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Microparticles mediated cross-talk between tumoral and endothelial cells promote the constitution of a pro-metastatic vascular niche through Arf6 up regulation.

Pasquier J, Thawadi HA, Ghiabi P, Abu-Kaoud N, Maleki M, Guerrouahen BS, Vidal F, Courderc B, Ferron G, Martinez A, Al Sulaiti H, Gupta R, Rafii S, Rafii A - Cancer Microenviron (2014)

Bottom Line: Our data show that MPs from mesenchymal-like cell lines (MDA-MB231, SKOV3 and APOCC) were able to promote an activation of endothelial cells through Akt phosphorylation, compared to MPs from epithelial-like cell lines (OVCAR3 and MCF7).The endothelial activation was associated to increased Arf6 expression and MPs secretion.Such cross-talk may play a role in perfusion independent role of the endothelium.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Education City, Qatar Foundation, Doha, Qatar.

ABSTRACT
The tumor stroma plays an essential role in tumor growth, resistance to therapy and occurrence of metastatic phenotype. Tumor vessels have been considered as passive conducts for nutrients but several studies have demonstrated secretion of pro-tumoral factors by endothelial cells. The failure of anti-angiogenic therapies to meet expectations raised by pre-clinical studies prompt us to better study the cross-talk between endothelial and cancer cells. Here, we hypothesized that tumor cells and the endothelium secrete bio-active microparticles (MPs) participating to a functional cross-talk. We characterized the cancer cells MPs, using breast and ovarian cancer cell lines (MCF7, MDA-MB231, SKOV3, OVCAR3 and a primary cell lines, APOCC). Our data show that MPs from mesenchymal-like cell lines (MDA-MB231, SKOV3 and APOCC) were able to promote an activation of endothelial cells through Akt phosphorylation, compared to MPs from epithelial-like cell lines (OVCAR3 and MCF7). The MPs from mesenchymal-like cells contained increased angiogenic molecules including PDGF, IL8 and angiogenin. The endothelial activation was associated to increased Arf6 expression and MPs secretion. Endothelial activation functionalized an MP dependent pro-tumoral vascular niche promoting cancer cells proliferation, invasiveness, stem cell phenotype and chemoresistance. MPs from cancer and endothelial cells displayed phenotypic heterogeneity, and participated to a functional cross-talk where endothelial activation by cancer MPs resulted in increased secretion of EC-MPs sustaining tumor cells. Such cross-talk may play a role in perfusion independent role of the endothelium.

No MeSH data available.


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Endothelial activation induces an Arf6 mediated increase in MPs secretion. a Budding of MPs was counted in HUVEC and E4+ECs treated or not with M-MPs. Before imaging by confocal microscopy, cells were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). 10 areas of the slides were captured and the number of budding by number of cells was quantified. The budding is more important in E4+ECs than HUVECs and is increased in presence of M-MPs. Scale bar: 10 μm. b SiRNA of Arf6 decreased the budding in endothelial cells. c Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is increased in HUVECs in presence of M-MPs. d Western blot analysis showed that Arf6 expression is dependent of Akt phosphorylation. f Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is more important in E4+ECs than HUVECs and decreased when the cells were treated with an Akt inhibitor (LY294002)
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Fig5: Endothelial activation induces an Arf6 mediated increase in MPs secretion. a Budding of MPs was counted in HUVEC and E4+ECs treated or not with M-MPs. Before imaging by confocal microscopy, cells were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). 10 areas of the slides were captured and the number of budding by number of cells was quantified. The budding is more important in E4+ECs than HUVECs and is increased in presence of M-MPs. Scale bar: 10 μm. b SiRNA of Arf6 decreased the budding in endothelial cells. c Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is increased in HUVECs in presence of M-MPs. d Western blot analysis showed that Arf6 expression is dependent of Akt phosphorylation. f Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is more important in E4+ECs than HUVECs and decreased when the cells were treated with an Akt inhibitor (LY294002)

