Limits...
Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype.

Connor Y, Tekleab S, Nandakumar S, Walls C, Tekleab Y, Husain A, Gadish O, Sabbisetti V, Kaushik S, Sehrawat S, Kulkarni A, Dvorak H, Zetter B, R Edelman E, Sengupta S - Nat Commun (2015)

Bottom Line: Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis.The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits.These results lead us to define the notion of 'metastatic hijack': cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits.

View Article: PubMed Central - PubMed

Affiliation: Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of 'metastatic hijack': cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.

Show MeSH

Related in: MedlinePlus

The nanoscale membrane bridges act as conduits for intercellular communication between cancer and endothelial cells(a) Confocal image of nanoscale membrane bridge-mediated transfer of cytoplasmic contents. CFSE (green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. Transfer of the CFSE dye was observed after 24-h co-culture. CFSE dye can be seen within HUVEC cells (yellow arrow). Tumour cells can form a nanobridge with a distal endothelial cell (EC1) than an endothelial cell (EC2) in close proximity. (b,c) Cartoon shows the experimental design, where dual cultures control for vesicle-mediated intercellular transfer. FACS plot show gating for sorting endothelial cells from the co-cultures using dual staining for DiI-Ac-LDL and PECAM-1, and then quantification for CFSE transfer in the isolated endothelial cells. (d) Graph shows quantification of FACS analysis, highlighting increased transfer of CFSE to endothelial cells in the co-culture. (N>100,000 events, n = 36 replicates, 3 replicates per study). (e) Graph shows the temporal kinetics of nanoscale connection-mediated intercellular transfer of CFSE from MDA-MB-231 cells to the endothelium (n = 2 studies, 3 replicates per study). (f) Effect of small molecule inhibitors of cytoskeletal components on membrane nanobridges. (g) Graphs show treatment with vehicle (control) or a low-dose combination of docetaxel and cytochalasin do not affect the exosome shedding (n = 2 independent studies). (h,i) Graphs show the effect of pharmacological inhibitors on the formation of heterotypic and homotypic nanoscale bridges (arrows). (n = 2 studies, 6 replicates per study). (j) Graph shows the effect of pharmacological inhibitors on intercellular transfer of CFSE to endothelial cells from cancer cells (n = 10 studies, 3 replicates per study). Data shown are mean±s.e.m. (*P<0.05, **P<0.01, ****P<0.001, analysis of variance followed by Bonferroni’s post-hoc test).
© Copyright Policy - open-access - permissions-link
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4697439&req=5

Figure 4: The nanoscale membrane bridges act as conduits for intercellular communication between cancer and endothelial cells(a) Confocal image of nanoscale membrane bridge-mediated transfer of cytoplasmic contents. CFSE (green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. Transfer of the CFSE dye was observed after 24-h co-culture. CFSE dye can be seen within HUVEC cells (yellow arrow). Tumour cells can form a nanobridge with a distal endothelial cell (EC1) than an endothelial cell (EC2) in close proximity. (b,c) Cartoon shows the experimental design, where dual cultures control for vesicle-mediated intercellular transfer. FACS plot show gating for sorting endothelial cells from the co-cultures using dual staining for DiI-Ac-LDL and PECAM-1, and then quantification for CFSE transfer in the isolated endothelial cells. (d) Graph shows quantification of FACS analysis, highlighting increased transfer of CFSE to endothelial cells in the co-culture. (N>100,000 events, n = 36 replicates, 3 replicates per study). (e) Graph shows the temporal kinetics of nanoscale connection-mediated intercellular transfer of CFSE from MDA-MB-231 cells to the endothelium (n = 2 studies, 3 replicates per study). (f) Effect of small molecule inhibitors of cytoskeletal components on membrane nanobridges. (g) Graphs show treatment with vehicle (control) or a low-dose combination of docetaxel and cytochalasin do not affect the exosome shedding (n = 2 independent studies). (h,i) Graphs show the effect of pharmacological inhibitors on the formation of heterotypic and homotypic nanoscale bridges (arrows). (n = 2 studies, 6 replicates per study). (j) Graph shows the effect of pharmacological inhibitors on intercellular transfer of CFSE to endothelial cells from cancer cells (n = 10 studies, 3 replicates per study). Data shown are mean±s.e.m. (*P<0.05, **P<0.01, ****P<0.001, analysis of variance followed by Bonferroni’s post-hoc test).

