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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.

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The nanoscale membrane bridges act as conduits for intercellular transfer of miRNA between cancer and endothelial cellsRepresentative confocal images show the transfer of Cy3-labelled miRNA from MDA-MB-231 cells (EPI) to endothelial cells (ENDO) at (a) 24 h and (b) 36 h of co-culture. Alexa Fluor 488-Ac-LDL (green)-labelled endothelial cells were co-cultured with Cy3-labelled miRNA-transfected MDA-MB-231. Co-cultures were counterstained with phalloidin (purple) and DAPI + WGA (blue). A 3D visualization shows the localization of miRNA within the nanoscale connections (white arrows), which act as conduits for horizontal transfer of miRNAs to endothelial cells. (c) Schema shows quantification of Cy3-labelled control miRNA and Cy3-labelled miR132 transfer between cancer cell and endothelium using flow cytometry. Endothelial cell populations were isolated from the co-cultures and percentage of miRNA + ve cells was determined. Dual cultures in Boyden chambers were included as controls. (d) Graph shows the effect of pharmacological disruption of nanoscale conduits on miRNA transfer. (e) Schema shows experimental design for reverse transcriptase–PCR-based detection of transferred miR-132 in endothelial cells under different experimental conditions. MDA-MB-231 cells transfected with miR-132 and α-miR-132 were co-cultured with endothelial tubes. FACS-isolated endothelial cell populations were analysed for the expression of miR-132. (f) Graph shows miR-132 + ve cell populations (solid red) show 5 × increase compared with miR-132 − ve populations (solid blue) (P<0.0001), whereas anti-miR-132 + ve cells (striped red) show 26 − decrease in miR-132 expression (P<0.0001) compared with α-miR-132 − ve cells (striped blue). Direct transfection of miR-132 (black) and α-miR-132 (light blue) in endothelial cells is used as positive and negative controls, respectively. Upregulation of miR-132 from baseline was observed in dual culture (solid green), which could be inhibited with anti-miR-132 (striped green). MiR-132 levels are increased compared with dual only in those cells that are positive for intercellular transfer. Fold change was determined compared with endothelial cell transfection with control miRNA (grey). (g) FACS analysis shows nanoscale bridges-mediated transfer of miRNAs leads to changes in p120RasGAP and pAkt (S473) expression downstream of the miR-132 pathway in endothelial cell populations isolated from co-cultures. (h) Graphs show p120RasGAP expression is decreased in the miR-132 + ve cell populations and increased in the α-miR-132 + ve cell populations, while further downstream miR-132 positively regulates pAkt expression. Data shown are mean±s.e.m. (N = 2–5 independent studies, with 3 replicates per study, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analysis of variance followed by Bonferroni’s post-hoc test).
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Figure 5: The nanoscale membrane bridges act as conduits for intercellular transfer of miRNA between cancer and endothelial cellsRepresentative confocal images show the transfer of Cy3-labelled miRNA from MDA-MB-231 cells (EPI) to endothelial cells (ENDO) at (a) 24 h and (b) 36 h of co-culture. Alexa Fluor 488-Ac-LDL (green)-labelled endothelial cells were co-cultured with Cy3-labelled miRNA-transfected MDA-MB-231. Co-cultures were counterstained with phalloidin (purple) and DAPI + WGA (blue). A 3D visualization shows the localization of miRNA within the nanoscale connections (white arrows), which act as conduits for horizontal transfer of miRNAs to endothelial cells. (c) Schema shows quantification of Cy3-labelled control miRNA and Cy3-labelled miR132 transfer between cancer cell and endothelium using flow cytometry. Endothelial cell populations were isolated from the co-cultures and percentage of miRNA + ve cells was determined. Dual cultures in Boyden chambers were included as controls. (d) Graph shows the effect of pharmacological disruption of nanoscale conduits on miRNA transfer. (e) Schema shows experimental design for reverse transcriptase–PCR-based detection of transferred miR-132 in endothelial cells under different experimental conditions. MDA-MB-231 cells transfected with miR-132 and α-miR-132 were co-cultured with endothelial tubes. FACS-isolated endothelial cell populations were analysed for the expression of miR-132. (f) Graph shows miR-132 + ve cell populations (solid red) show 5 × increase compared with miR-132 − ve populations (solid blue) (P<0.0001), whereas anti-miR-132 + ve cells (striped red) show 26 − decrease in miR-132 expression (P<0.0001) compared with α-miR-132 − ve cells (striped blue). Direct transfection of miR-132 (black) and α-miR-132 (light blue) in endothelial cells is used as positive and negative controls, respectively. Upregulation of miR-132 from baseline was observed in dual culture (solid green), which could be inhibited with anti-miR-132 (striped green). MiR-132 levels are increased compared with dual only in those cells that are positive for intercellular transfer. Fold change was determined compared with endothelial cell transfection with control miRNA (grey). (g) FACS analysis shows nanoscale bridges-mediated transfer of miRNAs leads to changes in p120RasGAP and pAkt (S473) expression downstream of the miR-132 pathway in endothelial cell populations isolated from co-cultures. (h) Graphs show p120RasGAP expression is decreased in the miR-132 + ve cell populations and increased in the α-miR-132 + ve cell populations, while further downstream miR-132 positively regulates pAkt expression. Data shown are mean±s.e.m. (N = 2–5 independent studies, with 3 replicates per study, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analysis of variance followed by Bonferroni’s post-hoc test).

