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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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Cancer cell–endothelial intercellular transfer alters the endogenous miRNA profile and phenotype of recipient endothelial cells(a) Schema shows the experimental design. CFSE(green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. A miRNA microarray was used to evaluate the transport of endogenous miRNAs. The intercellular CFSE-transfer − ve and -transfer + ve endothelial cells were sorted from the same pool. The heat map shows potential miRNA candidates that were significantly upregulated in the cells receiving transfer of intercellular contents from the cancer cells. HUVECs that were not exposed to cancer cells were used as a baseline control. (b) The volcano plot shows the statistically significant upregulated (red) and downregulated (green) miRNAs in the HUVEC cells that received intercellular transfer compared with those that did not receive transfer. (c,d) Sorting of the endothelial cells from the co-cultures with MDA-MB-231 cells reveal higher expression of tumour endothelial markers CD137 and CD276 in intercellular transfer + ve endothelial cell populations compared with intercellular transfer − ve cells. (e,f) Pharmacological inhibition of nanoscale tether formation reduced the expression of CD137 and CD276 in endothelial cells isolated from the co-cultures. Data shown are mean±s.e.m. (n = 5 studies, with 3 replicates per study, ****P<0.0001, ***P<0.001, *P<0.05. Analysis of variance followed by Bonferroni’s post-hoc test).
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Figure 6: Cancer cell–endothelial intercellular transfer alters the endogenous miRNA profile and phenotype of recipient endothelial cells(a) Schema shows the experimental design. CFSE(green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. A miRNA microarray was used to evaluate the transport of endogenous miRNAs. The intercellular CFSE-transfer − ve and -transfer + ve endothelial cells were sorted from the same pool. The heat map shows potential miRNA candidates that were significantly upregulated in the cells receiving transfer of intercellular contents from the cancer cells. HUVECs that were not exposed to cancer cells were used as a baseline control. (b) The volcano plot shows the statistically significant upregulated (red) and downregulated (green) miRNAs in the HUVEC cells that received intercellular transfer compared with those that did not receive transfer. (c,d) Sorting of the endothelial cells from the co-cultures with MDA-MB-231 cells reveal higher expression of tumour endothelial markers CD137 and CD276 in intercellular transfer + ve endothelial cell populations compared with intercellular transfer − ve cells. (e,f) Pharmacological inhibition of nanoscale tether formation reduced the expression of CD137 and CD276 in endothelial cells isolated from the co-cultures. Data shown are mean±s.e.m. (n = 5 studies, with 3 replicates per study, ****P<0.0001, ***P<0.001, *P<0.05. Analysis of variance followed by Bonferroni’s post-hoc test).

Mentions: Once we had established that the nanoscale membrane bridges do act as physical conduits for horizontal transfer of miRNA, we next tested whether such transfers can alter the endogenous miRNA profiles in the recipient endothelial cells. We set up a simple experiment, where CFSE-loaded MDA-MB-231 cancer cells were co-cultured with endothelial cells for 36 h and the latter were then sorted into CFSE + ve (recipient) and CFSE − ve (non-recipient) populations (Fig. 6a). The non-recipient (CFSE −ve) endothelial cells therefore served as an internal control, as both pools (recipient and non-recipient) were exposed to the same exogenous cancer cell-secreted factors. An additional control group was run, where endothelial cells were cultured without any exposure to tumour cells, and were considered as naive cells. miRNA profiling data did indicate that culturing tumour cells with endothelial cells can alter the miRNA signature of the latter. Both recipient and non-recipient endothelial cells from the co-cultures exhibited a distinct miRNA profile compared with naive endothelial cells (Fig. 6a), consistent with previous reports implicating cell-secreted growth factors, microvesicles and exosomes in modulating the miRNA regulome11,37. For example, miR-18a expression, which can be induced by vascular endothelial growth factor, was upregulated in endothelial cells isolated from the co-culture and predicts a poor prognosis in patients with breast cancer38,39. The interesting finding was the distinct miRNA profiles of the two pools of recipient and non-recipient endothelial cells. For example, the recipient endothelial cells exhibited a significant number of upregulated miRNAs (Fig. 6a,b), many of which have been implicated in activation of endothelium and/or metastasis40–42. For example, miR-92, belonging to the miR-17-92 cluster (Oncomir-1), has been implicated in cancer metastasis to lymph nodes43. Similarly, transfer of miR-210 from metastatic cancer cells to endothelial cells results in angiogenesis and metastasis41. Furthermore, upregulation of miRNA-182 and miR29b was observed in invasive and metastatic breast cancer44,45. We also observed several downregulated miRNAs in the recipient endothelial cells (Fig. 6b). For example, miR150, which can target vascular endothelial growth factor-A, and miR885, which has been implicated as a tumour suppressor, were downregulated in the recipient endothelial cells46,47. At a phenotypic level, the analysis of cell surface markers revealed an upregulation in the expression of CD276 (Fig. 6c) and CD137 (Fig. 6d) in the recipient endothelial cells as compared with non-recipient endothelial cells from the same co-culture. Pharmacological inhibition of the nanoscale conduit formation decreased the expression of CD137 and CD276 in endothelial cells in the co-culture (Fig. 6e,f).