Mentions: As the first step, to test the hypothesis that the nanoscale membrane bridges indeed facilitate intercellular communication from the cancer cells to endothelium, we loaded MDA-MB-231 breast cancer cells with a cell-impermeable dye, carboxyfluorescein succinimidyl ester (CFSE), before adding them to a co-culture with DiI-Ac-LDL-labelled endothelial cells. De-convolved volume 3D rendering of the co-cultures revealed transfer of cytoplasmic CFSE within the nanoscale membrane bridges connecting the cancer cells and endothelial cells (Fig. 4a and Supplementary Fig. 2B). Interestingly, although gap junctions have been reported to mediate intercellular transfers between cancer and endothelial cells33, here we observed intercellular transfer between distant cancer and endothelial cells that were not in direct physical contact except via the nanoscale membrane bridges (Fig. 4a). We next validated the intercellular transfer using flow cytometry. As a control, CFSE-loaded cancer cells and the endothelial cells were grown in the top and bottom chambers of a Boyden assay, respectively (dual culture), which allowed media contact and exosomes to cross through the 0.4-μm pores but did not allow direct physical contact, that is, no nanoscale membrane bridges could form between the cells (Fig. 4b and Supplementary Fig. 2C). In addition, we used a membrane with 3 μm pores, which allow both exosomes and larger vesicles to pass through (Supplementary Fig. 2C). The endothelial cells were first flow sorted from the dual/co-cultures using double labelling for DiI-Ac-LDL and platelet endothelial cell adhesion molecule-1 (PECAM-1) (Fig. 2c). The sorted endothelial cells were then analysed for CFSE and the subset of endothelial cells positive for CFSE was then quantified as a percentage of the total sorted endothelial cell population (Fig. 4c) as a measure of transfer from cancer cells. Indeed, we did observe intercellular transfer when the cells were separated in the Boyden assay (in both 0.4 and 3 μm pores), consistent with exosome- and extracellular vesicle-mediated transfer. Interestingly, as seen in Fig. 4d, <5% of the endothelial cells were found to be CSFE + ve in the dual culture (Boyden assay) as compared with ~30% of the endothelial cells being labelled as CFSE + ve when isolated from the co-cultures (after subtracting background signal from both conditions). It is possible that this transfer seen in the co-culture study includes the basal transfer arising from exosome- or gap-junction-mediated transfer. Similar results were described in the TNT-mediated transfer of p-glycoproteins, where two cells separated by a membrane with 0.4 μm pores exhibited a basal transfer consistent with exosome-mediated transfer as opposed to higher levels of transmission when TNTs were present26. Furthermore, the temporal quantification of intercellular transfer of CFSE revealed that the peak is reached between 24 and 36 h, lagging behind the kinetics of formation of these nanoscale structures (Fig. 4e). The lag in transfer kinetics is consistent with the notion that the nanoscale connections are not fully functional at the early stages of formation. A similar transfer was observed between the metastatic breast cancer cells and primary human vascular and lymphatic endothelial cells (Supplementary Fig. 3). In addition to CFSE, the nanostructures facilitated the transfer of nanoparticles (quantum dots) and proteins (green fluorescent protein) (Supplementary Fig. 4A–E). Interestingly, we did not observe a similar communication between metastatic tumour cells and vascular smooth muscle cells, further emphasizing the specificity of the communication between cancer cells and the endothelium (Supplementary Fig. 5). These results indicated that the nanoscale bridges act as conduits for communication from cancer cells to endothelial cells.


Physical nanoscale conduit-mediated communication between tumour cells and the endothelium modulates endothelial phenotype.

