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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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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.
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pcbi.1004050.g005: 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.

Mentions: Having identified the critical ligands and pathways controlling TEM plasticity, we next examined in TEM differentiated in vitro whether differential gene expression might also contribute to the molecular basis of TEM plastic behavior. This analysis may shed light on the molecular mechanisms underlying the observed TEM responses. To this end, we selected VEGF/TNF-α, ANG-2/TGF-β and PlGF/TGF-β treatments for gene expression profiling using Affimetrix whole genome microarrays, because these treatments were present in 17, 16 and 14, respectively of the 74 links (treatment/receptor/cytokine) retained in TEM regulatory network (S4 Table and Fig. 3). All the other treatments occurred less frequently. Hierarchical clustering demonstrated that TGF-β-based treatments (ANG-2/TGF-β and PlGF/TGF-β) clustered separately from VEGF/TNF-α and control treatments. A total of 398 genes were significantly (p<0.05) and differentially expressed between the two clusters among which 369 and 72 genes were altered by TGF-β/ANG-2 and TGF-β/PlGF treatments respectively (S5 Table, NT unique lists) while 43 were regulated in common (S5 Table, NT intersect list). Enrichment analyses of the gene expression data against known pathways and functional gene categories were conducted as described in Materials and Methods. No enrichment of specific pathways of interest was observed due to the fact that the gene annotations were too general and did not correspond to specific functions of monocytes. Therefore, the 398 differentially expressed genes were annotated and classified in categories manually (S5 Table). Similar expression profiles were obtained for untreated and TNF-α/VEGF treated cells consistent with their weak impact on TEM functional angiogenic phenotype (Fig. 2). By contrast, ANG-2/TGF-β and PlGF/TGF-β treatments inhibited TEM pro-angiogenic activity (Fig. 2) and down-modulated the expression of pro-angiogenic genes (Fig. 5 and S5 Table). Furthermore and interestingly, the expression of VASH1 (vasohibin 1) and UCN (urocortin) genes coding for anti-angiogenic proteins was simultaneously up-regulated (Fig. 5 and S5 Table). In response to both ANG-2/TGF-β and PlGF/TGF-β treatments, 95% of the genes functionally related to the cell cycle displayed a down-modulated expression indicating that TEM stopped proliferating with profound changes in their metabolism but without, however undergoing apoptosis (the expression of metabolism and apoptosis related genes was down-modulated for 76% and 88% of them respectively). TEM treated with TGF-β/ANG-2 or TGF-β/PlGF show the expression of some genes (P2RY12, TMCC3, NPDC1, IFFO1, UBASH3B, C11orf52, SLC4A7, TMEM87A, NPL, EMB, PCNA, DNA2, TMEM86A, MMP12, CTSD, AXL, RASGRP3, TUBB, FCGR1A, CR1, MX2) previously ascribed to mouse TAM [45,46,47]. However, in response to ANG-2/TGF-β and PlGF/TGF-β treatments, TEM down-modulated the expression of genes involved in macrophage differentiation (Fig. 5) and started to acquire the profile (RGS1, CXCL11, CXCL9, STAT1, IFIH1, ISG20, NT5C3, ADC, PDGFRL, TNF-ASF12, IFIT5, RGS10, TRAF3IP3, CIDEB, APOBEC3A, PYGL, RRM1, MAF, NLRC4, IL10, MYC, DUT, POLE4, CXCL17) of dendritic cells matured in vitro by exposure to lipopolysaccharide and interferon-gamma [48,49]. Along these lines, genes encoding for dendritic cell markers, antigen processing and adaptive immune response were upregulated while genes involved in immune suppression show markedly decreased expression (Fig. 5 and [38]). Finally the expression of genes related to adhesion and migration were up- and down-regulated respectively indicating that TEM mobility was strongly reduced; an observation consistent with the arrest of their cell cycle and the alteration of their differentiation program (Fig. 5 and S5 Table). Along these lines, we observed experimentally that ivdTEM treated with PlGF/TGF-β/TIE-2i display reduced mobility towards the human epithelial tumor cell line MDA-231 (S3A Fig.) and slowed down the growth of MDA-231 cells (S3B Fig.)


