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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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Nanoscale membrane bridge-mediated intercellular communication in vivo(a) Three-dimensional confocal reconstructions demonstrate the nanoscale membrane bridges from CFSE-loaded MDA-MB-231 cancer cells transferring CFSE to the vascular endothelial growth factor receptor 2 (VEGFR2)/PECAM1-labelled endothelial cells (white solid outline). Examples of intercellular transfer are indicated with yellow arrows. Animals were injected with CFSE-loaded MDA-MB-231 cancer cells via the tail vein and the lungs were excised at defined time points to monitor cancer cell–endothelial interactions. (b) Graph shows the effect of pre-treatment of MDA-MB-231 cancer cells with low-dose pharmacological inhibitors of nanoscale membrane bridges on the transfer of CFSE to lung endothelial cells in vivo. Endothelial cells were isolated from the mouse lungs 48 h post injection. Heterotypic intercellular transfer was quantified using FACS. (c) Confocal images of lung sections from balb/c mice treated with CFSE-loaded 4T1 metastatic breast cancer cells show transfer of CFSE to vWF-labelled lung endothelial cells in vehicle treated but not in the case of drug pretreatment that inhibits the formation of nanoscale connections. (d) Graph shows the number of CFSE + ve tumour cells in the lungs 24 h post injection, showing no significant (NS) difference between pretreated and vehicle-treated groups. (e) Graph shows a reduction in metastatic foci at day 7 in the pretreated, that is, inhibition of nanoscale connections, versus vehicle-treated groups. Data shown are mean±s.e.m. The total number of tumour cells over ten sections was quantified for each lung. Lungs were isolated from n = 4 mice in each treatment group (**P<0.01 versus vehicle treated). (f,g) Isolation of lung endothelial cells and quantification of CD137 and CD276 expression using FACS reveal decreased expressions in conditions where the injected MDA-MB-231 cells were pretreated with pharmacological inhibitors to disrupt the nanoscale connections. (1) Naive endothelial cells; (2) endothelial cells from cytochalasin + docetaxel-treated group; (3) docetaxel + Latrunculin A-treated group; and (4) endothelial cells from vehicle-treated MDA-MB-231 group. Data shown are mean±s.e.m. (n = 3, **P<0.01, ***P<0.001 versus control, analysis of variance followed by Bonferroni’s post-hoc test). (h) Schematic illustration of potential role of nanoscale conduit-mediated intercellular heterotypic communication in metastatic progression. The horizontal transfer of cellular material, including miRNA, can alter the endothelial regulon and switch the endothelial barrier to a dysfunctional ‘enabling’ pre-metastatic niche.
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Figure 7: Nanoscale membrane bridge-mediated intercellular communication in vivo(a) Three-dimensional confocal reconstructions demonstrate the nanoscale membrane bridges from CFSE-loaded MDA-MB-231 cancer cells transferring CFSE to the vascular endothelial growth factor receptor 2 (VEGFR2)/PECAM1-labelled endothelial cells (white solid outline). Examples of intercellular transfer are indicated with yellow arrows. Animals were injected with CFSE-loaded MDA-MB-231 cancer cells via the tail vein and the lungs were excised at defined time points to monitor cancer cell–endothelial interactions. (b) Graph shows the effect of pre-treatment of MDA-MB-231 cancer cells with low-dose pharmacological inhibitors of nanoscale membrane bridges on the transfer of CFSE to lung endothelial cells in vivo. Endothelial cells were isolated from the mouse lungs 48 h post injection. Heterotypic intercellular transfer was quantified using FACS. (c) Confocal images of lung sections from balb/c mice treated with CFSE-loaded 4T1 metastatic breast cancer cells show transfer of CFSE to vWF-labelled lung endothelial cells in vehicle treated but not in the case of drug pretreatment that inhibits the formation of nanoscale connections. (d) Graph shows the number of CFSE + ve tumour cells in the lungs 24 h post injection, showing no significant (NS) difference between pretreated and vehicle-treated groups. (e) Graph shows a reduction in metastatic foci at day 7 in the pretreated, that is, inhibition of nanoscale connections, versus vehicle-treated groups. Data shown are mean±s.e.m. The total number of tumour cells over ten sections was quantified for each lung. Lungs were isolated from n = 4 mice in each treatment group (**P<0.01 versus vehicle treated). (f,g) Isolation of lung endothelial cells and quantification of CD137 and CD276 expression using FACS reveal decreased expressions in conditions where the injected MDA-MB-231 cells were pretreated with pharmacological inhibitors to disrupt the nanoscale connections. (1) Naive endothelial cells; (2) endothelial cells from cytochalasin + docetaxel-treated group; (3) docetaxel + Latrunculin A-treated group; and (4) endothelial cells from vehicle-treated MDA-MB-231 group. Data shown are mean±s.e.m. (n = 3, **P<0.01, ***P<0.001 versus control, analysis of variance followed by Bonferroni’s post-hoc test). (h) Schematic illustration of potential role of nanoscale conduit-mediated intercellular heterotypic communication in metastatic progression. The horizontal transfer of cellular material, including miRNA, can alter the endothelial regulon and switch the endothelial barrier to a dysfunctional ‘enabling’ pre-metastatic niche.

