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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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Structure and function of the heterotypic intercellular nanoscale membrane bridges(a) Representative images show the heterotypic nanoscale membrane bridges are composed of both F-actin and α/β-tubulin cytoskeletal components. Co-cultures were stained with α/β-tubulin antibody (green) and phalloidin (purple) to label actin, and counterstained with DAPI (nuclear) + WGA (plasma membrane) (blue). Endothelial cells were labelled with DiL-Ac-LDL (red). (b) Mathematical modelling of the structure of the nanoscale connections. The physical properties of actin filaments necessitate microtubules for projections of certain length scales. The maximum projection length for a given minimum diameter at the buckling limit is plotted for actin-only nanoscale structures (purple line). This curve is overlaid with the experimental length and diameter measurements (red dots) from the observed thin projections measured in these studies. Projections containing only actin or projections containing both actin and tubulin can exist to the right of the curve (purple line). However, actin-only projections cannot exist to the left of the curve (green region). (c) The effect of incorporating tubulin in these projections. The maximum length is plotted against the minimum diameter for varying fractions of tubulin incorporated in the nanoscale projection. Addition of microtubules to the projections increases the overall flexural rigidity, shifting the curves left of the actin-only limit (purple line), thus allowing for longer and thinner nanoscale connections. However, owing to the larger radius of microtubules (4 × radius of actin filaments), there is an optimal fraction of tubulin (green line) that can be incorporated into the projection before the effect is reversed. (d) The optimal fraction of microtubules is about 6.6% (red dashed line) to maximize nanostructure flexural strength, while minimizing thickness. (e) Representative confocal image shows the presence of myosin V motor proteins within the intercellular nanostructure (inset shows higher magnification). Scale bar 10 μm.
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Figure 3: Structure and function of the heterotypic intercellular nanoscale membrane bridges(a) Representative images show the heterotypic nanoscale membrane bridges are composed of both F-actin and α/β-tubulin cytoskeletal components. Co-cultures were stained with α/β-tubulin antibody (green) and phalloidin (purple) to label actin, and counterstained with DAPI (nuclear) + WGA (plasma membrane) (blue). Endothelial cells were labelled with DiL-Ac-LDL (red). (b) Mathematical modelling of the structure of the nanoscale connections. The physical properties of actin filaments necessitate microtubules for projections of certain length scales. The maximum projection length for a given minimum diameter at the buckling limit is plotted for actin-only nanoscale structures (purple line). This curve is overlaid with the experimental length and diameter measurements (red dots) from the observed thin projections measured in these studies. Projections containing only actin or projections containing both actin and tubulin can exist to the right of the curve (purple line). However, actin-only projections cannot exist to the left of the curve (green region). (c) The effect of incorporating tubulin in these projections. The maximum length is plotted against the minimum diameter for varying fractions of tubulin incorporated in the nanoscale projection. Addition of microtubules to the projections increases the overall flexural rigidity, shifting the curves left of the actin-only limit (purple line), thus allowing for longer and thinner nanoscale connections. However, owing to the larger radius of microtubules (4 × radius of actin filaments), there is an optimal fraction of tubulin (green line) that can be incorporated into the projection before the effect is reversed. (d) The optimal fraction of microtubules is about 6.6% (red dashed line) to maximize nanostructure flexural strength, while minimizing thickness. (e) Representative confocal image shows the presence of myosin V motor proteins within the intercellular nanostructure (inset shows higher magnification). Scale bar 10 μm.

Mentions: To characterize the compositional structure of the nanoscale membrane bridges connections, we labelled the co-culture with phalloidin to delineate the actin filaments, 4,6-diamidino-2-phenylindole (DAPI) and wheat germ agglutinin (WGA) to stain the nucleus and cell membrane, respectively, and used immunolabelling for tubulin. As shown in Fig. 3, intercellular nanoscale connections were continuous membranous structures composed of actin supported by tubulin cytoskeletal components (Fig. 3a and Supplementary Fig. 2A). Analysis of a population of the nanoscale membrane bridges revealed that ~70% were composed of both actin and tubulin, while the remaining were composed of only actin (Supplementary Fig. 2A). Previous studies have reported that membrane bridges or nanotubes could comprise only actin, as well as both actin and tubulin19. Indeed, extrapolating an established ‘filopodial’ mathematical model (Supplementary Discussion) to the observed structures indicates that at the observed mean diameter, a majority of the nanoscale membrane bridges, and especially the longer ones, should comprise both actin and tubulin, consistent with the fact that tubulin is critical for increasing flexural rigidity of the nanostructures at the observed length and diameter scales (Fig. 3b–d). However, owing to the larger radius of tubulins (4 × radius of actin filaments), there is an optimal fraction of tubulin (~6.6%) to maximize nanostructure flexural strength while minimizing thickness. In addition, immunocytochemistry revealed a punctate Myosin V motor protein expression within these nanostructures (Fig. 3e), consistent with the previous observation of Myosin V in TNTs31. Myosin V motors are known to transport cargo progressively along actin filaments32. Taken together with the increased propensity of metastatic cells to form heterotypic nanoscale connections and the directionality of growth towards the endothelium, these compositional observations suggested that these membrane nanostructures could function as an intercellular highway enabling transfer of materials from the cancer cells to 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)