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi.1004050.g005: 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.
Mentions: Having identified the critical ligands and pathways controlling TEM plasticity, we next examined in TEM differentiated in vitro whether differential gene expression might also contribute to the molecular basis of TEM plastic behavior. This analysis may shed light on the molecular mechanisms underlying the observed TEM responses. To this end, we selected VEGF/TNF-α, ANG-2/TGF-β and PlGF/TGF-β treatments for gene expression profiling using Affimetrix whole genome microarrays, because these treatments were present in 17, 16 and 14, respectively of the 74 links (treatment/receptor/cytokine) retained in TEM regulatory network (S4 Table and Fig. 3). All the other treatments occurred less frequently. Hierarchical clustering demonstrated that TGF-β-based treatments (ANG-2/TGF-β and PlGF/TGF-β) clustered separately from VEGF/TNF-α and control treatments. A total of 398 genes were significantly (p<0.05) and differentially expressed between the two clusters among which 369 and 72 genes were altered by TGF-β/ANG-2 and TGF-β/PlGF treatments respectively (S5 Table, NT unique lists) while 43 were regulated in common (S5 Table, NT intersect list). Enrichment analyses of the gene expression data against known pathways and functional gene categories were conducted as described in Materials and Methods. No enrichment of specific pathways of interest was observed due to the fact that the gene annotations were too general and did not correspond to specific functions of monocytes. Therefore, the 398 differentially expressed genes were annotated and classified in categories manually (S5 Table). Similar expression profiles were obtained for untreated and TNF-α/VEGF treated cells consistent with their weak impact on TEM functional angiogenic phenotype (Fig. 2). By contrast, ANG-2/TGF-β and PlGF/TGF-β treatments inhibited TEM pro-angiogenic activity (Fig. 2) and down-modulated the expression of pro-angiogenic genes (Fig. 5 and S5 Table). Furthermore and interestingly, the expression of VASH1 (vasohibin 1) and UCN (urocortin) genes coding for anti-angiogenic proteins was simultaneously up-regulated (Fig. 5 and S5 Table). In response to both ANG-2/TGF-β and PlGF/TGF-β treatments, 95% of the genes functionally related to the cell cycle displayed a down-modulated expression indicating that TEM stopped proliferating with profound changes in their metabolism but without, however undergoing apoptosis (the expression of metabolism and apoptosis related genes was down-modulated for 76% and 88% of them respectively). TEM treated with TGF-β/ANG-2 or TGF-β/PlGF show the expression of some genes (P2RY12, TMCC3, NPDC1, IFFO1, UBASH3B, C11orf52, SLC4A7, TMEM87A, NPL, EMB, PCNA, DNA2, TMEM86A, MMP12, CTSD, AXL, RASGRP3, TUBB, FCGR1A, CR1, MX2) previously ascribed to mouse TAM [45,46,47]. However, in response to ANG-2/TGF-β and PlGF/TGF-β treatments, TEM down-modulated the expression of genes involved in macrophage differentiation (Fig. 5) and started to acquire the profile (RGS1, CXCL11, CXCL9, STAT1, IFIH1, ISG20, NT5C3, ADC, PDGFRL, TNF-ASF12, IFIT5, RGS10, TRAF3IP3, CIDEB, APOBEC3A, PYGL, RRM1, MAF, NLRC4, IL10, MYC, DUT, POLE4, CXCL17) of dendritic cells matured in vitro by exposure to lipopolysaccharide and interferon-gamma [48,49]. Along these lines, genes encoding for dendritic cell markers, antigen processing and adaptive immune response were upregulated while genes involved in immune suppression show markedly decreased expression (Fig. 5 and [38]). Finally the expression of genes related to adhesion and migration were up- and down-regulated respectively indicating that TEM mobility was strongly reduced; an observation consistent with the arrest of their cell cycle and the alteration of their differentiation program (Fig. 5 and S5 Table). Along these lines, we observed experimentally that ivdTEM treated with PlGF/TGF-β/TIE-2i display reduced mobility towards the human epithelial tumor cell line MDA-231 (S3A Fig.) and slowed down the growth of MDA-231 cells (S3B Fig.)

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