Connor Y, Tekleab S, Nandakumar S, Walls C, Tekleab Y, Husain A, Gadish O, Sabbisetti V, Kaushik S, Sehrawat S, Kulkarni A, Dvorak H, Zetter B, R Edelman E, Sengupta S - Nat Commun (2015)

The nanoscale membrane bridges act as conduits for intercellular communication between cancer and endothelial cells(a) Confocal image of nanoscale membrane bridge-mediated transfer of cytoplasmic contents. CFSE (green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. Transfer of the CFSE dye was observed after 24-h co-culture. CFSE dye can be seen within HUVEC cells (yellow arrow). Tumour cells can form a nanobridge with a distal endothelial cell (EC1) than an endothelial cell (EC2) in close proximity. (b,c) Cartoon shows the experimental design, where dual cultures control for vesicle-mediated intercellular transfer. FACS plot show gating for sorting endothelial cells from the co-cultures using dual staining for DiI-Ac-LDL and PECAM-1, and then quantification for CFSE transfer in the isolated endothelial cells. (d) Graph shows quantification of FACS analysis, highlighting increased transfer of CFSE to endothelial cells in the co-culture. (N>100,000 events, n = 36 replicates, 3 replicates per study). (e) Graph shows the temporal kinetics of nanoscale connection-mediated intercellular transfer of CFSE from MDA-MB-231 cells to the endothelium (n = 2 studies, 3 replicates per study). (f) Effect of small molecule inhibitors of cytoskeletal components on membrane nanobridges. (g) Graphs show treatment with vehicle (control) or a low-dose combination of docetaxel and cytochalasin do not affect the exosome shedding (n = 2 independent studies). (h,i) Graphs show the effect of pharmacological inhibitors on the formation of heterotypic and homotypic nanoscale bridges (arrows). (n = 2 studies, 6 replicates per study). (j) Graph shows the effect of pharmacological inhibitors on intercellular transfer of CFSE to endothelial cells from cancer cells (n = 10 studies, 3 replicates per study). Data shown are mean±s.e.m. (*P<0.05, **P<0.01, ****P<0.001, analysis of variance followed by Bonferroni’s post-hoc test).
© Copyright Policy - open-access - permissions-link
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4697439&req=5

