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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.


Related in: MedlinePlus

MPs from activated endothelial cells induce EMT in epithelial cancer cells and trigger expansion of cells from ovarian cancer metastatic nodules explants. a The relative quantification of EMT genes were performed by real-time qPCR on MCF7 and OVCAR3 after treatment with E4+ECs MPs. The mesenchymal genes (N-cadherin, Snail, Fibronectin and Vimentin) were increased compared to the control. E-cadherin expression was decreased. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. Phase contrast microscopy showing significant morphological change concordant with a mesenchymal phenotype (bottom panel). Scale bar 100 μm. b Western blot analysis of P-Smad 1/5, P-Smad 3 and Smad3 revealed an implication of P-Smad 3 in the EMT process induced by E4+ECs MPs in MCF7 and OVCAR3 cells. c Phase contrast imaging of ovarian cancer explant. Explants of metastatic ovarian cancer nodules were cultured in Petri dishes (top panel). Cells spreading from 3D explants after 7 days of cultured without (bottom left panel) or with E4-ECs MPs stained with AF594-WGA (bottom right panel). Top scale bar: 500 μm. Bottom scale bar: 100 μm. d Epcam+ outgrowths are observed in the EC-MPs treated group. Scale bar: 25 μm. e After 7 days of culture, explants treated with AF594-WGA MPs were collected and analyzed by confocal microscopy. AF594 staining in the explant demonstrated that the MPs have been uptake. Scale bar: 50 μm
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Fig7: MPs from activated endothelial cells induce EMT in epithelial cancer cells and trigger expansion of cells from ovarian cancer metastatic nodules explants. a The relative quantification of EMT genes were performed by real-time qPCR on MCF7 and OVCAR3 after treatment with E4+ECs MPs. The mesenchymal genes (N-cadherin, Snail, Fibronectin and Vimentin) were increased compared to the control. E-cadherin expression was decreased. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. Phase contrast microscopy showing significant morphological change concordant with a mesenchymal phenotype (bottom panel). Scale bar 100 μm. b Western blot analysis of P-Smad 1/5, P-Smad 3 and Smad3 revealed an implication of P-Smad 3 in the EMT process induced by E4+ECs MPs in MCF7 and OVCAR3 cells. c Phase contrast imaging of ovarian cancer explant. Explants of metastatic ovarian cancer nodules were cultured in Petri dishes (top panel). Cells spreading from 3D explants after 7 days of cultured without (bottom left panel) or with E4-ECs MPs stained with AF594-WGA (bottom right panel). Top scale bar: 500 μm. Bottom scale bar: 100 μm. d Epcam+ outgrowths are observed in the EC-MPs treated group. Scale bar: 25 μm. e After 7 days of culture, explants treated with AF594-WGA MPs were collected and analyzed by confocal microscopy. AF594 staining in the explant demonstrated that the MPs have been uptake. Scale bar: 50 μm

Mentions: Recently the role of cancer propagating cells has been illustrated in both breast and ovarian malignancies [42]. To investigate the effect of EC-MPs on the induction of a propagating phenotype, we used sphere formation assay and flow cytometry. EC-MPs treatment increased CCs sphere formation in all cell lines for both number and size, from 1.3 to 4.13 fold in a serum free 3D media (Fig. 6e and Supplementary figure 7A). To characterize tumor-propagating population by flow cytometry, we used previously described cell surface markers (CD44+CD24low for breast cancers, and CD44+CD117+ for ovarian cancers) [43,44]. Treatment of CCs with EC-MPs increased the putative stem cell population in both breast and ovarian cancer models by 12, 4.5, 2.29 and 1.71 fold for MCF7, MDA, SKOV3 and OVCAR3 respectively (Fig. 7f and Supplementary figure 7B). Interestingly when we performed similar experiments with HUVEC derived MPs normalized on protein quantity and could not demonstrate any significant increase in pro-metastatic phenotype (data not shown).Fig. 7


