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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

Characterization of mesenchymal and epithelial phenotype. a Phase contrast microscopy pictures show significant morphological difference concordant with an epithelial phenotype (MCF7 and OVCAR3) and a mesenchymal phenotype (MDA, SKOV3 and APOCC). Scale bar: 150 μm. Cells were stained for E-cadherin or N-cadherin and analyzed by confocal microscopy. Presence of N-cadherin and E-cadherin staining is concordant with the first observations of the cells body shape. Scale bar 50 μm. b Western blot analysis of the different cells lines for mesenchymal (Vimentin and Snail) and epithelial (E-cadherin) markers confirm the difference between MCF7 and OVCAR3 on one part (epithelial phenotype) and SKOV3, MDA and APOCC on the other part (mesenchymal phenotype). MDA, SKOV3 and APOCC expressed mesenchymal genes at levels significantly higher than MCF7 and OVCAR3, as determined by real-time qPCR. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. c eGFP-E4+ECs were co-cultured with tumor cells for 3 days. Before imaging by confocal microscopy, co-cultures were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). GFP-MPs and WGA-MPs are detected floating between cells and on cell membranes. Typical characteristics of MPs are observed: (i) particles smaller than 1 μm, (ii) budding at the membrane. Scale bar: 5 μm. Confocal imaging of MDA/eGFP-E4+ECs sphere. Spheroids of MDA and eGFP-E4+ECs were grown in 3D media for 5 days. MDA cells were stained with Pkh red before spheroids formation. Red or green MPs (arrow) are visible inside the sphere. Scale bar: 10 μm. d eGFP-E4+ECs were co-cultured with MCF7 or SKOV3 cells for 3 days. Before imaging by confocal microscopy, fixed cells were stained with DAPI and AlexaFluor 647 conjugated-phalloidin. Fixed cells were stained with WGA, DAPI and AlexaFluor 647 conjugated-phalloidin. Arrows demonstrate co-localization of eGFP-E4+ECs-MPs (green) and actin patches (red). Scale bar 10 μm. e MPs from E4+ECs were extracted from 80 % confluent cells and labeled with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). Each cancer cells lines were incubated with E4+ECs-MPs for 24 h at 37°c in presence or absence (control) of Annexin V (AnnV) or cytochalsine D (CytoD). MPs uptake quantification was made by flow cytometry. MPs uptake decrease in presence of cytoskeleton heckler or inhibitors
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Fig1: Characterization of mesenchymal and epithelial phenotype. a Phase contrast microscopy pictures show significant morphological difference concordant with an epithelial phenotype (MCF7 and OVCAR3) and a mesenchymal phenotype (MDA, SKOV3 and APOCC). Scale bar: 150 μm. Cells were stained for E-cadherin or N-cadherin and analyzed by confocal microscopy. Presence of N-cadherin and E-cadherin staining is concordant with the first observations of the cells body shape. Scale bar 50 μm. b Western blot analysis of the different cells lines for mesenchymal (Vimentin and Snail) and epithelial (E-cadherin) markers confirm the difference between MCF7 and OVCAR3 on one part (epithelial phenotype) and SKOV3, MDA and APOCC on the other part (mesenchymal phenotype). MDA, SKOV3 and APOCC expressed mesenchymal genes at levels significantly higher than MCF7 and OVCAR3, as determined by real-time qPCR. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. c eGFP-E4+ECs were co-cultured with tumor cells for 3 days. Before imaging by confocal microscopy, co-cultures were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). GFP-MPs and WGA-MPs are detected floating between cells and on cell membranes. Typical characteristics of MPs are observed: (i) particles smaller than 1 μm, (ii) budding at the membrane. Scale bar: 5 μm. Confocal imaging of MDA/eGFP-E4+ECs sphere. Spheroids of MDA and eGFP-E4+ECs were grown in 3D media for 5 days. MDA cells were stained with Pkh red before spheroids formation. Red or green MPs (arrow) are visible inside the sphere. Scale bar: 10 μm. d eGFP-E4+ECs were co-cultured with MCF7 or SKOV3 cells for 3 days. Before imaging by confocal microscopy, fixed cells were stained with DAPI and AlexaFluor 647 conjugated-phalloidin. Fixed cells were stained with WGA, DAPI and AlexaFluor 647 conjugated-phalloidin. Arrows demonstrate co-localization of eGFP-E4+ECs-MPs (green) and actin patches (red). Scale bar 10 μm. e MPs from E4+ECs were extracted from 80 % confluent cells and labeled with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). Each cancer cells lines were incubated with E4+ECs-MPs for 24 h at 37°c in presence or absence (control) of Annexin V (AnnV) or cytochalsine D (CytoD). MPs uptake quantification was made by flow cytometry. MPs uptake decrease in presence of cytoskeleton heckler or inhibitors

Mentions: We used 4 different cancer cells lines, 2 breast (MCF7 and MDA-MB231) and 2 ovarian (OVCAR3 and SKOV3), as well as a primary ovarian cancer cell line derived in our laboratory from ascites of a patient with Stage III serous adenocarcinoma (APOCC). All cell lines displayed different morphology (Fig. 1a). MCF7 and OVCAR3 were polygonal in shape with regular dimensions, and grew in discrete patches. MDA, SKOV3 and APOCC had mesenchymal-like features and grew with less interaction. Concordantly MDA, SKOV3 and APOCC expression of mesenchymal markers was confirmed by confocal microscopy (Fig. 1a), western blot and qPCR (Fig. 1b) while OVCAR3 and MCF7 expressed epithelial markers.Fig. 1


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)