Figure 4: The nanoscale membrane bridges act as conduits for intercellular communication between cancer and endothelial cells(a) Confocal image of nanoscale membrane bridge-mediated transfer of cytoplasmic contents. CFSE (green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. Transfer of the CFSE dye was observed after 24-h co-culture. CFSE dye can be seen within HUVEC cells (yellow arrow). Tumour cells can form a nanobridge with a distal endothelial cell (EC1) than an endothelial cell (EC2) in close proximity. (b,c) Cartoon shows the experimental design, where dual cultures control for vesicle-mediated intercellular transfer. FACS plot show gating for sorting endothelial cells from the co-cultures using dual staining for DiI-Ac-LDL and PECAM-1, and then quantification for CFSE transfer in the isolated endothelial cells. (d) Graph shows quantification of FACS analysis, highlighting increased transfer of CFSE to endothelial cells in the co-culture. (N>100,000 events, n = 36 replicates, 3 replicates per study). (e) Graph shows the temporal kinetics of nanoscale connection-mediated intercellular transfer of CFSE from MDA-MB-231 cells to the endothelium (n = 2 studies, 3 replicates per study). (f) Effect of small molecule inhibitors of cytoskeletal components on membrane nanobridges. (g) Graphs show treatment with vehicle (control) or a low-dose combination of docetaxel and cytochalasin do not affect the exosome shedding (n = 2 independent studies). (h,i) Graphs show the effect of pharmacological inhibitors on the formation of heterotypic and homotypic nanoscale bridges (arrows). (n = 2 studies, 6 replicates per study). (j) Graph shows the effect of pharmacological inhibitors on intercellular transfer of CFSE to endothelial cells from cancer cells (n = 10 studies, 3 replicates per study). Data shown are mean±s.e.m. (*P<0.05, **P<0.01, ****P<0.001, analysis of variance followed by Bonferroni’s post-hoc test).
Mentions: As the first step, to test the hypothesis that the nanoscale membrane bridges indeed facilitate intercellular communication from the cancer cells to endothelium, we loaded MDA-MB-231 breast cancer cells with a cell-impermeable dye, carboxyfluorescein succinimidyl ester (CFSE), before adding them to a co-culture with DiI-Ac-LDL-labelled endothelial cells. De-convolved volume 3D rendering of the co-cultures revealed transfer of cytoplasmic CFSE within the nanoscale membrane bridges connecting the cancer cells and endothelial cells (Fig. 4a and Supplementary Fig. 2B). Interestingly, although gap junctions have been reported to mediate intercellular transfers between cancer and endothelial cells33, here we observed intercellular transfer between distant cancer and endothelial cells that were not in direct physical contact except via the nanoscale membrane bridges (Fig. 4a). We next validated the intercellular transfer using flow cytometry. As a control, CFSE-loaded cancer cells and the endothelial cells were grown in the top and bottom chambers of a Boyden assay, respectively (dual culture), which allowed media contact and exosomes to cross through the 0.4-μm pores but did not allow direct physical contact, that is, no nanoscale membrane bridges could form between the cells (Fig. 4b and Supplementary Fig. 2C). In addition, we used a membrane with 3 μm pores, which allow both exosomes and larger vesicles to pass through (Supplementary Fig. 2C). The endothelial cells were first flow sorted from the dual/co-cultures using double labelling for DiI-Ac-LDL and platelet endothelial cell adhesion molecule-1 (PECAM-1) (Fig. 2c). The sorted endothelial cells were then analysed for CFSE and the subset of endothelial cells positive for CFSE was then quantified as a percentage of the total sorted endothelial cell population (Fig. 4c) as a measure of transfer from cancer cells. Indeed, we did observe intercellular transfer when the cells were separated in the Boyden assay (in both 0.4 and 3 μm pores), consistent with exosome- and extracellular vesicle-mediated transfer. Interestingly, as seen in Fig. 4d, <5% of the endothelial cells were found to be CSFE + ve in the dual culture (Boyden assay) as compared with ~30% of the endothelial cells being labelled as CFSE + ve when isolated from the co-cultures (after subtracting background signal from both conditions). It is possible that this transfer seen in the co-culture study includes the basal transfer arising from exosome- or gap-junction-mediated transfer. Similar results were described in the TNT-mediated transfer of p-glycoproteins, where two cells separated by a membrane with 0.4 μm pores exhibited a basal transfer consistent with exosome-mediated transfer as opposed to higher levels of transmission when TNTs were present26. Furthermore, the temporal quantification of intercellular transfer of CFSE revealed that the peak is reached between 24 and 36 h, lagging behind the kinetics of formation of these nanoscale structures (Fig. 4e). The lag in transfer kinetics is consistent with the notion that the nanoscale connections are not fully functional at the early stages of formation. A similar transfer was observed between the metastatic breast cancer cells and primary human vascular and lymphatic endothelial cells (Supplementary Fig. 3). In addition to CFSE, the nanostructures facilitated the transfer of nanoparticles (quantum dots) and proteins (green fluorescent protein) (Supplementary Fig. 4A–E). Interestingly, we did not observe a similar communication between metastatic tumour cells and vascular smooth muscle cells, further emphasizing the specificity of the communication between cancer cells and the endothelium (Supplementary Fig. 5). These results indicated that the nanoscale bridges act as conduits for communication from cancer cells to endothelial cells.

Bottom Line: Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis.The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits.These results lead us to define the notion of 'metastatic hijack': cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits.

View Article: PubMed Central - PubMed

Affiliation: Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts 02139, USA.

ABSTRACT
Metastasis is a major cause of mortality and remains a hurdle in the search for a cure for cancer. Not much is known about metastatic cancer cells and endothelial cross-talk, which occurs at multiple stages during metastasis. Here we report a dynamic regulation of the endothelium by cancer cells through the formation of nanoscale intercellular membrane bridges, which act as physical conduits for transfer of microRNAs. The communication between the tumour cell and the endothelium upregulates markers associated with pathological endothelium, which is reversed by pharmacological inhibition of these nanoscale conduits. These results lead us to define the notion of 'metastatic hijack': cancer cell-induced transformation of healthy endothelium into pathological endothelium via horizontal communication through the nanoscale conduits. Pharmacological perturbation of these nanoscale membrane bridges decreases metastatic foci in vivo. Targeting these nanoscale membrane bridges may potentially emerge as a new therapeutic opportunity in the management of metastatic cancer.

Show MeSH
Related in: MedlinePlus