Mentions: Although our study revealed the nanoscale membrane bridges could act as conduits for intercellular transfer, we rationalized that communication via the transfer of miRNAs from the cancer cells to the endothelium could result in the maximal amplification of signalling. Indeed, multiple studies have highlighted the role of miRNAs as signalling regulators in tumour cell migration and invasion36. For example, miR-132 was reported to be highly expressed in the endothelium of human tumours but was undetectable in normal endothelium. Furthermore, conditioned media from MDA-MB-231 cells was shown to upregulate miR-132 in endothelial cells37. As a proof-of-concept, we assessed whether the nanoscale membrane bridges can act as a physical conduit for transfer of miR-132 from metastatic cancer cells into endothelial cells. Cy3-labelled control miRNA or miR-132 was transfected in the metastatic MDA-MB-231 cells, which were then used to establish the co-cultures with endothelial cells. As shown in Fig. 5a,b (Supplementary Fig. 8), Cy3-labelled miRNAs were detected within the nanoscale bridges and were transferred to the endothelial cells. To further validate the transfer, we quantified Cy3-labelled miRNA in endothelial cells by flow cytometry (Fig. 5c,d). As a control, the cancer and endothelial cells were separated in dual chambers of a Boyden assay, which revealed a baseline transfer of Cy3-labelled miRNAs from the cancer cells to the endothelium that remained constant between 24 and 36 h. Indeed, a previous study reported that the kinetics of exosome-mediated miRNA transfer between MDA-MB-231 and endothelial cells starts by 4 h and peaks by 24 h17. In contrast, a significant increase in Cy3-labelled miRNAs in the endothelial cells was observed over baseline by 36 h of co-culture (Fig. 5d). Interestingly, pretreating the cancer cells with a combination of docetaxel and cytochalasin or latrunculin A, at concentrations previously established to inhibit the formation of the nanoscale membrane connections without affecting exosome shedding, reduced the elevated miRNA levels in the endothelial cells in the co-cultures but had no effect on basal transfer (Fig. 5d). This further validated that basal transfer is probably mediated by exosomes, whereas the nanoscale membrane bridges play a critical role as conduits for enhancing miRNA-mediated communication between the metastatic cancer cells and the endothelium.


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 transfer of miRNA between cancer and endothelial cellsRepresentative confocal images show the transfer of Cy3-labelled miRNA from MDA-MB-231 cells (EPI) to endothelial cells (ENDO) at (a) 24 h and (b) 36 h of co-culture. Alexa Fluor 488-Ac-LDL (green)-labelled endothelial cells were co-cultured with Cy3-labelled miRNA-transfected MDA-MB-231. Co-cultures were counterstained with phalloidin (purple) and DAPI + WGA (blue). A 3D visualization shows the localization of miRNA within the nanoscale connections (white arrows), which act as conduits for horizontal transfer of miRNAs to endothelial cells. (c) Schema shows quantification of Cy3-labelled control miRNA and Cy3-labelled miR132 transfer between cancer cell and endothelium using flow cytometry. Endothelial cell populations were isolated from the co-cultures and percentage of miRNA + ve cells was determined. Dual cultures in Boyden chambers were included as controls. (d) Graph shows the effect of pharmacological disruption of nanoscale conduits on miRNA transfer. (e) Schema shows experimental design for reverse transcriptase–PCR-based detection of transferred miR-132 in endothelial cells under different experimental conditions. MDA-MB-231 cells transfected with miR-132 and α-miR-132 were co-cultured with endothelial tubes. FACS-isolated endothelial cell populations were analysed for the expression of miR-132. (f) Graph shows miR-132 + ve cell populations (solid red) show 5 × increase compared with miR-132 − ve populations (solid blue) (P<0.0001), whereas anti-miR-132 + ve cells (striped red) show 26 − decrease in miR-132 expression (P<0.0001) compared with α-miR-132 − ve cells (striped blue). Direct transfection of miR-132 (black) and α-miR-132 (light blue) in endothelial cells is used as positive and negative controls, respectively. Upregulation of miR-132 from baseline was observed in dual culture (solid green), which could be inhibited with anti-miR-132 (striped green). MiR-132 levels are increased compared with dual only in those cells that are positive for intercellular transfer. Fold change was determined compared with endothelial cell transfection with control miRNA (grey). (g) FACS analysis shows nanoscale bridges-mediated transfer of miRNAs leads to changes in p120RasGAP and pAkt (S473) expression downstream of the miR-132 pathway in endothelial cell populations isolated from co-cultures. (h) Graphs show p120RasGAP expression is decreased in the miR-132 + ve cell populations and increased in the α-miR-132 + ve cell populations, while further downstream miR-132 positively regulates pAkt expression. Data shown are mean±s.e.m. (N = 2–5 independent studies, with 3 replicates per study, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analysis of variance followed by Bonferroni’s post-hoc test).