Mentions: We next studied whether the nanoscale membrane bridge-mediated intercellular communication between cancer cells and the endothelium occurs in vivo. We used well-established in vivo models that capture the extravasation step of metastasis7,48. MDA-MB-231 cells, which metastasize to the lungs, were injected intravenously (i.v.) in mice. The animals were killed at the specified time points and the interaction between the CFSE-loaded cancer cells and the endothelium of the lung vasculature was studied by confocal microscopy after immunolabelling the endothelial cells. As seen in Fig. 7a and Supplementary Fig. 10A, CFSE + ve cancer cells were visualized adjacent to the lung endothelium as early as 18 h post injection and an increasing number of cancer cells in close proximity were evident by 48 h, consistent with the in vitro observation that the cancer cells tended to cluster around an endothelial niche. Interestingly, intercellular transfer of CFSE to the endothelial cells was detected by 18 h (Fig. 7a). By 72 h, micrometastases were found in the lung parenchyma. To validate that the transfer of CFSE from cancer cells to the endothelial cells is indeed mediated by the nanoscale conduits, we pre-treated the CFSE-loaded cancer cells with low-dose pharmacological inhibitors to block nanobridge formation. Treatment-naive and pharmacological inhibitor-treated cancer cells were then injected i.v. into mice, which were killed at 48 h post injection. The lung endothelial cells were isolated using magnetic separation and were sorted into CFSE + ve and −ve population. As shown in Fig. 7b, pharmacological inhibition of nanoscale conduit formation resulted in a decrease in the intercellular transfer of CFSE compared with treatment-naive cancer 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)

Nanoscale membrane bridge-mediated intercellular communication in vivo(a) Three-dimensional confocal reconstructions demonstrate the nanoscale membrane bridges from CFSE-loaded MDA-MB-231 cancer cells transferring CFSE to the vascular endothelial growth factor receptor 2 (VEGFR2)/PECAM1-labelled endothelial cells (white solid outline). Examples of intercellular transfer are indicated with yellow arrows. Animals were injected with CFSE-loaded MDA-MB-231 cancer cells via the tail vein and the lungs were excised at defined time points to monitor cancer cell–endothelial interactions. (b) Graph shows the effect of pre-treatment of MDA-MB-231 cancer cells with low-dose pharmacological inhibitors of nanoscale membrane bridges on the transfer of CFSE to lung endothelial cells in vivo. Endothelial cells were isolated from the mouse lungs 48 h post injection. Heterotypic intercellular transfer was quantified using FACS. (c) Confocal images of lung sections from balb/c mice treated with CFSE-loaded 4T1 metastatic breast cancer cells show transfer of CFSE to vWF-labelled lung endothelial cells in vehicle treated but not in the case of drug pretreatment that inhibits the formation of nanoscale connections. (d) Graph shows the number of CFSE + ve tumour cells in the lungs 24 h post injection, showing no significant (NS) difference between pretreated and vehicle-treated groups. (e) Graph shows a reduction in metastatic foci at day 7 in the pretreated, that is, inhibition of nanoscale connections, versus vehicle-treated groups. Data shown are mean±s.e.m. The total number of tumour cells over ten sections was quantified for each lung. Lungs were isolated from n = 4 mice in each treatment group (**P<0.01 versus vehicle treated). (f,g) Isolation of lung endothelial cells and quantification of CD137 and CD276 expression using FACS reveal decreased expressions in conditions where the injected MDA-MB-231 cells were pretreated with pharmacological inhibitors to disrupt the nanoscale connections. (1) Naive endothelial cells; (2) endothelial cells from cytochalasin + docetaxel-treated group; (3) docetaxel + Latrunculin A-treated group; and (4) endothelial cells from vehicle-treated MDA-MB-231 group. Data shown are mean±s.e.m. (n = 3, **P<0.01, ***P<0.001 versus control, analysis of variance followed by Bonferroni’s post-hoc test). (h) Schematic illustration of potential role of nanoscale conduit-mediated intercellular heterotypic communication in metastatic progression. The horizontal transfer of cellular material, including miRNA, can alter the endothelial regulon and switch the endothelial barrier to a dysfunctional ‘enabling’ pre-metastatic niche.
