic effect of ANG-2 and PIGF to promote CD14-mediated angiogenesis and the corresponding impact on patient relapse free survival. (E) Shows the survival curve of lower expression patients for the three cases.
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Grants and funding

This work was supported by grants from Oncosuisse (MAD project 02069-04-2007), the Swiss National Foundation (MAD project 310030-120473 and JFD project CR32I3_135073), the Medic foundation (MAD) and the Experimental Network for Functional Integration FP6 Program (IX project LSHG-CT-2005-518254). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.