Structure and function of the heterotypic intercellular nanoscale membrane bridges(a) Representative images show the heterotypic nanoscale membrane bridges are composed of both F-actin and α/β-tubulin cytoskeletal components. Co-cultures were stained with α/β-tubulin antibody (green) and phalloidin (purple) to label actin, and counterstained with DAPI (nuclear) + WGA (plasma membrane) (blue). Endothelial cells were labelled with DiL-Ac-LDL (red). (b) Mathematical modelling of the structure of the nanoscale connections. The physical properties of actin filaments necessitate microtubules for projections of certain length scales. The maximum projection length for a given minimum diameter at the buckling limit is plotted for actin-only nanoscale structures (purple line). This curve is overlaid with the experimental length and diameter measurements (red dots) from the observed thin projections measured in these studies. Projections containing only actin or projections containing both actin and tubulin can exist to the right of the curve (purple line). However, actin-only projections cannot exist to the left of the curve (green region). (c) The effect of incorporating tubulin in these projections. The maximum length is plotted against the minimum diameter for varying fractions of tubulin incorporated in the nanoscale projection. Addition of microtubules to the projections increases the overall flexural rigidity, shifting the curves left of the actin-only limit (purple line), thus allowing for longer and thinner nanoscale connections. However, owing to the larger radius of microtubules (4 × radius of actin filaments), there is an optimal fraction of tubulin (green line) that can be incorporated into the projection before the effect is reversed. (d) The optimal fraction of microtubules is about 6.6% (red dashed line) to maximize nanostructure flexural strength, while minimizing thickness. (e) Representative confocal image shows the presence of myosin V motor proteins within the intercellular nanostructure (inset shows higher magnification). Scale bar 10 μm.
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Figure 3: Structure and function of the heterotypic intercellular nanoscale membrane bridges(a) Representative images show the heterotypic nanoscale membrane bridges are composed of both F-actin and α/β-tubulin cytoskeletal components. Co-cultures were stained with α/β-tubulin antibody (green) and phalloidin (purple) to label actin, and counterstained with DAPI (nuclear) + WGA (plasma membrane) (blue). Endothelial cells were labelled with DiL-Ac-LDL (red). (b) Mathematical modelling of the structure of the nanoscale connections. The physical properties of actin filaments necessitate microtubules for projections of certain length scales. The maximum projection length for a given minimum diameter at the buckling limit is plotted for actin-only nanoscale structures (purple line). This curve is overlaid with the experimental length and diameter measurements (red dots) from the observed thin projections measured in these studies. Projections containing only actin or projections containing both actin and tubulin can exist to the right of the curve (purple line). However, actin-only projections cannot exist to the left of the curve (green region). (c) The effect of incorporating tubulin in these projections. The maximum length is plotted against the minimum diameter for varying fractions of tubulin incorporated in the nanoscale projection. Addition of microtubules to the projections increases the overall flexural rigidity, shifting the curves left of the actin-only limit (purple line), thus allowing for longer and thinner nanoscale connections. However, owing to the larger radius of microtubules (4 × radius of actin filaments), there is an optimal fraction of tubulin (green line) that can be incorporated into the projection before the effect is reversed. (d) The optimal fraction of microtubules is about 6.6% (red dashed line) to maximize nanostructure flexural strength, while minimizing thickness. (e) Representative confocal image shows the presence of myosin V motor proteins within the intercellular nanostructure (inset shows higher magnification). Scale bar 10 μm.
Mentions: To characterize the compositional structure of the nanoscale membrane bridges connections, we labelled the co-culture with phalloidin to delineate the actin filaments, 4,6-diamidino-2-phenylindole (DAPI) and wheat germ agglutinin (WGA) to stain the nucleus and cell membrane, respectively, and used immunolabelling for tubulin. As shown in Fig. 3, intercellular nanoscale connections were continuous membranous structures composed of actin supported by tubulin cytoskeletal components (Fig. 3a and Supplementary Fig. 2A). Analysis of a population of the nanoscale membrane bridges revealed that ~70% were composed of both actin and tubulin, while the remaining were composed of only actin (Supplementary Fig. 2A). Previous studies have reported that membrane bridges or nanotubes could comprise only actin, as well as both actin and tubulin19. Indeed, extrapolating an established ‘filopodial’ mathematical model (Supplementary Discussion) to the observed structures indicates that at the observed mean diameter, a majority of the nanoscale membrane bridges, and especially the longer ones, should comprise both actin and tubulin, consistent with the fact that tubulin is critical for increasing flexural rigidity of the nanostructures at the observed length and diameter scales (Fig. 3b–d). However, owing to the larger radius of tubulins (4 × radius of actin filaments), there is an optimal fraction of tubulin (~6.6%) to maximize nanostructure flexural strength while minimizing thickness. In addition, immunocytochemistry revealed a punctate Myosin V motor protein expression within these nanostructures (Fig. 3e), consistent with the previous observation of Myosin V in TNTs31. Myosin V motors are known to transport cargo progressively along actin filaments32. Taken together with the increased propensity of metastatic cells to form heterotypic nanoscale connections and the directionality of growth towards the endothelium, these compositional observations suggested that these membrane nanostructures could function as an intercellular highway enabling transfer of materials from the cancer cells to 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