Characterization of mesenchymal and epithelial phenotype. a Phase contrast microscopy pictures show significant morphological difference concordant with an epithelial phenotype (MCF7 and OVCAR3) and a mesenchymal phenotype (MDA, SKOV3 and APOCC). Scale bar: 150 μm. Cells were stained for E-cadherin or N-cadherin and analyzed by confocal microscopy. Presence of N-cadherin and E-cadherin staining is concordant with the first observations of the cells body shape. Scale bar 50 μm. b Western blot analysis of the different cells lines for mesenchymal (Vimentin and Snail) and epithelial (E-cadherin) markers confirm the difference between MCF7 and OVCAR3 on one part (epithelial phenotype) and SKOV3, MDA and APOCC on the other part (mesenchymal phenotype). MDA, SKOV3 and APOCC expressed mesenchymal genes at levels significantly higher than MCF7 and OVCAR3, as determined by real-time qPCR. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. c eGFP-E4+ECs were co-cultured with tumor cells for 3 days. Before imaging by confocal microscopy, co-cultures were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). GFP-MPs and WGA-MPs are detected floating between cells and on cell membranes. Typical characteristics of MPs are observed: (i) particles smaller than 1 μm, (ii) budding at the membrane. Scale bar: 5 μm. Confocal imaging of MDA/eGFP-E4+ECs sphere. Spheroids of MDA and eGFP-E4+ECs were grown in 3D media for 5 days. MDA cells were stained with Pkh red before spheroids formation. Red or green MPs (arrow) are visible inside the sphere. Scale bar: 10 μm. d eGFP-E4+ECs were co-cultured with MCF7 or SKOV3 cells for 3 days. Before imaging by confocal microscopy, fixed cells were stained with DAPI and AlexaFluor 647 conjugated-phalloidin. Fixed cells were stained with WGA, DAPI and AlexaFluor 647 conjugated-phalloidin. Arrows demonstrate co-localization of eGFP-E4+ECs-MPs (green) and actin patches (red). Scale bar 10 μm. e MPs from E4+ECs were extracted from 80 % confluent cells and labeled with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). Each cancer cells lines were incubated with E4+ECs-MPs for 24 h at 37°c in presence or absence (control) of Annexin V (AnnV) or cytochalsine D (CytoD). MPs uptake quantification was made by flow cytometry. MPs uptake decrease in presence of cytoskeleton heckler or inhibitors
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Characterization of mesenchymal and epithelial phenotype. a Phase contrast microscopy pictures show significant morphological difference concordant with an epithelial phenotype (MCF7 and OVCAR3) and a mesenchymal phenotype (MDA, SKOV3 and APOCC). Scale bar: 150 μm. Cells were stained for E-cadherin or N-cadherin and analyzed by confocal microscopy. Presence of N-cadherin and E-cadherin staining is concordant with the first observations of the cells body shape. Scale bar 50 μm. b Western blot analysis of the different cells lines for mesenchymal (Vimentin and Snail) and epithelial (E-cadherin) markers confirm the difference between MCF7 and OVCAR3 on one part (epithelial phenotype) and SKOV3, MDA and APOCC on the other part (mesenchymal phenotype). MDA, SKOV3 and APOCC expressed mesenchymal genes at levels significantly higher than MCF7 and OVCAR3, as determined by real-time qPCR. Relative transcript levels are represented as the log10 of ratios between the 2 subpopulations of their 2–ΔΔCp real-time PCR values. c eGFP-E4+ECs were co-cultured with tumor cells for 3 days. Before imaging by confocal microscopy, co-cultures were stained with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). GFP-MPs and WGA-MPs are detected floating between cells and on cell membranes. Typical characteristics of MPs are observed: (i) particles smaller than 1 μm, (ii) budding at the membrane. Scale bar: 5 μm. Confocal imaging of MDA/eGFP-E4+ECs sphere. Spheroids of MDA and eGFP-E4+ECs were grown in 3D media for 5 days. MDA cells were stained with Pkh red before spheroids formation. Red or green MPs (arrow) are visible inside the sphere. Scale bar: 10 μm. d eGFP-E4+ECs were co-cultured with MCF7 or SKOV3 cells for 3 days. Before imaging by confocal microscopy, fixed cells were stained with DAPI and AlexaFluor 647 conjugated-phalloidin. Fixed cells were stained with WGA, DAPI and AlexaFluor 647 conjugated-phalloidin. Arrows demonstrate co-localization of eGFP-E4+ECs-MPs (green) and actin patches (red). Scale bar 10 μm. e MPs from E4+ECs were extracted from 80 % confluent cells and labeled with Alexa Fluor 594 conjugated-wheat germ agglutinin (WGA). Each cancer cells lines were incubated with E4+ECs-MPs for 24 h at 37°c in presence or absence (control) of Annexin V (AnnV) or cytochalsine D (CytoD). MPs uptake quantification was made by flow cytometry. MPs uptake decrease in presence of cytoskeleton heckler or inhibitors
Mentions: We used 4 different cancer cells lines, 2 breast (MCF7 and MDA-MB231) and 2 ovarian (OVCAR3 and SKOV3), as well as a primary ovarian cancer cell line derived in our laboratory from ascites of a patient with Stage III serous adenocarcinoma (APOCC). All cell lines displayed different morphology (Fig. 1a). MCF7 and OVCAR3 were polygonal in shape with regular dimensions, and grew in discrete patches. MDA, SKOV3 and APOCC had mesenchymal-like features and grew with less interaction. Concordantly MDA, SKOV3 and APOCC expression of mesenchymal markers was confirmed by confocal microscopy (Fig. 1a), western blot and qPCR (Fig. 1b) while OVCAR3 and MCF7 expressed epithelial markers.Fig. 1

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