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)

MPs from activated endothelial cells induce EMT in epithelial cancer cells and trigger expansion of cells from ovarian cancer metastatic nodules explants. a The relative quantification of EMT genes were performed by real-time qPCR on MCF7 and OVCAR3 after treatment with E4+ECs MPs. The mesenchymal genes (N-cadherin, Snail, Fibronectin and Vimentin) were increased compared to the control. E-cadherin expression was decreased. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. Phase contrast microscopy showing significant morphological change concordant with a mesenchymal phenotype (bottom panel). Scale bar 100 μm. b Western blot analysis of P-Smad 1/5, P-Smad 3 and Smad3 revealed an implication of P-Smad 3 in the EMT process induced by E4+ECs MPs in MCF7 and OVCAR3 cells. c Phase contrast imaging of ovarian cancer explant. Explants of metastatic ovarian cancer nodules were cultured in Petri dishes (top panel). Cells spreading from 3D explants after 7 days of cultured without (bottom left panel) or with E4-ECs MPs stained with AF594-WGA (bottom right panel). Top scale bar: 500 μm. Bottom scale bar: 100 μm. d Epcam+ outgrowths are observed in the EC-MPs treated group. Scale bar: 25 μm. e After 7 days of culture, explants treated with AF594-WGA MPs were collected and analyzed by confocal microscopy. AF594 staining in the explant demonstrated that the MPs have been uptake. Scale bar: 50 μm
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Related In: Results  -  Collection

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Fig7: MPs from activated endothelial cells induce EMT in epithelial cancer cells and trigger expansion of cells from ovarian cancer metastatic nodules explants. a The relative quantification of EMT genes were performed by real-time qPCR on MCF7 and OVCAR3 after treatment with E4+ECs MPs. The mesenchymal genes (N-cadherin, Snail, Fibronectin and Vimentin) were increased compared to the control. E-cadherin expression was decreased. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. Phase contrast microscopy showing significant morphological change concordant with a mesenchymal phenotype (bottom panel). Scale bar 100 μm. b Western blot analysis of P-Smad 1/5, P-Smad 3 and Smad3 revealed an implication of P-Smad 3 in the EMT process induced by E4+ECs MPs in MCF7 and OVCAR3 cells. c Phase contrast imaging of ovarian cancer explant. Explants of metastatic ovarian cancer nodules were cultured in Petri dishes (top panel). Cells spreading from 3D explants after 7 days of cultured without (bottom left panel) or with E4-ECs MPs stained with AF594-WGA (bottom right panel). Top scale bar: 500 μm. Bottom scale bar: 100 μm. d Epcam+ outgrowths are observed in the EC-MPs treated group. Scale bar: 25 μm. e After 7 days of culture, explants treated with AF594-WGA MPs were collected and analyzed by confocal microscopy. AF594 staining in the explant demonstrated that the MPs have been uptake. Scale bar: 50 μm
Mentions: Recently the role of cancer propagating cells has been illustrated in both breast and ovarian malignancies [42]. To investigate the effect of EC-MPs on the induction of a propagating phenotype, we used sphere formation assay and flow cytometry. EC-MPs treatment increased CCs sphere formation in all cell lines for both number and size, from 1.3 to 4.13 fold in a serum free 3D media (Fig. 6e and Supplementary figure 7A). To characterize tumor-propagating population by flow cytometry, we used previously described cell surface markers (CD44+CD24low for breast cancers, and CD44+CD117+ for ovarian cancers) [43,44]. Treatment of CCs with EC-MPs increased the putative stem cell population in both breast and ovarian cancer models by 12, 4.5, 2.29 and 1.71 fold for MCF7, MDA, SKOV3 and OVCAR3 respectively (Fig. 7f and Supplementary figure 7B). Interestingly when we performed similar experiments with HUVEC derived MPs normalized on protein quantity and could not demonstrate any significant increase in pro-metastatic phenotype (data not shown).Fig. 7

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