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Figure 7: Nanoscale membrane bridge-mediated intercellular communication in vivo(a) Three-dimensional confocal reconstructions demonstrate the nanoscale membrane bridges from CFSE-loaded MDA-MB-231 cancer cells transferring CFSE to the vascular endothelial growth factor receptor 2 (VEGFR2)/PECAM1-labelled endothelial cells (white solid outline). Examples of intercellular transfer are indicated with yellow arrows. Animals were injected with CFSE-loaded MDA-MB-231 cancer cells via the tail vein and the lungs were excised at defined time points to monitor cancer cell–endothelial interactions. (b) Graph shows the effect of pre-treatment of MDA-MB-231 cancer cells with low-dose pharmacological inhibitors of nanoscale membrane bridges on the transfer of CFSE to lung endothelial cells in vivo. Endothelial cells were isolated from the mouse lungs 48 h post injection. Heterotypic intercellular transfer was quantified using FACS. (c) Confocal images of lung sections from balb/c mice treated with CFSE-loaded 4T1 metastatic breast cancer cells show transfer of CFSE to vWF-labelled lung endothelial cells in vehicle treated but not in the case of drug pretreatment that inhibits the formation of nanoscale connections. (d) Graph shows the number of CFSE + ve tumour cells in the lungs 24 h post injection, showing no significant (NS) difference between pretreated and vehicle-treated groups. (e) Graph shows a reduction in metastatic foci at day 7 in the pretreated, that is, inhibition of nanoscale connections, versus vehicle-treated groups. Data shown are mean±s.e.m. The total number of tumour cells over ten sections was quantified for each lung. Lungs were isolated from n = 4 mice in each treatment group (**P<0.01 versus vehicle treated). (f,g) Isolation of lung endothelial cells and quantification of CD137 and CD276 expression using FACS reveal decreased expressions in conditions where the injected MDA-MB-231 cells were pretreated with pharmacological inhibitors to disrupt the nanoscale connections. (1) Naive endothelial cells; (2) endothelial cells from cytochalasin + docetaxel-treated group; (3) docetaxel + Latrunculin A-treated group; and (4) endothelial cells from vehicle-treated MDA-MB-231 group. Data shown are mean±s.e.m. (n = 3, **P<0.01, ***P<0.001 versus control, analysis of variance followed by Bonferroni’s post-hoc test). (h) Schematic illustration of potential role of nanoscale conduit-mediated intercellular heterotypic communication in metastatic progression. The horizontal transfer of cellular material, including miRNA, can alter the endothelial regulon and switch the endothelial barrier to a dysfunctional ‘enabling’ pre-metastatic niche.
Mentions: We next studied whether the nanoscale membrane bridge-mediated intercellular communication between cancer cells and the endothelium occurs in vivo. We used well-established in vivo models that capture the extravasation step of metastasis7,48. MDA-MB-231 cells, which metastasize to the lungs, were injected intravenously (i.v.) in mice. The animals were killed at the specified time points and the interaction between the CFSE-loaded cancer cells and the endothelium of the lung vasculature was studied by confocal microscopy after immunolabelling the endothelial cells. As seen in Fig. 7a and Supplementary Fig. 10A, CFSE + ve cancer cells were visualized adjacent to the lung endothelium as early as 18 h post injection and an increasing number of cancer cells in close proximity were evident by 48 h, consistent with the in vitro observation that the cancer cells tended to cluster around an endothelial niche. Interestingly, intercellular transfer of CFSE to the endothelial cells was detected by 18 h (Fig. 7a). By 72 h, micrometastases were found in the lung parenchyma. To validate that the transfer of CFSE from cancer cells to the endothelial cells is indeed mediated by the nanoscale conduits, we pre-treated the CFSE-loaded cancer cells with low-dose pharmacological inhibitors to block nanobridge formation. Treatment-naive and pharmacological inhibitor-treated cancer cells were then injected i.v. into mice, which were killed at 48 h post injection. The lung endothelial cells were isolated using magnetic separation and were sorted into CFSE + ve and −ve population. As shown in Fig. 7b, pharmacological inhibition of nanoscale conduit formation resulted in a decrease in the intercellular transfer of CFSE compared with treatment-naive cancer 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