Mentions: In order to understand the effect of Akt phosphorylation on endothelial cells microparticles machinery we used a model of endothelial cells with autonomous Akt-activation surviving in the absence of FBS and cytokines (HUVECs-E4ORF1, referred to as E4+ECs, Supplementary figure 6) [38]. Live cell imaging showed increased budding of MPs at the membrane of E4+ECs compared to the HUVECs (Fig. 5a). Several genes have been implicated in MPs release such as Rab27, Rab11, Arf6, P53, TSAP6 and DGKA [7,20,39–41]. We performed transcriptomic analysis comparing HUVEC and E4+ECs. There was a moderate increase in expression of Rab27 and Rab11 (1.2 to 1.5 fold respectively). P53, TSAP6 and DGKA expression were not modified. Arf6 expression was increased by 2.1 fold; therefore we investigated the role of Arf6 in our experimental conditions. Arf6 is a GTPase of the Ras superfamily playing a major role in membrane trafficking and MPs secretion [20]. Si-RNA mediated inhibition of Arf6 (Supplementary figure 7 for inhibition efficiency) dramatically reduced endothelial and cancer cells MPs secretion (data not shown). Live cell imaging concordantly showed decreased budding at the membrane of both HUVECArf6- and E4+ECArf6- cells (Fig. 5b). We then showed that Arf6 expression was increased in HUVECs treated with M-MPs compared to E-MPs (Fig. 5c). Finally, as M-MPs induced Akt activation in HUVEC, we investigated the role of Akt activation in Arf6 expression. First we showed that FGF2-mediated EC activation induced concomitant Akt activation and Arf6 expression (Fig. 5d). Concordantly, inhibition of P-Akt by LY294002, in E4+ECs resulted in a decreased expression of Arf6 (Fig. 5d-e).Fig. 5


Microparticles mediated cross-talk between tumoral and endothelial cells promote the constitution of a pro-metastatic vascular niche through Arf6 up regulation.

Pasquier J, Thawadi HA, Ghiabi P, Abu-Kaoud N, Maleki M, Guerrouahen BS, Vidal F, Courderc B, Ferron G, Martinez A, Al Sulaiti H, Gupta R, Rafii S, Rafii A - Cancer Microenviron (2014)

Endothelial activation induces an Arf6 mediated increase in MPs secretion. a Budding of MPs was counted in HUVEC and E4+ECs treated or not with M-MPs. Before imaging by confocal microscopy, cells were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). 10 areas of the slides were captured and the number of budding by number of cells was quantified. The budding is more important in E4+ECs than HUVECs and is increased in presence of M-MPs. Scale bar: 10 μm. b SiRNA of Arf6 decreased the budding in endothelial cells. c Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is increased in HUVECs in presence of M-MPs. d Western blot analysis showed that Arf6 expression is dependent of Akt phosphorylation. f Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is more important in E4+ECs than HUVECs and decreased when the cells were treated with an Akt inhibitor (LY294002)
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Related In: Results  -  Collection