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)

Cancer cell–endothelial intercellular transfer alters the endogenous miRNA profile and phenotype of recipient endothelial cells(a) Schema shows the experimental design. CFSE(green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. A miRNA microarray was used to evaluate the transport of endogenous miRNAs. The intercellular CFSE-transfer − ve and -transfer + ve endothelial cells were sorted from the same pool. The heat map shows potential miRNA candidates that were significantly upregulated in the cells receiving transfer of intercellular contents from the cancer cells. HUVECs that were not exposed to cancer cells were used as a baseline control. (b) The volcano plot shows the statistically significant upregulated (red) and downregulated (green) miRNAs in the HUVEC cells that received intercellular transfer compared with those that did not receive transfer. (c,d) Sorting of the endothelial cells from the co-cultures with MDA-MB-231 cells reveal higher expression of tumour endothelial markers CD137 and CD276 in intercellular transfer + ve endothelial cell populations compared with intercellular transfer − ve cells. (e,f) Pharmacological inhibition of nanoscale tether formation reduced the expression of CD137 and CD276 in endothelial cells isolated from the co-cultures. Data shown are mean±s.e.m. (n = 5 studies, with 3 replicates per study, ****P<0.0001, ***P<0.001, *P<0.05. Analysis of variance followed by Bonferroni’s post-hoc test).
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Figure 6: Cancer cell–endothelial intercellular transfer alters the endogenous miRNA profile and phenotype of recipient endothelial cells(a) Schema shows the experimental design. CFSE(green)-loaded MDA-MB-231 cells were co-cultured with the Dil-Ac-LDL (red)-labelled HUVECs. A miRNA microarray was used to evaluate the transport of endogenous miRNAs. The intercellular CFSE-transfer − ve and -transfer + ve endothelial cells were sorted from the same pool. The heat map shows potential miRNA candidates that were significantly upregulated in the cells receiving transfer of intercellular contents from the cancer cells. HUVECs that were not exposed to cancer cells were used as a baseline control. (b) The volcano plot shows the statistically significant upregulated (red) and downregulated (green) miRNAs in the HUVEC cells that received intercellular transfer compared with those that did not receive transfer. (c,d) Sorting of the endothelial cells from the co-cultures with MDA-MB-231 cells reveal higher expression of tumour endothelial markers CD137 and CD276 in intercellular transfer + ve endothelial cell populations compared with intercellular transfer − ve cells. (e,f) Pharmacological inhibition of nanoscale tether formation reduced the expression of CD137 and CD276 in endothelial cells isolated from the co-cultures. Data shown are mean±s.e.m. (n = 5 studies, with 3 replicates per study, ****P<0.0001, ***P<0.001, *P<0.05. Analysis of variance followed by Bonferroni’s post-hoc test).
Mentions: Once we had established that the nanoscale membrane bridges do act as physical conduits for horizontal transfer of miRNA, we next tested whether such transfers can alter the endogenous miRNA profiles in the recipient endothelial cells. We set up a simple experiment, where CFSE-loaded MDA-MB-231 cancer cells were co-cultured with endothelial cells for 36 h and the latter were then sorted into CFSE + ve (recipient) and CFSE − ve (non-recipient) populations (Fig. 6a). The non-recipient (CFSE −ve) endothelial cells therefore served as an internal control, as both pools (recipient and non-recipient) were exposed to the same exogenous cancer cell-secreted factors. An additional control group was run, where endothelial cells were cultured without any exposure to tumour cells, and were considered as naive cells. miRNA profiling data did indicate that culturing tumour cells with endothelial cells can alter the miRNA signature of the latter. Both recipient and non-recipient endothelial cells from the co-cultures exhibited a distinct miRNA profile compared with naive endothelial cells (Fig. 6a), consistent with previous reports implicating cell-secreted growth factors, microvesicles and exosomes in modulating the miRNA regulome11,37. For example, miR-18a expression, which can be induced by vascular endothelial growth factor, was upregulated in endothelial cells isolated from the co-culture and predicts a poor prognosis in patients with breast cancer38,39. The interesting finding was the distinct miRNA profiles of the two pools of recipient and non-recipient endothelial cells. For example, the recipient endothelial cells exhibited a significant number of upregulated miRNAs (Fig. 6a,b), many of which have been implicated in activation of endothelium and/or metastasis40–42. For example, miR-92, belonging to the miR-17-92 cluster (Oncomir-1), has been implicated in cancer metastasis to lymph nodes43. Similarly, transfer of miR-210 from metastatic cancer cells to endothelial cells results in angiogenesis and metastasis41. Furthermore, upregulation of miRNA-182 and miR29b was observed in invasive and metastatic breast cancer44,45. We also observed several downregulated miRNAs in the recipient endothelial cells (Fig. 6b). For example, miR150, which can target vascular endothelial growth factor-A, and miR885, which has been implicated as a tumour suppressor, were downregulated in the recipient endothelial cells46,47. At a phenotypic level, the analysis of cell surface markers revealed an upregulation in the expression of CD276 (Fig. 6c) and CD137 (Fig. 6d) in the recipient endothelial cells as compared with non-recipient endothelial cells from the same co-culture. Pharmacological inhibition of the nanoscale conduit formation decreased the expression of CD137 and CD276 in endothelial cells in the co-culture (Fig. 6e,f).

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