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Figure 5: The nanoscale membrane bridges act as conduits for intercellular transfer of miRNA between cancer and endothelial cellsRepresentative confocal images show the transfer of Cy3-labelled miRNA from MDA-MB-231 cells (EPI) to endothelial cells (ENDO) at (a) 24 h and (b) 36 h of co-culture. Alexa Fluor 488-Ac-LDL (green)-labelled endothelial cells were co-cultured with Cy3-labelled miRNA-transfected MDA-MB-231. Co-cultures were counterstained with phalloidin (purple) and DAPI + WGA (blue). A 3D visualization shows the localization of miRNA within the nanoscale connections (white arrows), which act as conduits for horizontal transfer of miRNAs to endothelial cells. (c) Schema shows quantification of Cy3-labelled control miRNA and Cy3-labelled miR132 transfer between cancer cell and endothelium using flow cytometry. Endothelial cell populations were isolated from the co-cultures and percentage of miRNA + ve cells was determined. Dual cultures in Boyden chambers were included as controls. (d) Graph shows the effect of pharmacological disruption of nanoscale conduits on miRNA transfer. (e) Schema shows experimental design for reverse transcriptase–PCR-based detection of transferred miR-132 in endothelial cells under different experimental conditions. MDA-MB-231 cells transfected with miR-132 and α-miR-132 were co-cultured with endothelial tubes. FACS-isolated endothelial cell populations were analysed for the expression of miR-132. (f) Graph shows miR-132 + ve cell populations (solid red) show 5 × increase compared with miR-132 − ve populations (solid blue) (P<0.0001), whereas anti-miR-132 + ve cells (striped red) show 26 − decrease in miR-132 expression (P<0.0001) compared with α-miR-132 − ve cells (striped blue). Direct transfection of miR-132 (black) and α-miR-132 (light blue) in endothelial cells is used as positive and negative controls, respectively. Upregulation of miR-132 from baseline was observed in dual culture (solid green), which could be inhibited with anti-miR-132 (striped green). MiR-132 levels are increased compared with dual only in those cells that are positive for intercellular transfer. Fold change was determined compared with endothelial cell transfection with control miRNA (grey). (g) FACS analysis shows nanoscale bridges-mediated transfer of miRNAs leads to changes in p120RasGAP and pAkt (S473) expression downstream of the miR-132 pathway in endothelial cell populations isolated from co-cultures. (h) Graphs show p120RasGAP expression is decreased in the miR-132 + ve cell populations and increased in the α-miR-132 + ve cell populations, while further downstream miR-132 positively regulates pAkt expression. Data shown are mean±s.e.m. (N = 2–5 independent studies, with 3 replicates per study, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, analysis of variance followed by Bonferroni’s post-hoc test).
Mentions: Although our study revealed the nanoscale membrane bridges could act as conduits for intercellular transfer, we rationalized that communication via the transfer of miRNAs from the cancer cells to the endothelium could result in the maximal amplification of signalling. Indeed, multiple studies have highlighted the role of miRNAs as signalling regulators in tumour cell migration and invasion36. For example, miR-132 was reported to be highly expressed in the endothelium of human tumours but was undetectable in normal endothelium. Furthermore, conditioned media from MDA-MB-231 cells was shown to upregulate miR-132 in endothelial cells37. As a proof-of-concept, we assessed whether the nanoscale membrane bridges can act as a physical conduit for transfer of miR-132 from metastatic cancer cells into endothelial cells. Cy3-labelled control miRNA or miR-132 was transfected in the metastatic MDA-MB-231 cells, which were then used to establish the co-cultures with endothelial cells. As shown in Fig. 5a,b (Supplementary Fig. 8), Cy3-labelled miRNAs were detected within the nanoscale bridges and were transferred to the endothelial cells. To further validate the transfer, we quantified Cy3-labelled miRNA in endothelial cells by flow cytometry (Fig. 5c,d). As a control, the cancer and endothelial cells were separated in dual chambers of a Boyden assay, which revealed a baseline transfer of Cy3-labelled miRNAs from the cancer cells to the endothelium that remained constant between 24 and 36 h. Indeed, a previous study reported that the kinetics of exosome-mediated miRNA transfer between MDA-MB-231 and endothelial cells starts by 4 h and peaks by 24 h17. In contrast, a significant increase in Cy3-labelled miRNAs in the endothelial cells was observed over baseline by 36 h of co-culture (Fig. 5d). Interestingly, pretreating the cancer cells with a combination of docetaxel and cytochalasin or latrunculin A, at concentrations previously established to inhibit the formation of the nanoscale membrane connections without affecting exosome shedding, reduced the elevated miRNA levels in the endothelial cells in the co-cultures but had no effect on basal transfer (Fig. 5d). This further validated that basal transfer is probably mediated by exosomes, whereas the nanoscale membrane bridges play a critical role as conduits for enhancing miRNA-mediated communication between the metastatic cancer cells and the endothelium.

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