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Fig5: Endothelial activation induces an Arf6 mediated increase in MPs secretion. a Budding of MPs was counted in HUVEC and E4+ECs treated or not with M-MPs. Before imaging by confocal microscopy, cells were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). 10 areas of the slides were captured and the number of budding by number of cells was quantified. The budding is more important in E4+ECs than HUVECs and is increased in presence of M-MPs. Scale bar: 10 μm. b SiRNA of Arf6 decreased the budding in endothelial cells. c Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is increased in HUVECs in presence of M-MPs. d Western blot analysis showed that Arf6 expression is dependent of Akt phosphorylation. f Arf6 relative expression was evaluated by real-time qPCR. Arf6 expression is more important in E4+ECs than HUVECs and decreased when the cells were treated with an Akt inhibitor (LY294002)
Mentions: In order to understand the effect of Akt phosphorylation on endothelial cells microparticles machinery we used a model of endothelial cells with autonomous Akt-activation surviving in the absence of FBS and cytokines (HUVECs-E4ORF1, referred to as E4+ECs, Supplementary figure 6) [38]. Live cell imaging showed increased budding of MPs at the membrane of E4+ECs compared to the HUVECs (Fig. 5a). Several genes have been implicated in MPs release such as Rab27, Rab11, Arf6, P53, TSAP6 and DGKA [7,20,39–41]. We performed transcriptomic analysis comparing HUVEC and E4+ECs. There was a moderate increase in expression of Rab27 and Rab11 (1.2 to 1.5 fold respectively). P53, TSAP6 and DGKA expression were not modified. Arf6 expression was increased by 2.1 fold; therefore we investigated the role of Arf6 in our experimental conditions. Arf6 is a GTPase of the Ras superfamily playing a major role in membrane trafficking and MPs secretion [20]. Si-RNA mediated inhibition of Arf6 (Supplementary figure 7 for inhibition efficiency) dramatically reduced endothelial and cancer cells MPs secretion (data not shown). Live cell imaging concordantly showed decreased budding at the membrane of both HUVECArf6- and E4+ECArf6- cells (Fig. 5b). We then showed that Arf6 expression was increased in HUVECs treated with M-MPs compared to E-MPs (Fig. 5c). Finally, as M-MPs induced Akt activation in HUVEC, we investigated the role of Akt activation in Arf6 expression. First we showed that FGF2-mediated EC activation induced concomitant Akt activation and Arf6 expression (Fig. 5d). Concordantly, inhibition of P-Akt by LY294002, in E4+ECs resulted in a decreased expression of Arf6 (Fig. 5d-e).Fig. 5

Bottom Line: Our data show that MPs from mesenchymal-like cell lines (MDA-MB231, SKOV3 and APOCC) were able to promote an activation of endothelial cells through Akt phosphorylation, compared to MPs from epithelial-like cell lines (OVCAR3 and MCF7).The endothelial activation was associated to increased Arf6 expression and MPs secretion.Such cross-talk may play a role in perfusion independent role of the endothelium.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Microenvironment Laboratory, Weill Cornell Medical College in Qatar, Education City, Qatar Foundation, Doha, Qatar.

ABSTRACT
The tumor stroma plays an essential role in tumor growth, resistance to therapy and occurrence of metastatic phenotype. Tumor vessels have been considered as passive conducts for nutrients but several studies have demonstrated secretion of pro-tumoral factors by endothelial cells. The failure of anti-angiogenic therapies to meet expectations raised by pre-clinical studies prompt us to better study the cross-talk between endothelial and cancer cells. Here, we hypothesized that tumor cells and the endothelium secrete bio-active microparticles (MPs) participating to a functional cross-talk. We characterized the cancer cells MPs, using breast and ovarian cancer cell lines (MCF7, MDA-MB231, SKOV3, OVCAR3 and a primary cell lines, APOCC). Our data show that MPs from mesenchymal-like cell lines (MDA-MB231, SKOV3 and APOCC) were able to promote an activation of endothelial cells through Akt phosphorylation, compared to MPs from epithelial-like cell lines (OVCAR3 and MCF7). The MPs from mesenchymal-like cells contained increased angiogenic molecules including PDGF, IL8 and angiogenin. The endothelial activation was associated to increased Arf6 expression and MPs secretion. Endothelial activation functionalized an MP dependent pro-tumoral vascular niche promoting cancer cells proliferation, invasiveness, stem cell phenotype and chemoresistance. MPs from cancer and endothelial cells displayed phenotypic heterogeneity, and participated to a functional cross-talk where endothelial activation by cancer MPs resulted in increased secretion of EC-MPs sustaining tumor cells. Such cross-talk may play a role in perfusion independent role of the endothelium.

No MeSH data available.


Related in: MedlinePlus